Oligonucleotide therapeutic agents and uses thereof

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 oligonucleotide therapeutics, particularly to oligonucleotides conjugated with dodecylamine, which may further comprise polyethylene glycol (PEG) conjugated to the dodecylamine, or even a peptide linker between the dodecylamine and the PEG. [Background technology]

[0002] According to the World Health Organization (WHO) statistics for 2023, more than 55 million people worldwide will suffer from dementia, with an additional 10 million cases expected each year. Alzheimer's disease (AD) is the most common neurodegenerative disease and the leading cause of dementia. AD is caused by the deposition of β-amyloid (Aβ), abnormal phosphorylation of tau protein, and excessive inflammation in neurons. Numerous studies have shown that β-amyloid contributes to plaque accumulation, further inducing neurotoxicity in the brain. Furthermore, excessive phosphorylation of tau protein leads to the formation of neurofibrillary tangles (NFTs), further leading to 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). This neuroinflammatory phenomenon, enhanced by Aβ deposition, stimulates microglia 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 reduce inflammatory responses, especially neuroinflammation, is crucial for preventing dementia and neurodegenerative diseases such as Alzheimer's disease. Summary of the Invention [Means for solving the problem]

[0004] The present invention is based, at least in part, on the following discovery: Dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (referred to as C12-miR) unexpectedly increases PD-L1 expression in cells, contrary to previous reports of the inhibitory effect of microRNA 200c-3p mimics or microRNA 200c-3p expression vectors on PD-L1 expression in cells (Anastasiadou et al., 2021; Zhang et al., 2023). Polyethylenically modifying C12-miR and adding a peptide linker (SEQ ID NO: 2) between PEG and C12-miR can further induce greater 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) of the present invention, pegylated C12-miRs, and C12-miRs bound by pegylated peptides can be used as oligonucleotide therapeutic agents to alleviate and suppress inflammatory responses, particularly neuroinflammatory responses.

[0005] Thus, in some specific embodiments, the present invention provides an oligonucleotide therapeutic agent having at least one oligonucleotide conjugated to dodecylamine, where the oligonucleotide is conjugated to the dodecylamine at its 5' end. In other specific embodiments, the oligonucleotide therapeutic agent can further comprise polyethylene glycol (PEG) conjugated to the dodecylamine at the amino terminus of the dodecylamine. In other specific embodiments, the oligonucleotide therapeutic agent can further comprise a peptide linker between the dodecylamine and the PEG.

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

[0007] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific exemplary embodiments of the invention described herein which equivalents are intended to be encompassed by the following exemplary embodiments.

[0008] Specific Example 1. An oligonucleotide therapeutic agent comprising an oligonucleotide and dodecylamine, wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5' end of the oligonucleotide and the amino group of the dodecylamine is on the 12th carbon atom of the dodecylamine.

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

[0010] Specific Example 3. An oligonucleotide therapeutic agent according to specific example 1 or 2, wherein the oligonucleotide consists of the sequence shown in SEQ ID NO: 1.

[0011] Specific Example 4. The oligonucleotide therapeutic agent according to any one of specific examples 1 to 3, further comprising polyethylene glycol (PEG) conjugated to the dodecylamine at the amino terminus of the dodecylamine.

[0012] Specific Example 5. The oligonucleotide therapeutic of 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. The oligonucleotide therapeutic agent described in any of specific examples 1 to 3 further comprises a peptide linker conjugated to the dodecylamine at the amino terminus of the dodecylamine, and polyethylene glycol (PEG) conjugated to the peptide linker at the amino terminus of the peptide linker.

[0014] Specific Example 7. The oligonucleotide therapeutic agent of specific example 6, wherein the peptide linker consists of the sequence shown in SEQ ID NO: 2.

[0015] Specific Example 8. The oligonucleotide therapeutic of 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 as described in any of specific Examples 1-8, selected from the group consisting of: an oligonucleotide therapeutic agent consisting of an oligonucleotide and dodecylamine, wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5' end of the oligonucleotide and the amino group of the dodecylamine is on the 12th carbon atom of the dodecylamine; an oligonucleotide therapeutic agent consisting of an oligonucleotide, dodecylamine, and polyethylene glycol (PEG), wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5' end of the oligonucleotide, the amino group of the dodecylamine is on the 12th carbon atom of the dodecylamine, and the polyethylene glycol (PEG) is conjugated to the dodecylamine at the amino end of the dodecylamine; and An oligonucleotide therapeutic agent comprising an oligonucleotide, dodecylamine, a peptide linker, and polyethylene glycol (PEG), wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5' end of the oligonucleotide, the amino group of the dodecylamine is on the 12th carbon atom of the dodecylamine, the peptide linker is conjugated to the dodecylamine at the amino end of the dodecylamine, and the polyethylene glycol (PEG) is conjugated to the peptide linker at the amino end of the peptide linker.

