Oligonucleotide therapeutic agents and their use

Abstract

The present invention relates to an oligonucleotide therapeutic agent. The oligonucleotide therapeutic agent has at least one oligonucleotide conjugated to dodecylamine, where the oligonucleotide is conjugated to the dodecylamine at its 5' end. The oligonucleotide therapeutic agent may further comprise polyethylene glycol (PEG) conjugated to the dodecylamine at the amino end of the dodecylamine, and may further comprise a peptide linker between the dodecylamine and the PEG.

Description

[Technical Field]

[0001] The present invention relates to an oligonucleotide therapeutic agent, and more particularly to an oligonucleotide conjugated with dodecylamine. The oligonucleotide therapeutic agent may further comprise polyethylene glycol (PEG) conjugated with the dodecylamine, and may even further comprise a peptide linker between the dodecylamine and the PEG. [Background technology]

[0002] According to 2023 statistics from the World Health Organization (WHO), more than 55 million people worldwide suffer from dementia, with an additional 10 million cases reported annually. Alzheimer's disease (AD) is the most common neurodegenerative disease and the primary cause of dementia. This disease is caused by the accumulation of beta-amyloid (Aβ), abnormal phosphorylation of the Tau protein, and excessive inflammation of nerves. Numerous studies have shown that beta-amyloid contributes to plaque buildup and further exacerbates neurotoxicity in the brain. Furthermore, excessive phosphorylation of the tau protein leads to the formation of neurofibrillary tangles (NFTs), further causing irreversible neuronal cell death (Bloom, 2014).

[0003] Recent studies have shown that neuroinflammation is one of the primary causes of Alzheimer's disease (Kinney et al., 2018). Such neuroinflammatory phenomena, enhanced by Aβ deposition, stimulate microglial cells to release highly neurotoxic inflammatory factors, further promoting the development of cerebral inflammatory responses (Lueg et al., 2015; Hansen et al., 2018). Therefore, how to suppress and mitigate inflammatory responses, particularly neuroinflammation, is crucial for the prevention of neurodegenerative diseases such as dementia and Alzheimer's disease. [Overview of the project] [Means for solving the problem]

[0004] At least part of this invention is based on the following findings: Dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (referred to as C12-miR) unexpectedly increases PD-L1 expression in cells, which is the opposite of previous reports (Anastasiadou et al., 2021; Zhang et al., 2023) regarding the inhibitory effect of microRNA 200c-3p mimics or microRNA 200c-3p expression vectors on PD-L1 expression in cells. Polyethylene glycolation of C12-miR and the addition of a peptide linker (SEQ ID NO: 2) between PEG and C12-miR can further induce more PD-L1 expression in cells. Based on the PD-1 / PD-L1 receptor-ligand axis, the dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR), polyethylene glycolated C12-miRs, and C12-miRs conjugated with polyethylene glycolated peptides according to the present invention can be used as oligonucleotide therapeutic agents to reduce and suppress inflammatory responses, particularly neuroinflammatory responses.

[0005] Accordingly, in some specific embodiments, the present invention provides an oligonucleotide therapeutic agent having at least one oligonucleotide that conjugates with dodecylamine, wherein the oligonucleotide conjugates with the dodecylamine at its 5' end. In other specific embodiments, the oligonucleotide therapeutic agent may further comprise polyethylene glycol (PEG) conjugating to the dodecylamine at its amino group end. In other specific embodiments, the oligonucleotide therapeutic agent may further comprise a peptide linker between the dodecylamine and the PEG.

[0006] The present invention also provides uses or applications of the oligonucleotide therapeutic agent. In some specific embodiments, the oligonucleotide therapeutic agent according to the present invention can be used to increase PD-L1 expression in cells. In other specific embodiments, the oligonucleotide therapeutic agent according to the present invention can be used to increase PD-L1 expression in the body of a test subject. In other specific embodiments, the oligonucleotide therapeutic agent according to the present invention can be used to prevent, reduce, suppress, or treat inflammatory responses in the body of a test subject.

[0007] Those skilled in the art will recognize, or can determine by ordinary experiment alone, many equivalents in the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the following specific embodiments.

[0008] Specific Example 1. An oligonucleotide therapeutic agent comprising an oligonucleotide and a dodecylamine, wherein the first carbon atom of the dodecylamine is conjugated with the oligonucleotide at the 5' end of the oligonucleotide, and the amino group of the dodecylamine is located on the twelfth carbon atom of the dodecylamine.

[0009] Specific Example 2. The oligonucleotide therapeutic agent described in Specific Example 1, wherein the oligonucleotide is a microRNA.

[0010] Specific Example 3. The oligonucleotide therapeutic agent according to Specific Example 1 or 2, wherein the oligonucleotide in question consists of the sequence indicated by SEQ ID NO: 1.

[0011] Specific Example 4. An oligonucleotide therapeutic agent according to any one of Specific Examples 1 to 3, further comprising polyethylene glycol (PEG) conjugated with the dodecylamine at the amino group end of the dodecylamine.

[0012] Specific Example 5. The oligonucleotide therapeutic agent described in Specific Example 4, wherein the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

[0013] Specific Example 6. An oligonucleotide therapeutic agent according to any one of Specific Examples 1 to 3, further comprising a peptide linker that conjugates with the dodecylamine at the amino group end of the dodecylamine, and polyethylene glycol (PEG) that conjugates with the peptide linker at the amino group end of the peptide linker.

[0014] Specific Example 7. The oligonucleotide therapeutic agent according to Specific Example 6, wherein the peptide linker consists of the sequence indicated by SEQ ID NO: 2.

[0015] Specific Example 8. The oligonucleotide therapeutic agent according to Specific Example 6 or 7, wherein the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

[0016] Specific Example 9. An oligonucleotide therapeutic agent selected from the group consisting of the following, as described in any of Specific Examples 1 to 8. An oligonucleotide therapeutic agent comprising an oligonucleotide and a dodecylamine, wherein the first carbon atom of the dodecylamine is conjugated with the oligonucleotide at its 5' end, and the amino group of the dodecylamine is located on the twelfth carbon atom of the dodecylamine; An oligonucleotide therapeutic agent comprising an oligonucleotide, dodecylamine, and polyethylene glycol (PEG), wherein the first carbon atom of the dodecylamine is conjugated with the oligonucleotide at its 5' end, the amino group of the dodecylamine is located on the twelfth carbon atom of the dodecylamine, and the polyethylene glycol (PEG) is conjugated with the dodecylamine at the amino group end of the dodecylamine; and An oligonucleotide therapeutic agent comprising an oligonucleotide, dodecylamine, a peptide linker, and polyethylene glycol (PEG), wherein at the 5' end of the oligonucleotide, the first carbon atom of the dodecylamine is conjugated with the oligonucleotide, the amino group of the dodecylamine is located on the twelfth carbon atom of the dodecylamine, the peptide linker is conjugated with the dodecylamine at the amino group end of the dodecylamine, and the polyethylene glycol (PEG) is conjugated with the peptide linker at the amino group end of the peptide linker.

[0017] Specific Example 10. An oligonucleotide therapeutic agent according to any one of Specific Examples 1 to 9, comprising an oligonucleotide and a dodecylamine, wherein the first carbon atom of the dodecylamine is conjugated with the oligonucleotide at its 5' end, and the amino group of the dodecylamine is located on the twelfth carbon atom of the dodecylamine.

[0018] Specific Example 11. An oligonucleotide therapeutic agent comprising an oligonucleotide, dodecylamine, and polyethylene glycol (PEG), wherein at the 5'-end of the oligonucleotide, the first carbon atom of the dodecylamine is conjugated to the oligonucleotide, the amino group of the dodecylamine is on the 12th carbon atom of the dodecylamine, and at the amino group end of the dodecylamine, the polyethylene glycol (PEG) is conjugated to the dodecylamine, and which is the oligonucleotide therapeutic agent according to any one of Specific Examples 1 to 9.

[0019] Specific Example 12. An oligonucleotide therapeutic agent comprising an oligonucleotide, dodecylamine, a peptide linker, and polyethylene glycol (PEG), wherein at the 5'-end of the oligonucleotide, the first carbon atom of the dodecylamine is conjugated to the oligonucleotide, the amino group of the dodecylamine is on the 12th carbon atom of the dodecylamine, at the amino group end of the dodecylamine, the peptide linker is conjugated to the dodecylamine, and at the amino group end of the peptide linker, the polyethylene glycol (PEG) is conjugated to the peptide linker, and which is the oligonucleotide therapeutic agent according to any one of Specific Examples 1 to 9.

[0020] Specific Example 13. The oligonucleotide therapeutic agent according to any one of claims 9 to 12, wherein the oligonucleotide is a microRNA.

[0021] Specific Example 14. The oligonucleotide therapeutic agent according to any one of claims 9 to 13, wherein the oligonucleotide consists of the sequence represented by SEQ ID NO: 1.

[0022] Specific Example 15. The oligonucleotide therapeutic agent according to any one of Specific Examples 9 to 14, wherein the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 208000.

[0023] Specific Example 16. An oligonucleotide therapeutic agent according to any of Specific Examples 9 to 15, wherein the peptide linker consists of the sequence indicated by SEQ ID NO: 2.

[0024] Specific Example 17. A composition comprising an oligonucleotide therapeutic agent described in any of Specific Examples 1 to 16 and a pharmaceutically acceptable carrier or excipient.

[0025] Specific Example 18. A method for increasing PD-L1 expression in cells, comprising contacting the cells with an oligonucleotide therapeutic agent described in any of Specific Examples 1 to 16 or a composition described in Specific Example 17.

[0026] Specific Example 19. A method for increasing PD-L1 expression in a subject's body, comprising administering to the subject an oligonucleotide therapeutic agent described in any of Specific Examples 1 to 16 or the composition described in Specific Example 17.

[0027] Specific Example 20. A method for preventing, reducing, suppressing, or treating an inflammatory response in a subject's body, comprising administering to the subject a pharmaceutically effective amount of an oligonucleotide therapeutic agent described in any of Specific Examples 1 to 16 or the composition described in Specific Example 17.

[0028] Specific Example 21. The method according to Specific Example 20, wherein the inflammatory response in the subject's body is a neuroinflammatory response.

[0029] Specific Example 22. Use of an oligonucleotide therapeutic agent described in any of Specific Examples 1 to 16 or the composition described in Specific Example 17 in increasing PD-L1 expression in cells.

[0030] Specific Example 23. The use of an oligonucleotide therapeutic agent described in any of Specific Examples 1 to 16 or the composition described in Specific Example 17 in increasing PD-L1 expression in the body of a test subject.

[0031] Specific Example 24. Use of an oligonucleotide therapeutic agent described in any of Specific Examples 1 to 16 or the composition described in Specific Example 17 in preventing, reducing, suppressing, or treating inflammatory responses in the body of a test subject.

[0032] Specific Example 25. The use described in Specific Example 24, wherein the inflammatory response in the subject's body is a neuroinflammatory response.

[0033] Specific Example 26. Use of an oligonucleotide therapeutic agent described in any of Specific Examples 1 to 16 or the composition described in Specific Example 17 in the preparation of a drug that increases PD-L1 expression in cells.

[0034] Specific Example 27. The use of an oligonucleotide therapeutic agent described in any of Specific Examples 1 to 16 or the composition described in Specific Example 17 in the preparation of a drug that increases PD-L1 expression in the body of a test subject.

[0035] Specific Example 28. The use of an oligonucleotide therapeutic agent described in any of Specific Examples 1 to 16 or the composition described in Specific Example 17 in the preparation of a drug for preventing, reducing, suppressing, or treating an inflammatory response in the body of a test subject.

[0036] Specific Example 29. The use described in Specific Example 28, wherein the inflammatory response in the subject's body is a neuroinflammatory response.

[0037] The following description of preferred specific embodiments, when combined with the drawings, will clarify these and other aspects.