[0017] Specific Example 10. An oligonucleotide therapeutic agent as described in any of Specific Examples 1 to 9, which comprises an oligonucleotide and dodecylamine, wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5' end of the oligonucleotide, and the amino group of the dodecylamine is on the 12th carbon atom of the dodecylamine.

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

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

[0020] Specific Example 13. 13. The oligonucleotide therapeutic agent according to claim 9, wherein the oligonucleotide is a microRNA.

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

[0022] Specific Example 15. The oligonucleotide therapeutic agent according to any of specific Examples 9-14, wherein the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

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

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

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

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

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

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

[0029] Specific Example 22. Use of an oligonucleotide therapeutic agent as described in any of specific Examples 1-16 or a composition as described in specific Example 17 in increasing PD-L1 expression in a cell.

[0030] Specific Example 23. Use of an oligonucleotide therapeutic agent as described in any of specific Examples 1-16 or a composition as described in specific Example 17 in increasing PD-L1 expression in a subject.

[0031] Specific Example 24. Use of an oligonucleotide therapeutic agent as set forth in any of specific Examples 1-16 or a composition as set forth in specific Example 17 in the prevention, reduction, suppression or treatment of an inflammatory response in a subject.

[0032] Specific Example 25. The use according to 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 as described in any of specific Examples 1-16 or a composition as described in specific Example 17 in the preparation of a medicament for increasing PD-L1 expression in a cell.

[0034] Specific Example 27. Use of an oligonucleotide therapeutic agent as described in any of specific Examples 1-16 or a composition as described in specific Example 17 in the preparation of a medicament for increasing PD-L1 expression in a subject.

[0035] Specific Example 28. Use of an oligonucleotide therapeutic agent as described in any of specific examples 1-16 or a composition as described in specific example 17 in the preparation of a medicament for preventing, reducing, suppressing or treating an inflammatory response in a subject.

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

[0037] These and other aspects will become apparent from the following description of the preferred specific embodiments, taken in conjunction with the drawings.

[0038] The drawings illustrate one or more illustrative embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Where possible, the same drawing reference numerals will be used throughout the drawings to refer to the same or similar parts in the illustrative embodiments. [Brief explanation of the drawings]

[0039] [Figure 1] 1 shows a schematic diagram of the design of oligonucleotide therapeutic agents according to the present invention, where miR, C12, and Linker represent microRNA, dodecylamine, and peptide linker (SEQ ID NO: 2), respectively. [Figure 2] Figure 2 shows the results of a characterization analysis of the oligonucleotide therapeutic agent prepared in Example 1. The characterization 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. Arrows indicate the position of each size (7 kDa and 8 kDa) on the gel. [Figure 3] Figure 2 shows the expression levels of PD-L1 in SH-SY5Y human neuroblastoma cells (ATCC® CRL-2266) treated with phosphate-buffered saline (PBS) (blank control), C12-miR 2 μM, P.5-C12-miR 2 μM, or P.5-L-C12-miR 2 μM for 48 hours. 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, with error bars representing standard deviations. Statistical significance was calculated using the 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. † p<0.05 vs. P.5-C12-miR group. [Figure 4]Example 3 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). PD-L1 expression was analyzed by flow cytometry using an anti-human PD-L1 surface antibody. Results are expressed as mean values, with error bars representing standard deviations, and statistical significance was calculated using 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] Example 4 shows the effect of conjugates of different sizes of PEG and C12-miR on PD-L1 expression in SH-SY5Y human neuroblastoma cells (ATCC® CRL-2266). 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 was calculated using Student's t-test. *p<0.05 compared to the positive control group (C12-miR group). [Figure 6] This figure shows the size reduction of PEGylated peptide-linked C12-miR (P.5-L-C12-miR) enzymatically cleaved by lysosomal cathepsin D in Example 5. P.5-L-C12-miR (control group) and P.5-L-C12-miR enzymatically cleaved by 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. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention is based, at least in part, on the discovery that dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (referred to as C12-miR) unexpectedly increases PD-L1 expression in cells, and pegylation of C12-miR and addition of a peptide linker (SEQ ID NO: 2) between PEG and C12-miR can further induce greater PD-L1 expression in cells.