[0038] The drawings illustrate one or more specific embodiments of the present invention and are used together with the written description to interpret the principles of the present invention. Where possible, the same reference numerals in the drawings are used throughout to refer to the same or similar components in the specific embodiments. [Brief explanation of the drawing]

[0039] [Figure 1] A schematic diagram of the design of the oligonucleotide therapeutic agent according to the present invention is shown. miR, C12, and Linker represent microRNA, dodecylamine, and peptide linker (SEQ ID NO: 2), respectively. [Figure 2] Figure 2 shows the results of the characteristic analysis of the oligonucleotide therapeutic agent prepared in Example 1. The characteristic analysis was performed by electrophoresis on a 15% (v / v) polyacrylamide gel containing 8 M urea, followed by staining with 0.2% (w / v) methylene blue. The arrows indicate the positions of each size (7 kDa and 8 kDa) on the gel. [Figure 3] The expression levels of PD-L1 in SH-SY5Y human neuroblastoma cells (ATCC® CRL-2266) treated for 48 hours with phosphate-buffered saline (PBS) (blank control group), C12-miR 2 μM, P.5-C12-miR 2 μM, or P.5-L-C12-miR 2 μM in Example 2 are shown. In Example 2, PD-L1 expression was analyzed by flow cytometry using an anti-human PD-L1 surface antibody. Results are expressed as mean values, error bars represent standard deviations, and statistical significance is calculated by Student's t-test. Compared to the blank control group, * p<0.05, ** p<0.01, *** p<0.001. Compared to the C12-miR group, # p<0.05, ### p<0.001. For the P.5-C12-miR group, † p<0.05. [Figure 4]This study shows the effects of different concentrations (0.5 μM, 2 μM, and 4 μM) of C12-miR and P.5-C12-miR on PD-L1 expression in SH-SY5Y human neuroblastoma cells (ATCC® CRL-2266) in Example 3. PD-L1 expression was analyzed by flow cytometry using an anti-human PD-L1 surface antibody. Results are expressed as mean values, error bars represent standard deviations, and statistical significance is calculated by Student's t-test. Compared to the blank control group (0 μM), ** p<0.01, *** p<0.001. Compared to the C12-miR group, # p<0.05, ## p<0.01. [Figure 5] This example shows the effect of different sized PEG and C12-miR conjugates on PD-L1 expression in SH-SY5Y human neuroblastoma cells (ATCC® CRL-2266) in Example 4. PD-L1 expression was analyzed by flow cytometry using an anti-human PD-L1 surface antibody. Results are expressed as mean values, error bars as standard deviations, and statistical significance is calculated by Student's t-test. Compared to the positive control group (C12-miR group), * p < 0.05. [Figure 6] This shows the size reduction of polyethylene glycolated peptide-linked C12-miR (P.5-L-C12-miR) enzymatically cleaved with lysosomal cathepsin D in Example 5. P.5-L-C12-miR (control group) and P.5-L-C12-miR enzymatically cleaved with lysosomal cathepsin D were analyzed by electrophoresis on a 15% (v / v) polyacrylamide gel containing 8 M urea, followed by staining with 0.2% (w / v) methylene blue. The lines indicate the size of each band. [Modes for carrying out the invention]

[0040] At least part of this invention is based on the following findings: Dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (referred to as C12-miR) unexpectedly increases PD-L1 expression in cells. Furthermore, polyethylene glycolation of C12-miR and the addition of a peptide linker (SEQ ID NO: 2) between PEG and C12-miR can further induce more PD-L1 expression in cells.

[0041] Accordingly, the present invention provides an oligonucleotide therapeutic agent having at least one oligonucleotide that conjugates with decylamine, wherein the oligonucleotide conjugates with the dodecylamine at its 5' end. Preferably, in some specific embodiments, the oligonucleotide is a microRNA. More preferably, in some preferred specific embodiments, the oligonucleotide consists of the sequence indicated by SEQ ID NO: 1. Preferably, in some specific embodiments, the oligonucleotide therapeutic agent may further contain polyethylene glycol (PEG) that conjugates with the dodecylamine at its amino group end. Preferably, in some specific embodiments, the oligonucleotide therapeutic agent may further contain a peptide linker between the dodecylamine and the PEG. More preferably, in some preferred specific embodiments, the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000. Even more preferably, in some more preferred specific embodiments, the polyethylene glycol (PEG) is PEG 500. More preferably, in some preferred specific embodiments, the peptide linker is conjugated with the dodecylamine at its amino terminal, and polyethylene glycol (PEG) is conjugated with the peptide linker at its amino terminal. More preferably, in some preferred specific embodiments, the peptide linker consists of the sequence shown in SEQ ID NO: 2.

[0042] The present invention also provides a composition comprising at least one oligonucleotide therapeutic agent according to the present invention and a pharmaceutically acceptable carrier or excipient.

[0043] The present invention also provides a method for increasing PD-L1 expression in cells, comprising contacting the cells with the oligonucleotide therapeutic agent according to the present invention. The present invention also provides a method for increasing PD-L1 expression in a subject's body, comprising administering the oligonucleotide therapeutic agent according to the present invention to the subject. The present invention also provides a method for preventing, mitigating, suppressing, or treating an inflammatory response in a subject's body, comprising administering the oligonucleotide therapeutic agent according to the present invention to the subject in a pharmaceutically effective amount.

[0044] The terms "programmed cell death protein 1," "PD-1," and "CD279 (differentiation cluster 279)" used in this text refer to cell surface receptor proteins present on certain immune cells (particularly T cells). These proteins regulate the immune system's response to human cells through downregulation of the immune system and promote self-tolerance by suppressing the inflammatory activity of T cells.

[0045] The terms "programmed cell death ligand 1," "PD-L1," and "CD274 (differentiation cluster 274)" used in this text refer to 40 kDa type I transmembrane proteins that are present on the surface of some cells, including cancer cells and immune cells, and that can interact with inhibitory checkpoint molecules.

[0046] The term "PD-1 / PD-L1 axis" used in this text refers to a crucial immunomodulatory pathway involving the interaction of PD-1 and PD-L1 in the human body. Binding of PD-1 on the surface of T cells to PD-L1 on the surface of other cells transmits inhibitory signals, reducing the proliferation of antigen-specific T cells in lymph nodes while decreasing the cell death of regulatory T cells (anti-inflammatory, suppressive T cells). The primary role of PD-1 / PD-L1 interaction is to prevent overactive immune responses that could lead to autoimmunity or excessive tissue damage. However, some cancers utilize this interaction as a way to evade the immune system. Cancer cells can express PD-L1, and when they bind to PD-1 on T cells, they can effectively suppress the tumor-fighting ability of T cells. This mechanism is one way cancer cells evade immunological surveillance. Furthermore, the PD-1 / PD-L1 axis has been identified as an important pathway for regulating the cerebral immune system, maintaining Aβ uptake by microglial cells, and mitigating chronic neuroinflammation. Studies have shown that both PD-L1 expression in astrocytes surrounding Aβ plaques and PD-1 expression in microglial cells are upregulated, and that astrocytes secrete a soluble form of PD-L1 that binds to PD-1 in microglial cells. The binding of PD-L1 and PD-1 increases Aβ uptake and removal by microglial cells, suppresses the sustained dilation of Aβ, and further suppresses neuroinflammation (Kummer et al., 2021). Therefore, increased PD-L1 expression in neurons has an effect of suppressing neuroinflammation.

[0047] The term "dodecylamine" used in this text refers to C 12 H 27 Organic compounds having the chemical formula NH2 and the following chemical structural formulas: This refers to TIFF0007883067000001.tif13145. Dodecylamines belong to the amine class, and among them, the functional group (-NH2) of primary amines is C 12It is bonded to a carbon alkyl chain. The first carbon atom of the dodecylamine used in this text is conjugated with the oligonucleotide at its 5' end, and the amino group of the dodecylamine is located on the 12th carbon atom of the dodecylamine.

[0048] The terms "polyethylene glycol" or "PEG" used in this text refer to H-(O-CH2-CH2) n Polymer compounds having the chemical formula -OH and the following chemical structural formulas: This refers to TIFF0007883067000002.tif20145. After PEG, a number representing the average molecular weight can be added; for example, PEG 500, PEG 1000, and PEG 2000 indicate average molecular weights of 500, 1000, and 2000, respectively.

[0049] As used in this text, the term "nucleotide" refers to an element in which a nitrogen-containing base is bonded to a sugar phosphate, and such sugar phosphates include those in which one or more phosphate groups are bonded to a sugar such as ribose or 2'-deoxyribose. "Polynucleotide" and "nucleic acid" refer to polymers of one or more nucleotide elements, where the elements are usually linked by sugar-phosphate bonds in a sugar-phosphate backbone. A polynucleotide does not necessarily contain only one type of nucleotide element. For example, a given polynucleotide may contain only ribonucleotides, only 2'-deoxyribonucleotides, or a combination of ribonucleotides and 2'-deoxyribonucleotides. Polynucleotides encompass naturally occurring nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as nucleic acid analogs containing one or more unnatural elements. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" usually refers to a large polynucleotide. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it will be understood that this also includes an RNA sequence (i.e., A, U, G, C) in which "T" is replaced by "U". The term "cDNA" refers to DNA that is complementary to or identical to mRNA, and may be in single-stranded or double-stranded form, in which "T" is replaced by "U". The term "recombinant nucleic acid" refers to a polynucleotide or nucleic acid that has sequences that are unnaturally bound together. Recombinant nucleic acids can exist in the form of a vector.

[0050] The term "oligonucleotide" as used in this text typically refers to short DNA or RNA molecules with a length of 13 to 25 nucleotides. The maximum length of an oligonucleotide is approximately 200 nucleotide residues.

[0051] The terms "microribonucleic acid," "microRNA," and "miRNA" used in this text refer to short, non-coding, single-stranded RNA sequences consisting of 18 to 22 nucleotides. MicroRNAs regulate the expression of target genes by binding to the complementary untranslated region (3'-UTR) of messenger RNA (mRNA), leading to the suppression or degradation of the target gene's translation. A single microRNA can regulate many, even hundreds, different mRNA molecules, and multiple microRNAs can regulate the same mRNA. MicroRNAs participate in many biological functions, including growth, differentiation, proliferation, and apoptosis.

[0052] In this text, the terms "microRNA 200c-3p" or "miRNA 200c-3p" refer to a specific microRNA molecule belonging to the microRNA-200 family that has the sequence 5'-UAAUACUGCCGGGUAAUGAUGGA-3' (SEQ ID NO: 1). Previous studies have shown that miR-200c-3p can reduce the expression of PD-L1, c-Myc, and β-catenin in ovarian cancer, and that miR-200c-3p can be a tumor inhibitor of epithelial ovarian cancer (Anastasiadou et al., 2021). Other studies have also shown that miR-200c can suppress the expression of PD-L1 mRNA in mouse lung tumor cells, further exhibiting antitumor effects (Zhang et al., 2023).

[0053] The nomenclature used in this text to describe the peptides of the present invention follows common practice, with the amino group (N-terminus) and / or 5' terminus on the left and the carboxyl group (C-terminus) and / or 3' terminus on the right.

[0054] As used in this text, the term "peptide" refers to a molecular chain of amino acids, including L- and D-type amino acids. If necessary, amino acids may be modified in or out of the body, for example, through mannosylation, glycosylation, amidation (especially C-terminal amide), carbosylation, or phosphorylation, and these modifications must maintain the biological activity of the original molecule. Furthermore, peptides may be part of fusion proteins.

[0055] As used in this text, the term "peptide linker" refers to a short-chain amino acid (peptide fragment) used to bind or link different functional components in various biological or chemical molecules.

[0056] Functional derivatives of peptides are also included in the present invention. Functional derivatives refer to peptides that have one or more amino acid deletions, substitutions, inversions, or additions throughout the sequence. Amino acid substitutions that are not expected to fundamentally alter biological and immunological activity have already been described. Amino acid substitutions between related amino acids, or substitutions that have occurred frequently in evolution, include Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, and Ile / Val, among others.

[0057] The peptides according to the present invention can be produced by synthesis or DNA recombination technology. Methods for producing synthetic peptides are well known in the art.