[0041] Thus, the present invention provides an oligonucleotide therapeutic agent having at least one oligonucleotide conjugated to decylamine, wherein the oligonucleotide is conjugated to the dodecylamine at its 5' end. In some preferred specific embodiments, the oligonucleotide is a microRNA. More preferably, in some preferred specific embodiments, the oligonucleotide consists of the sequence set forth in SEQ ID NO: 1. In some preferred specific embodiments, the oligonucleotide therapeutic agent can further comprise polyethylene glycol (PEG) conjugated to the dodecylamine at the amino terminus of the dodecylamine. In some preferred specific embodiments, the oligonucleotide therapeutic agent can further comprise 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. In some preferred embodiments, the peptide linker is conjugated to the dodecylamine at its amino terminus, and the polyethylene glycol (PEG) is conjugated to the peptide linker at its amino terminus. In some preferred embodiments, the peptide linker consists of the sequence set forth in SEQ ID NO: 2.

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

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

[0044] As used herein, the terms "programmed cell death protein 1," "PD-1," and "CD279 (cluster of differentiation 279)" refer to cell surface receptor proteins present on some immune cells, particularly T cells, that modulate the immune system's response to human cells by downregulating the immune system and promote self-tolerance by suppressing T cell inflammatory activity.

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

[0046] The term "PD-1 / PD-L1 axis" used in this study refers to an important immune regulatory pathway mediated by the interaction of PD-1 and PD-L1 in the human body. The 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 and reducing the cell death of regulatory T cells (anti-inflammatory, suppressive T cells). The primary role of PD-1 / PD-L1 interaction is to prevent an overactive immune response that could lead to autoimmunity or excessive tissue damage. However, some cancers use it 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 ability of T cells to attack tumors. This mechanism is one way cancer cells evade immunosurveillance. The PD-1 / PD-L1 axis has also been found to be an important pathway for regulating the cerebral immune system, maintaining Aβ uptake in microglial cells, and reducing chronic neuroinflammation. Studies have shown that both PD-L1 expression in astrocytes and PD-1 expression in microglia surrounding Aβ plaques are upregulated, and that astrocytes secrete a soluble form of PD-L1 that binds to PD-1 in microglia. Binding of PD-L1 to PD-1 increases the uptake and clearance of Aβ by microglial cells and inhibits the sustained spread of Aβ, further suppressing neuroinflammation (Kummer et al., 2021). Therefore, increased PD-L1 expression in neurons has the effect of suppressing neuroinflammation.

[0047] The term "dodecylamine" used in this text refers to C 12 H 27 An organic compound with the chemical formula NH2 and the following chemical structure: TIFF2025538662000002.tif13145. Dodecylamine belongs to the amine family, and among them, the primary amine functional group (-NH2) is C 12As used herein, the first carbon atom of dodecylamine is conjugated to the oligonucleotide at the 5' end of the oligonucleotide, and the amino group of dodecylamine is on the 12th carbon atom of the dodecylamine.

[0048] As used herein, the term "polyethylene glycol" or "PEG" refers to a compound of the formula H-(O-CH2-CH2) n A polymeric compound having the chemical formula -OH and the following chemical structure: TIFF2025538662000003.tif20145. PEG can be followed by a number representing the average molecular weight, for example, PEG 500, PEG 1000, and PEG 2000 represent average molecular weights of 500, 1000, and 2000, respectively.

[0049] As used herein, the term "nucleotide" refers to a nitrogenous base attached to a sugar phosphate, including sugars such as ribose or 2'-deoxyribose, with one or more phosphate groups attached to the sugar. "Polynucleotide" and "nucleic acid" refer to polymers of more than one nucleotide unit, where the units are typically joined by sugar-phosphate bonds in a sugar-phosphate backbone. A polynucleotide does not necessarily contain a single type of nucleotide unit. For example, a given polynucleotide may contain only ribonucleotides, only 2'-deoxyribonucleotides, or a combination of ribonucleotides and 2'-deoxyribonucleotides. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as nucleic acid analogs containing one or more non-natural units. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" generally refers to large polynucleotides. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this will be understood to also include RNA sequences (i.e., A, U, G, C) in which "U" replaces "T." The term "cDNA" refers to DNA complementary to or identical to mRNA, in which "T" replaces "U," and may be in single- or double-stranded form. The term "recombinant nucleic acid" refers to a polynucleotide or nucleic acid having non-naturally linked sequences. Recombinant nucleic acids may be in the form of a vector.

[0050] As used herein, the term "oligonucleotide" refers to a short DNA or RNA molecule, typically 13 to 25 nucleotides in length. The maximum length of an oligonucleotide is about 200 nucleotide residues.

[0051] As used herein, the terms "microribonucleic acid," "microRNA," and "miRNA" 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 of translation or degradation of the target gene. A single microRNA can regulate many, even hundreds, of different mRNA molecules, and multiple microRNAs can regulate the same mRNA. MicroRNAs are involved in many biological functions, including growth, differentiation, proliferation, and apoptosis.

[0052] As used herein, the term "microRNA 200c-3p" or "miRNA 200c-3p" refers to a specific microRNA molecule belonging to the microRNA-200 family and having 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, suggesting that miR-200c-3p can be a tumor inhibitor in epithelial ovarian cancer (Anastasiadou et al., 2021). Other studies have also shown that miR-200c can suppress PD-L1 mRNA expression in mouse lung tumor cells, further exerting antitumor effects (Zhang et al., 2023).

[0053] The nomenclature used herein to describe the peptides of the invention follows normal 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] The term "peptide" as used herein refers to a molecular chain of amino acids, including L- and D-forms. If necessary, amino acids can be modified in vivo or in vitro, for example, by mannosylation, glycosylation, amidation (especially the C-terminal amide), carboxylation, or phosphorylation, provided that these modifications maintain the biological activity of the original molecule. Peptides can also be part of fusion proteins.

[0055] As used herein, the term "peptide linker" refers to a short chain of amino acids (peptide fragment) for joining or linking components of different functions in various biological or chemical molecules.

[0056] Functional derivatives of peptides are also encompassed by the present invention. Functional derivatives encompass peptides with 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 been described. Amino acid substitutions between related amino acids, or substitutions that have commonly occurred in evolution, include Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, and Ile / Val.

[0057] The peptides of the present invention can be produced synthetically or by recombinant DNA techniques. Methods for producing synthetic peptides are well known in the art.

[0058] Organic chemistry methods used in peptide synthesis are recognized to include coupling the required amino acids by condensation reactions, whether in a homogeneous phase or by so-called solid phase. Condensation reactions can be carried out in the following manner: A compound (amino acid, peptide) having a free carboxy group and a protected reactive group is condensed with a compound (amino acid, peptide) having a free amino group and a protected reactive group in the presence of a condensing agent; A compound (amino acid, peptide) having an activated carboxy group and a free or protected reactive group is condensed with a compound (amino acid, peptide) having a free amino group and a free or protected reactive group; Activation of the carboxy group is achieved 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" includes all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption enhancing or delaying agents, and other physiologically acceptable excipients or additives. In certain specific embodiments, the carrier is adapted for nasal, intravenous, intramuscular, intradermal, subcutaneous, parenteral, oral, transmucosal, or transdermal administration. Depending on the route of administration, the active compound may be enclosed in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The application of such media and agents to pharmaceutical active substances is well known in the art.

[0060] Formulations suitable for administration according to the present invention can include aqueous and non-aqueous solutions, antioxidants, bacteriostats, buffers, solutes that affect isotonicity, preservatives, solubilizers, stabilizers, suspending agents, thickening agents, or combinations thereof, among other factors known to those skilled in the art.

[0061] Additionally or alternatively, formulations suitable for administration of the present invention can include gels, PEG (e.g., PEG 400), propylene glycol, saline, packets, water, other suitable liquids known in the art, or combinations thereof, among other circumstances known to those skilled in the art.

[0062] Additionally or alternatively, formulations suitable for administration of the present invention can 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, humectants, paraffin hydrocarbons, tablets, polyethylene glycol, preservatives, sorbitol, starch (e.g., corn starch, potato starch, or combinations thereof), stearic acid, sucrose, talc, triglycerides, or combinations thereof, among other circumstances known to those of skill in the art.

[0063] Additionally or alternatively, formulations suitable for administration of the present invention can include alcohol (e.g., phenylmethanol or ethanol), benzalkonium chloride, a buffer (e.g., phosphate buffer, acetate buffer, citrate buffer, or a combination thereof), carboxymethylcellulose or microcrystalline cellulose, cholesterol, glucose, fruit juice (e.g., yuzu juice), milk, phospholipids (e.g., lecithin), oil (e.g., vegetable oil, fish oil, or mineral oil, or a combination thereof); other pharmaceutically acceptable carriers known in the art; or combinations thereof, among other circumstances known to those of skill in the art.

[0064] Additionally or alternatively, formulations suitable for administration of the present invention can include biodegradable materials (e.g., polylactic-co-glycolic acid (PLGA) polymers), other substantial degradation products that can be rapidly removed from a biological system, or combinations thereof, among other circumstances known to those skilled in the art.