[0058] Organic chemical methods used in peptide synthesis are considered to encompass the coupling of required amino acids by condensation reactions, whether in a homogeneous phase or relying on a so-called solid phase. Condensation reactions can be carried out according to the following methods: A compound (amino acid, peptide) having a free carboxyl group and other protected reactive groups is condensed with a compound (amino acid, peptide) having a free amino group and other protected reactive groups in the presence of a coupling agent. A compound (amino acid, peptide) having an activated carboxyl group and other free or protected reactive groups is condensed with a compound (amino acid, peptide) having a free amino group and other free or protected reactive groups. The carboxyl group is activated by converting it to an acyl halide, azide, acid anhydride, imidazoline, activated ester (e.g., N-hydroxysuccinimide, N-hydroxybenzotriazole), or p-nitrophenyl.

[0059] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” encompasses all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption enhancers or retarders, and other physiologically acceptable excipients or additives. In certain specific embodiments, the carrier is applied to nasal, intravenous, intramuscular, intradermal, subcutaneous, parenteral, oral, mucosal, or transdermal administration. Depending on the route of administration, the active compound can be protected from the action of acids and other natural conditions that may inactivate the compound by coating it within the material. The application of such media and reagents to drug-active substances is well known in the art.

[0060] Formulations suitable for administration of the present invention may, in other circumstances well known to those skilled in the art, include aqueous and non-aqueous solutions, antioxidants, bacteriostatic agents, buffers, solutes affecting isotonicity, preservatives, solubilizers, stabilizers, suspending agents, thickeners, or combinations thereof.

[0061] Otherwise or as an alternative, formulations suitable for administration of the present invention may include, in other circumstances well known to those skilled in the art, gels, PEG (e.g., PEG400), propylene glycol, saline solution, sachets, water, other suitable liquids well known in the art, or combinations thereof.

[0062] Otherwise or as an alternative, formulations suitable for administration of the present invention may, in other circumstances well known to those skilled in the art, include adhesives, buffers, tricalcium phosphate, cellulose, colloids (e.g., colloidal silicon dioxide), colorants, diluents, disintegrants, dyes, fillers, flavorings, gelatin, lactose, magnesium stearate, mannitol, microcrystalline gelatin, wetting agents, paraffinic hydrocarbons, tablets, polyethylene glycol, preservatives, sorbitol, starches (e.g., corn starch, potato starch, or a combination thereof), stearic acid, sucrose, talc, triglycerides, or a combination thereof.

[0063] Otherwise or as an alternative, formulations suitable for administration of the present invention may, in other circumstances well known to those skilled in the art, include alcohols (e.g., phenylmethanol or ethanol), benzalkonium chloride, buffers (e.g., phosphate buffers, acetate buffers, citrate buffers, or combinations thereof), carboxymethylcellulose or microcrystalline cellulose, cholesterol, glucose, fruit juices (e.g., yuzu juice), milk, phospholipids (e.g., lecithin), oils (e.g., vegetable oils, fish oils, or mineral oils, or combinations thereof); other pharmaceutically acceptable carriers well known in the art; or combinations thereof.

[0064] Otherwise or as an alternative, formulations suitable for administration of the present invention may, in other circumstances well known to those skilled in the art, include biodegradable substances (e.g., polylactic acid-glycolic acid (PLGA) polymers), other substantial degradation products that can be rapidly removed from the biological system or combination thereof.

[0065] The formulations according to the present invention can be administered in single-dose form, multi-dose form, or a combination thereof. Such formulations can be packaged in single-dose containers, multi-dose containers, or a combination thereof. The present invention can exist in the form of ampoules, cachets, capsules, granules, lozenges, powders, tablets, vials, emulsions (including, but not limited to, gum arabic emulsions), suspensions, or a combination thereof.

[0066] As used in this text, the terms “effective dose” or “sufficient dose” of a substance refer to an amount sufficient to achieve a beneficial or desired outcome (including clinical outcomes). Therefore, the “effective dose” is determined by the context in which it is applied. When administering an immunogenic composition, the effective dose is an immunogenically effective dose, containing a sufficient amount of the immunogenic composition according to the present invention to elicit an immune response. When administering a drug composition, the effective dose is a pharmaceutically effective dose, containing a sufficient amount of the drug composition according to the present invention to maintain or produce the desired physiological outcome. One or more effective doses may be administered.

[0067] As used in this text, the term "pharmaceutical effective dose" means an amount that is possible or sufficient to maintain or produce a desired physiological outcome, and includes, but is not limited to, treating, reducing, mitigating, resolving, inhibiting, essentially preventing, or remediating a disease, condition, or combination thereof. The pharmaceutical effective dose may include one or more doses administered sequentially or simultaneously. Those skilled in the art will understand that the doses according to the present invention are adjusted to suit each type of formulation, including, but not limited to, sustained-release formulations. As used in this text, the term "preventive" means that the composition is essentially able to prevent or remediate any aspect of a disease, condition, or combination thereof. As used in this text, the term "therapeutic" means that the composition is able to treat, reduce, prevent, mitigate, beneficially modify, resolve, or remediate any aspect of a disease, condition, or combination thereof.

[0068] In this text, the term "dosage" for a composition refers to the measured portion of the composition taken (administered or received) by the examinee at any given time.

[0069] As used in this text, the term "test subject" refers to an animal, more specifically, a non-human mammal and a human organism. Non-human animal test subjects may also include prenatal forms of animals, such as embryos or fetuses. Non-limiting examples of non-human animals include horses, cattle, camels, goats, sheep, dogs, cats, non-human primates, mice, rats, rabbits, hamsters, guinea pigs, and pigs. In some specific embodiments, the test subject is a human. Human test subjects may also include fetuses.

[0070] As used in this text, the term "test subject" refers to any test subject who requires treatment, particularly mammalian test subjects, such as humans.

[0071] As used in this text, the terms “treat,” “treating,” or “treatment” include reducing the severity of at least one symptom, or preventing its worsening. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be an effective treatment, a useful composition in this text should reduce the severity of a disease, condition, or symptom, reduce the severity of related symptoms, or improve the quality of life of the patient or test subject.

[0072] As used in this text, the terms "prevent," "preventing," or "prevention" refer to the ability to eliminate, avoid, evade, prevent, block, hinder, or combine any aspect of a disease, symptom, or combination thereof, primarily through proactive action.