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

[0066] As used herein, the term "effective amount" or "sufficient amount" of a substance refers to an amount sufficient to achieve a beneficial or desired result (including a clinical result). Thus, an "effective amount" is determined by the context in which it is applied. When administering an immunogenic composition, the effective amount is an immunogenically effective amount, including an amount of the immunogenic composition of the present invention sufficient to elicit an immune response. When administering a drug composition, the effective amount is a pharmaceutically effective amount, including an amount of the drug composition of the present invention sufficient to maintain or produce the desired physiological result. An effective amount can be administered in one or more doses.

[0067] As used herein, the term "pharmaceutically effective amount" refers to an amount capable or sufficient to maintain or produce a desired physiological result, including, but not limited to, treating, reducing, alleviating, eliminating, suppressing, essentially preventing, or preventing a disease, condition, or combination thereof. A pharmaceutically effective amount may include one or more doses administered before, after, or simultaneously. One of ordinary skill in the art will understand how to adjust the dosages of the present invention to suit each type of formulation, including, but not limited to, sustained release formulations. As used herein, the term "prophylactic" refers to a composition that is capable of essentially preventing or preventing all aspects of a disease, condition, or combination thereof. As used herein, the term "therapeutic" refers to a composition that is capable of treating, reducing, preventing the progression of, alleviating the progression of, beneficially altering, eliminating, or a combination thereof, all aspects of a disease, condition, or combination thereof.

[0068] As used herein, the term "dose" of a composition refers to a measured portion of that composition taken (administered or received) by a subject at any given time.

[0069] As used herein, the term "subject" refers to animals, and more specifically, to non-human mammals and human organisms. Non-human animal subjects can also include prenatal forms of animals, such as embryos or fetuses. Non-limiting examples of non-human animals include horses, cows, camels, goats, sheep, dogs, cats, non-human primates, mice, rats, rabbits, hamsters, guinea pigs, and pigs. In some specific embodiments, the subject is a human. Human subjects can also include fetuses.

[0070] As used herein, the term "subject" refers to any subject in need of treatment, particularly mammalian subjects, such as humans.

[0071] As used herein, the terms "treat," "treating," or "treatment" include alleviating at least one symptom, reducing its severity, or inhibiting its worsening. Treating does not necessarily indicate a complete cure of the disease, disorder, or condition. To be an effective treatment, a composition useful herein may reduce the severity of the disease, condition, or condition, reduce the severity of symptoms associated therewith, or improve the quality of life of the patient or subject.

[0072] As used herein, the terms "prevent", "preventing" or "prevention" refer to the ability to essentially eliminate, avoid, evade, forestall, arrest, hinder, impede, or any combination thereof the occurrence of any aspect of a disease, symptom or combination thereof, especially by taking action in advance.

[0073] In some specific embodiments, oligonucleotide therapeutic agents and / or compositions of the present invention can be administered to a subject by multiple routes, including intradermal, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, intraperitoneal, parenteral, oral, rectal, nasal, intrapulmonary, and transdermal administration, or topical administration to the eye, ear, skin, or mucous membranes. Alternatively, the antigen can be optionally carried in a biologically suitable liquid or solid carrier and administered ex vivo by direct contact with cells, tissues, or organs from the subject (autologous) or another subject (alien).

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

[0075] As used herein, the terms "about," "approximately," or "generally," when used in conjunction with a numerical value, refer to plus or minus 10% of the referenced numerical value. For example, a length of about 1000 nanometers (nm) refers to a length in the range of 900 nm to 1100 nm.

[0076] As used herein, the term "comprising" is open, indicating that such embodiment may include additional elements. Conversely, the term "consisting of" is closed, indicating that such embodiment does not include additional elements (except for trace impurities). The words "consisting essentially of" are part-closed, indicating that such embodiment may also include elements that do not materially alter the basic characteristics of such embodiment.

[0077] It will be readily understood that when an applicant defines an invention or part thereof using an open conjunction such as "comprising," the specification should be construed as describing the invention also using the conjunctions "consisting essentially of" or "consisting of" (unless otherwise stated).

[0078] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a "polynucleotide" includes a plurality of such polynucleotides; a reference to "the polynucleotide" includes one or more of the polynucleotides referenced, as well as equivalents known to those of skill in the art. It should further be noted that the claims may be drafted to exclude all optional elements. Thus, the text is intended to precede the recitation of elements in the claims or, when using a "negative" limitation, the use of exclusive terminology such as "solely," "only," etc.

[0079] In some situations, when a convention similar to "at least one of A, B, and C, etc." is used, such construction is typically intended to ensure that one of ordinary skill in the art understands the meaning of that convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A only, B only, C only, A and B, A and C, B and C, and / or A, B and C, etc.). Those of ordinary skill in the art will further understand that all disjunctions and / or words that actually represent two or more alternative terms, whether in the description, claims, or drawings, should be considered to encompass the possibilities of one, either, or both of those terms. For example, the words "A or B" are understood to encompass the possibilities of "A," "B," or "A or B."

[0080] The present invention is further illustrated by the following examples. The presentation of these examples is intended to be illustrative and not limiting. Those skilled in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific examples disclosed and still obtain like or similar results without departing from the spirit and scope of the present invention. [Example]

[0081] Example 1 Preparation of Oligonucleotide Therapeutic Agents Materials and Methods Design and Preparation of Oligonucleotide Therapeutic Agents. A schematic diagram of the design of the oligonucleotide therapeutic agents described in the Examples is shown in Figure 1. The oligonucleotide therapeutic agents were prepared 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) was synthesized by solid-phase synthesis, and the 5'-ribose end of the microRNA was ligated with dodecylamine (C 12 H 27 The oligonucleotide therapeutic agent was modified with C12-miR, in which the first carbon atom of the dodecylamine was conjugated to the 5' end of the microRNA and the amino group of the dodecylamine was on the 12th 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 to polyethylene glycol (PEG) of different sizes (0.5, 1, and 2 kDa) at the amino terminus of the dodecylamine. The PEGylation of C12-miR was as follows: 4.15 nM of C12-miR and 4.15 nM of PEG (0.5, 1, and 2 kDa) were mixed and reacted 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. Then, the microspin TMThe mixture was purified using a G-25 column (Sigma-Aldrich, MO, USA) to obtain higher amounts of the oligonucleotide therapeutic agent in this example. C12-miR modified with PEG 500 (0.5 kDa), PEG 1000 (1 kDa), and PEG 2000 (2 kDa) were designated P.5-C12-miR, P1-C12-miR, and P2-C12-miR, respectively.

[0083] In addition, the dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR) was modified with a peptide linker (KGDGG; SEQ ID NO: 2) at the amino terminus of the dodecylamine. The amino terminus of the peptide linker (SEQ ID NO: 2) was further conjugated with PEG 500 (0.5 kDa) to form another oligonucleotide therapeutic agent, designated P.5-L-C12-miR. Briefly, 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 and reacted in MES buffer containing 4.15 nM EDC for 3 hours at room temperature. The mixture was then incubated with Microspin®. TM The mixture was purified on a G-25 column to obtain the oligonucleotide therapeutic agent P.5-L-C12-miR.

[0084] The pegylated 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 260 nm using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, MA, USA). Nonpegylated and pegylated 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] result As shown in Figure 2, unpegylated C12-miR has the expected size of approximately 7 kDa. The sizes of the pegylated C12-miR (P.5-C12-miR, P1-C12-miR, and P2-C12-miR) increase with the size of the conjugated PEG. Furthermore, due to the addition of the peptide linker (SEQ ID NO: 2), the pegylated peptide-linked C12-miR (P.5-L-C12-miR) has a larger size than P.5-C12-miR.

[0086] Example 2 Biological Functional Analysis of Pegylated Oligonucleotide Therapeutic Agents Modified or Unmodified with Peptide Linkers Materials and Methods Cell treatment. Approximately 5x10 SH-SY5Y human neuroblastoma cells (ATCC® CRL-2266) suspended in MEM / F12K medium (1:1, v / v) were cultured. 3Cells were seeded into a 96-well plate at a density of 1 cell / well. The cells were cultured at 37°C in a 5% CO environment for 16 hours and then treated with 2 μM of the C12-miR, P.5-C12-miR, and P.5-L-C12-miR obtained in Example 1. The treated cells were then cultured for an additional 48 hours at 37°C in a 5% CO environment. Cells treated with phosphate-buffered saline (PBS) served as a blank control.

[0087] Flow cytometry analysis. Cells were harvested and washed with PBS. The harvested cells were stained with anti-human PD-L1 surface antibody (No. 329706, Biolegend, California, USA) for 30 minutes at 4°C, protected from light. The cells were then washed twice with cold FACS buffer, resuspended in FACS buffer, and analyzed by flow cytometry (BD LSR Fortessa). TM X20, New Jersey, USA).

[0088] Statistical analysis: All data were analyzed using a one-tailed Student's t-test with the TTEST function in Microsoft Excel (Washington, USA), and a p-value of less than 0.05 between groups was considered to be statistically significant.