[0073] In some specific embodiments, the oligonucleotide therapeutic agent and / or composition according to the present invention can be administered to a test subject by a plurality of administration methods, including intradermal, intramuscular, subcutaneous, intravenous, intraatrial, intra-articular, intraperitoneal, parenteral, oral, rectal, nasal, intrapulmonary, and transdermal administration, or topical administration to the eyes, ears, skin, or mucous membranes. Alternatively, the antigen may be selectively immobilized on a biocompatible liquid or solid carrier and administered in vitro by direct contact with cells, tissues, or organs derived from the test subject (itself) or another test subject (another body).

[0074] The meanings of technical and scientific terms used in this text should be clearly understood by those skilled in the art.

[0075] When the terms "approximately," "roughly," or "about" are used in this text in combination with numerical values, they refer to a range of plus or minus 10% of the given reference value. For example, a length of approximately 1000 nanometers (nm) refers to a length in the range of 900 nm to 1100 nm.

[0076] As used in this text, the term "contains" is open, indicating that such embodiments may contain additional elements. Conversely, the term "consisting of" is closed, indicating that such embodiments do not contain additional elements (except for trace impurities). The phrase "essentially consisting of" is partially closed, indicating that such embodiments may also contain elements that do not substantially alter the fundamental characteristics of such embodiments.

[0077] When an applicant defines an invention or part thereof using an open conjunction such as “encompassing,” it will be readily understood that the specification should also be interpreted as describing the invention using conjunctions such as “essentially consisting of” or “consisting of” (unless otherwise stated).

[0078] It should be noted that the singular forms "a," "an," and "the" used in the text and attached claims encompass multiple references unless otherwise explicitly stated in the surrounding text. Therefore, for example, a reference to "polynucleotide" encompasses multiple such polynucleotides, and a reference to "the polynucleotide" encompasses one or more polynucleotides mentioned, as well as equivalents well known to those skilled in the art. Furthermore, it should be noted that claims can be written to exclude all selective elements. Therefore, the text is intended to precede the reference of elements in the claims, or, in the case of using "negative" limitations, the use of exclusive terms such as "alone" or "only."

[0079] In some contexts, when using a promise similar to "at least one of A, B, and C," such a structure is usually intended to be understood by those skilled in the art as meaning the promise (for example, "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, A and B, A and C, B and C, and / or A, B and C, etc.). Those skilled in the art will further understand that all separable words and / or words that actually represent two or more alternative terms should be considered to encompass the possibility of one of those terms, either of the terms, or both of the terms, whether in the detailed description of the invention, the claims, or the drawings. For example, the word "A or B" is understood to encompass the possibilities of "A," "B," or "A or B."

[0080] The present invention will be further illustrated by the following embodiments. These embodiments are presented for illustrative purposes only and not to limit them. Those skilled in the art will understand that, in light of the disclosure of the present invention, many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, and similar or comparable results can be obtained. [Examples]

[0081] Example 1: Preparation of an oligonucleotide therapeutic agent Materials and methods Design and preparation of oligonucleotide therapeutic agents. A schematic diagram of the design of the oligonucleotide therapeutic agent described in the examples is shown in Figure 1. The method for preparing the oligonucleotide therapeutic agent is as follows. First, microRNA 200c-3p (5'-UAAUACUGCCGGGUAAUGAUGGA-3'; SEQ ID NO: 1) (Genomics Biotech Co., Ltd., New Taipei City, Taiwan; GenScript Biotech, Inc., New Jersey, USA) is synthesized by solid-phase synthesis, and the 5'-ribose end of the microRNA is converted to dodecylamine (C 12 H 27 The microRNA is modified with N), and the first carbon atom of the dodecylamine is conjugated to the 5' end of the microRNA, and the amino group of the dodecylamine is located on the twelfth carbon atom of the dodecylamine. The resulting oligonucleotide therapeutic agent was named C12-miR.

[0082] The dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR) was further conjugated with polyethylene glycol (PEG) of different sizes (0.5, 1, and 2 kDa) at the amino group terminus of the dodecylamine. The polyethylene glycolation activity of C12-miR is as follows: 4.15 nM of C12-miR and 4.15 nM of PEG (0.5, 1, and 2 kDa) were mixed in 2(N-morpholino)ethanesulfonic acid (MES) buffer containing 4.15 nM of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) at room temperature for 3 hours. Subsequently, Microspin TMThe mixture was purified using a G-25 column (Sigma-Aldrich, Missouri, USA) to obtain a larger amount of the oligonucleotide therapeutic agent used in this example. C12-miR modified with PEG 500 (0.5 kDa), PEG 1000 (1 kDa), and PEG 2000 (2 kDa) were named P.5-C12-miR, P1-C12-miR, and P2-C12-miR, respectively.

[0083] Furthermore, the dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1)(C12-miR) was modified by modifying the amino group terminus of the dodecylamine with a peptide linker (KGDGG; SEQ ID NO: 2), and the amino group terminus of the peptide linker (SEQ ID NO: 2) was further conjugated with PEG 500 (0.5 kDa) to form another oligonucleotide therapeutic agent, which was named P.5-L-C12-miR. In short, 4.15 nM of C12-miR, 4.15 nM of the peptide linker (SEQ ID NO: 2), and 4.15 nM of PEG 500 were mixed in MES buffer containing 4.15 nM of EDC at room temperature for 3 hours. Subsequently, Microspin TM The oligonucleotide therapeutic agent P.5-L-C12-miR was obtained by purifying the mixture using a G-25 column.

[0084] The polyethylene glycolated C12-miR products (P.5-C12-miR, P1-C12-miR, P2-C12-miR, and P.5-L-C12-miR) were quantified by measuring the optical density at a wavelength of 260 nm using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA). Non-polyethylene glycolated and polyethylene glycolated C12-miR (C12-miR, P.5-C12-miR, P1-C12-miR, P2-C12-miR, and P.5-L-C12-miR) were characterized by electrophoresis on a 15% (v / v) polyacrylamide gel containing 8 M urea, followed by staining with 0.2% (w / v) methylene blue.

[0085] Results As shown in Figure 2, non-polyethylene glycolated C12-miR has an expected size of approximately 7 kDa. As the size of the conjugated PEG increases, the size of the polyethylene glycolated C12-miR (P.5-C12-miR, P1-C12-miR, and P2-C12-miR) increases. Also, due to the addition of the peptide linker (SEQ ID NO: 2), the polyethylene glycolated peptide-linked C12-miR (P.5-L-C12-miR) has a larger size than P.5-C12-miR.

[0086] <^ Example 2 Biological Function Analysis of Polyethylene Glycolated Oligonucleotide Therapeutics Modified or Unmodified with a Peptide Linker Materials and Methods Cell treatment. SH-SY5Y human neuroblastoma cells (ATCC® CRL-2266) suspended in MEM / F12K medium (1:1, v / v) were approximately 5x10 3Cells were inoculated into 96-well plates at a density of individual cells / well. The cells were cultured at 37°C in 5% CO2 for 16 hours, and then treated with 2 μM C12-miR, P.5-C12-miR, and P.5-L-C12-miR obtained in Example 1, respectively. The treated cells were then cultured for a further 48 hours at 37°C in 5% CO2. Cells treated with phosphate-buffered saline (PBS) served as a blank control group.

[0087] Flow cytometry analysis. Cells were collected and washed with PBS. The collected cells were stained with anti-human PD-L1 surface antibody (number: 329706, Biolegend, California, USA) and allowed to react for 30 minutes at 4°C, away from light. Subsequently, the cells were washed twice with cold FACS buffer, resuspended in FACS buffer, and further analyzed by flow cytometry (BD LSRFortessa). TM The analysis was conducted using X20 (New Jersey, USA).

[0088] Statistical analysis. All data were tested using the TTEST function in Microsoft Excel (Washington, USA) to perform a one-sided Student t-test. A group p-value of less than 0.05 was considered statistically significant.

[0089] result The oligonucleotide therapeutic agents according to the present invention induced PD-L1 expression. As shown in Figure 3, compared to the blank control group, the three oligonucleotide therapeutic agents used in this example—C12-miR, P.5-C12-miR, and P.5-L-C12-miR—significantly increased PD-L1 expression in SH-SY5Y cells to 23% (p<0.01), 39% (p<0.01), and 58% (p<0.001), respectively. In particular, compared to non-polyethylene glycolated C12-miR, both polyethylene glycolated C12-miR (P.5-C12-miR) (p<0.05) and polyethylene glycolated peptide-linked C12-miR (P.5-L-C12-miR) (p<0.001) significantly increased PD-L1 expression in SH-SY5Y cells. More specifically, polyethylene glycolated peptide-linked C12-miR (P.5-L-C12-miR) significantly increased PD-L1 expression in SH-SY5Y cells compared to polyethylene glycolated C12-miR (P.5-C12-miR) (p<0.05).

[0090] The results above show that dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR) unexpectedly increases PD-L1 expression in cells, which is the opposite of previous reports (Anastasiadou et al., 2021; Zhang et al., 2023) regarding the inhibitory effect of microRNA 200c-3p mimics or microRNA 200c-3p expression vectors on PD-L1 expression in cells. The results above also show that polyethylene glycolation of dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (P.5-C12-miR) induced more PD-L1 expression in cells than dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR). These results further demonstrate that the addition of a peptide linker (SEQ ID NO: 2) (P.5-L-C12-miR) between PEG and dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR) induced cells to express even more PD-L1 than the dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR).

[0091] Example 3: Biofunctional analysis of polyethylene glycolated oligonucleotide therapeutic agents at different doses. Materials and methods Cell treatment. SH-SY5Y human neuroblastoma cells (ATCC® CRL-2266) are used in this example. Cell culture is as described in Example 2, except that in this example the cells are treated with 0.5 μM, 2 μM, and 4 μM C12-miR or P.5-C12-miR. Cells treated with PBS are used as a blank control group (i.e., treated with 0 μM C12-miR or P.5-C12-miR).

[0092] Flow cytometry analysis. The method for flow cytometry analysis is as described in Example 2.

[0093] Statistical analysis. The method of statistical analysis is as described in Example 2.

[0094] result The oligonucleotide therapeutic agents according to the present invention induced PD-L1 expression in a dose-dependent manner. As shown in Figure 4, compared to the blank control group (0 μM), 0.5 μM, 2 μM, and 4 μM of dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR) significantly increased PD-L1 expression in SH-SY5Y cells to 16%, 23% (p<0.01), and 29% (p<0.01), respectively. Similarly, compared to the blank control group (0 μM), 0.5 μM, 2 μM, and 4 μM of polyethylene glycolated C12-miR (P.5-C12-miR) significantly increased PD-L1 expression in SH-SY5Y cells to 46% (p<0.001), 39% (p<0.001), and 59% (p<0.001), respectively. Furthermore, 0.5 μM, 2 μM, and 4 μM P.5-C12-miR significantly induced greater PD-L1 expression in SH-SY5Y cells than 0.5 μM, 2 μM, and 4 μM C12-miR, respectively (p<0.05 or p<0.01). This demonstrates that polyethylene glycolation of C12-miR (P.5-C12-miR) is more effective than C12-miR in inducing PD-L1 expression in cells. These results show that both C12-miR and polyethylene glycolated C12-miR (P.5-C12-miR) dose-dependently induced PD-L1 expression in neuroblastoma cells.

[0095] Example 4: Biofunctional analysis of polyethylene glycolated oligonucleotide therapeutic agents using PEG of different sizes Materials and methods Cell treatment. SH-SY5Y human neuroblastoma cells (ATCC® CRL-2266) are used in this example. Cell culture is as described in Example 2, except in this example where the cells are treated with 2 μM C12-miR, P.5-C12-miR, P1-C12-miR, or P2-C12-miR. Cells treated with 2 μM C12-miR are used as the positive control group.

[0096] Flow cytometry analysis. The method for flow cytometry analysis is as described in Example 2.

[0097] Statistical analysis. The method of statistical analysis is as described in Example 2.