[0089] result The oligonucleotide therapeutic agents of the present invention induced PD-L1 expression. As shown in Figure 3, compared with 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 by 23% (p<0.01), 39% (p<0.01), and 58% (p<0.001), respectively. In particular, compared with non-pegylated C12-miR, pegylated C12-miR (P.5-C12-miR) (p<0.05) and pegylated peptide-linked C12-miR (P.5-L-C12-miR) (p<0.001) both significantly increased PD-L1 expression in SH-SY5Y cells. More specifically, compared with pegylated C12-miR (P.5-C12-miR), pegylated peptide-linked C12-miR (P.5-L-C12-miR) also significantly increased the expression of PD-L1 in SH-SY5Y cells (p<0.05).

[0090] These results unexpectedly demonstrated that dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR) increased PD-L1 expression in cells, contrary to previous reports of the inhibition of PD-L1 expression in cells by microRNA 200c-3p mimics or microRNA 200c-3p expression vectors (Anastasiadou et al., 2021; Zhang et al., 2023). These results also demonstrated that PEG-modified microRNA 200c-3p (SEQ ID NO: 1) (P.5-C12-miR) induced greater PD-L1 expression in cells than dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR). The results further showed that the addition of a peptide linker (SEQ ID NO: 2) between PEG and dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (P.5-L-C12-miR) induced cells to express even more PD-L1 than dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR).

[0091] Example 3 Biological Function Analysis of Different Doses of Pegylated Oligonucleotide Therapeutic Agents Materials and Methods Cell Treatment. SH-SY5Y human neuroblastoma cells (ATCC® CRL-2266) were used in this example. Cell culture was as described in Example 2, except that in this example, cells were treated with 0.5 μM, 2 μM, and 4 μM C12-miR or P.5-C12-miR. PBS-treated cells served as the blank control (i.e., treated with 0 μM C12-miR or P.5-C12-miR).

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

[0093] Statistical Analysis. The statistical analysis method was as described in Example 2.

[0094] result The oligonucleotide therapeutic agents of 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 by 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 pegylated C12-miR (P.5-C12-miR) significantly increased PD-L1 expression in SH-SY5Y cells by 46% (p<0.001), 39% (p<0.001), and 59% (p<0.001), respectively. Furthermore, P.5-C12-miR at 0.5 μM, 2 μM, and 4 μM significantly induced greater PD-L1 expression in SH-SY5Y cells than C12-miR at 0.5 μM, 2 μM, and 4 μM, respectively (p<0.05 or p<0.01), indicating that PEGylated C12-miR (P.5-C12-miR) has a better effect than C12-miR in inducing PD-L1 expression in cells. These results demonstrate that both C12-miR and PEGylated C12-miR (P.5-C12-miR) induced PD-L1 expression in neuroblastoma cells in a dose-dependent manner.

[0095] Example 4 Biological Functional Analysis of Pegylated Oligonucleotide Therapeutic Agents with Different Sizes of PEG Materials and Methods Cell Treatment. SH-SY5Y human neuroblastoma cells (ATCC® CRL-2266) were used in this example. Cell culture was as described in Example 2, except that in this example, cells were treated with 2 μM C12-miR, P.5-C12-miR, P1-C12-miR, or P2-C12-miR. Cells treated with 2 μM C12-miR served as a positive control.

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

[0097] Statistical Analysis. The statistical analysis method was as described in Example 2.

[0098] result All pegylated oligonucleotide therapeutics with PEGs of different sizes can 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 by 10% (p<0.05), 2%, and 3%, respectively. Among these pegylated C12-miRs, pegylated C12-miR with PEG 500 (P.5-C12-miR) significantly induced the highest level of PD-L1 expression (p<0.05). These results indicate that pegylation of C12-miR with PEGs of different sizes, especially PEG 500, has a favorable effect on the induction of PD-L1 expression in cells.

[0099] Example 5 Analysis of peptide linker degradation Materials and Methods Degradation analysis. The pegylated peptide-linked C12-miR (P.5-L-C12-miR) obtained in Example 1 was enzymatically cleaved in a test tube with lysosomal cathepsin D (Cathepsin D). Briefly, 10 μL of P.5-L-C12-miR (4.15 nM) and 2 μL of cathepsin D (C8696, Sigma-Aldrich, MO, USA) were added to a final volume of 20 μL in 250 mM sodium acetate solution (pH 3.7). The reaction mixture was incubated at 37°C for 5 hours. The reaction products were analyzed by electrophoresis at 200 volts for 1 hour on a 15% (v / v) polyacrylamide gel containing 8 M urea. The gel was then stained with 0.2% (w / v) methylene blue for 20–30 minutes and imaged using Gel Doc EZ (Bio-Rad, California, USA) in conjunction with image analysis software (Image Lab, Bio-Rad). All bands stained for nucleic acid were analyzed, and the band size was determined as the point with the highest density.