[0098] result Polyethylene glycolated oligonucleotide therapeutic agents using PEG of different sizes can all induce PD-L1 expression. As shown in Figure 5, compared to 2 μM C12-miR, 2 μM P.5-C12-miR, P1-C12-miR, and P2-C12-miR increased PD-L1 expression in SH-SY5Y cells to 10% (p<0.05), 2%, and 3%, respectively. Among these polyethylene glycolated C12-miRs, C12-miR polyethylene glycolated with PEG 500 (P.5-C12-miR) significantly induced the highest PD-L1 expression in cells (p<0.05). These results demonstrate that polyethylene glycolation of C12-miRs using PEG of different sizes, particularly polyethylene glycolation with PEG 500, has a favorable effect on inducing PD-L1 expression in cells.

[0099] Example 5 Analysis of peptide linker degradation Materials and methods Decomposition analysis. The polyethylene glycolated peptide-linked C12-miR (P.5-L-C12-miR) obtained in Example 1 was enzymatically cleaved with lysosomal cathepsin D in a test tube. Briefly, 10 μL of P.5-L-C12-miR (4.15 nM) and 2 μL of cathepsin D (number C8696, Sigma-Aldrich, Missouri, USA) were added to a 250 mM sodium acetate solution (pH 3.7) until the final volume was 20 μL. The reaction mixture was allowed to react at 37°C for 5 hours. The reaction product was analyzed by electrophoresis at a voltage of 200 volts for 1 hour in a 15% (v / v) polyacrylamide gel containing 8 M urea. Subsequently, the gel was stained with 0.2% (w / v) methylene blue for 20-30 minutes, and imaging was performed using Gel Doc EZ (Bio-Rad, California, USA), integrated with image analysis software (Image Lab, Bio-Rad). All bands stained in the nucleic acid region were analyzed, and the band with the highest density was defined as the size of that band.

[0100] result Peptide linkers can be cleaved by cathepsin D. As shown in Figure 6, polyethylene glycolated peptide-linked C12-miR (P.5-L-C12-miR) was cleaved by lysosomal cathepsin D, generating small fragments. These results indicate that after the oligonucleotide therapeutic agent P.5-L-C12-miR was taken up into cells via phagocytosis, the peptide linker was enzymatically cleaved by enzymes in lysosomes, releasing the dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR) into the cells, thereby enhancing the effect of C12-miR on cells.

[0101] In summary, the dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR) unexpectedly increased PD-L1 expression in cells, and further polyethylene glycolation of C12-miR can induce even more PD-L1 expression. Adding a peptide linker (SEQ ID NO: 2) between PEG and C12-miR can induce even more PD-L1 expression in cells. Enzymatic degradation of this peptide linker (SEQ ID NO: 2) can further release the dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR) into cells. These results demonstrate that the oligonucleotide therapeutic agent according to the present invention can be used for the prevention, reduction, suppression, and treatment of inflammatory responses, particularly neuroinflammatory responses.

[0102] Of course, various changes and modifications can be made to the above embodiments of the present invention without departing from the scope of the invention. Therefore, the present invention is disclosed and limited only to the scope set forth in the appended claims, in order to promote progress in science and useful fields.

[0103] [References] (1)GS Bloom, Amyloid- and Tau. The trigger and bullet in Alzheimer disease pathogenesis. JAMA Neurology. 2014; 71: 505-508; doi: 10.1001 / jamaneurol.2013.5847. (2)JW Kinney et al., Inflammation as a central mechanism in Alzheimer's disease. Alzheimers Dement (NY). 2018; 4: 575-590; doi: 10.1016 / j.trci.2018.06.014. (3)G. Lueg. et al., Clinical relevance of specific T-cell activation in the blood and cerebrospinal fluid of patients with mild Alzheimer’s disease. Neurobiol. Aging. 2015; 36: 81-89; doi: 10.1016 / j.neurobiolaging.2014.08.008. (4)D. V. Hansen et al., Microglia in Alzheimer’s disease. J. Cell. Biol. 2018; 217: 459-472; doi: 10.1083 / jcb.201709069. (5)E. Anastasiadou et al., MiR-200c-3p contrasts PD-L1 induction by combinatorial therapies and slows proliferation of epithelial ovarian cancer through downregulation of -catenin and c-Myc. Cells 2021; 10: 519; doi: 10.3390 / cells10030519. (6)Q. Zhang et al., Aerosolized miR-138-5p and miR-200c targets PD-L1 for lung cancer prevention. Front. Immunol., 2023; 14:1166951; doi: 10.3389 / fimmu.2023.1166951. Kummer et al., Microglial PD-1 stimulation by astrocytic PD-L1 suppresses neuroinflammation and Alzheimer’s desease pathology. EMBO J. 2021; 40:e108662; doi: 10.15252 / embj.2021108662.

Claims

1. An oligonucleotide therapeutic agent comprising an oligonucleotide and dodecylamine, wherein at the 5' end of the oligonucleotide, the first carbon atom of the dodecylamine is conjugated with the oligonucleotide, the amino group of the dodecylamine is located on the twelfth carbon atom of the dodecylamine, and the oligonucleotide consists of a sequence indicated by SEQ ID NO:

1.

2. The oligonucleotide therapeutic agent according to claim 1, further comprising polyethylene glycol (PEG) conjugated with the dodecylamine at the amino group end of the dodecylamine, wherein the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

3. The oligonucleotide therapeutic agent according to claim 1, further comprising a peptide linker that conjugates with the dodecylamine at the amino end of the dodecylamine, and polyethylene glycol (PEG) that conjugates with the peptide linker at the amino end of the peptide linker, wherein the peptide linker consists of the sequence shown in SEQ ID NO: 2, and the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

4. A composition comprising an oligonucleotide therapeutic agent according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.

5. A pharmaceutical composition for increasing PD-L1 expression in cells, comprising an oligonucleotide therapeutic agent according to any one of claims 1 to 3 or the composition according to claim 4.

6. A pharmaceutical composition for reducing inflammatory responses in the body of a test subject, comprising an oligonucleotide therapeutic agent according to any one of claims 1 to 3 or the composition according to claim 4.

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