[0100] result The peptide linker can be cleaved by cathepsin D. As shown in Figure 6, the pegylated peptide-linked C12-miR (P.5-L-C12-miR) was cleaved by lysosomal cathepsin D to generate smaller fragments. The results indicate that after the oligonucleotide therapeutic P.5-L-C12-miR was taken up into cells by pinocytosis / phagocytosis, the peptide linker was enzymatically cleaved by an enzyme 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 the cells.

[0101] In summary, dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR) unexpectedly increased PD-L1 expression in cells. Furthermore, PEGylation of C12-miR can induce even greater PD-L1 expression. Furthermore, the addition of a peptide linker (SEQ ID NO: 2) between PEG and C12-miR can induce even greater PD-L1 expression in cells. Furthermore, enzymatic degradation of the 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 suggest that the oligonucleotide therapeutic agents of the present invention can be used to prevent, reduce, inhibit, or treat inflammatory responses, particularly neuroinflammatory responses.

[0102] Of course, various changes and modifications can be made in the above-described embodiments of the invention without departing from the scope of the invention. Therefore, in order to promote the progress of science and useful arts, it is the intention to disclose the invention and to limit it only as set forth in the appended claims.

[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. 1. An oligonucleotide therapeutic agent comprising an oligonucleotide and dodecylamine, wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5' end of the oligonucleotide and the amino group of the dodecylamine is on the 12th carbon atom of the dodecylamine.

2. The oligonucleotide therapeutic agent of claim 1, wherein the oligonucleotide is a microRNA.

3. The oligonucleotide therapeutic agent of claim 1, wherein the oligonucleotide consists of the sequence shown in SEQ ID NO:

1.

4. The oligonucleotide therapeutic agent of claim 1, further comprising polyethylene glycol (PEG) conjugated to the dodecylamine at the amino terminus of the dodecylamine.

5. The oligonucleotide therapeutic agent of claim 4, wherein the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

6. The oligonucleotide therapeutic agent of claim 1, further comprising a peptide linker conjugated to the dodecylamine at the amino terminus of the dodecylamine, and polyethylene glycol (PEG) conjugated to the peptide linker at the amino terminus of the peptide linker.

7. The oligonucleotide therapeutic agent of claim 6, wherein the peptide linker consists of the sequence shown in SEQ ID NO:

2.

8. The oligonucleotide therapeutic agent of claim 6, wherein the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

9. 2. The oligonucleotide therapeutic agent of claim 1, selected from the group consisting of: an oligonucleotide therapeutic agent consisting of an oligonucleotide and dodecylamine, wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5' end of the oligonucleotide and the amino group of the dodecylamine is on the 12th carbon atom of the dodecylamine; an oligonucleotide therapeutic agent consisting of an oligonucleotide, dodecylamine, and polyethylene glycol (PEG), wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5' end of the oligonucleotide, the amino group of the dodecylamine is on the 12th carbon atom of the dodecylamine, and the polyethylene glycol (PEG) is conjugated to the dodecylamine at the amino end of the dodecylamine; and An oligonucleotide therapeutic agent comprising an oligonucleotide, dodecylamine, a peptide linker, and polyethylene glycol (PEG), wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5' end of the oligonucleotide, the amino group of the dodecylamine is on the 12th carbon atom of the dodecylamine, the peptide linker is conjugated to the dodecylamine at the amino end of the dodecylamine, and the polyethylene glycol (PEG) is conjugated to the peptide linker at the amino end of the peptide linker.

10. The oligonucleotide therapeutic agent of claim 9, wherein the oligonucleotide is a microRNA.

11. The oligonucleotide therapeutic agent of claim 9, wherein the oligonucleotide consists of the sequence shown in SEQ ID NO:

1.

12. The oligonucleotide therapeutic agent of claim 9, wherein the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

13. The oligonucleotide therapeutic agent of claim 9, wherein the peptide linker consists of the sequence shown in SEQ ID NO:

2.

14. A composition comprising the oligonucleotide therapeutic agent of claim 1 and a pharmaceutically acceptable carrier or excipient.

15. 16. A method of increasing PD-L1 expression in a cell, comprising contacting the cell with the oligonucleotide therapeutic agent of claim 1 or the composition of claim 14.

16. Use of the oligonucleotide therapeutic agent of claim 1 or the composition of claim 14 in increasing PD-L1 expression in cells.

17. Use of the oligonucleotide therapeutic agent of claim 1 or the composition of claim 14 in increasing PD-L1 expression in a subject.

18. Use of an oligonucleotide therapeutic agent according to claim 1 or a composition according to claim 14 in reducing an inflammatory response in a subject.

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