Amphiregulin gene-specific double-stranded oligonucleotide and composition for preventing and treating fibrosis-related diseases and respiratory diseases comprising the same

Abstract

The present invention provides a double-stranded oligonucleotide capable of inhibiting amphiregulin expression with high specificity and high efficiency, preferably a double-stranded oligonucleotide comprising an RNA / RNA, DNA / DNA, or DNA / RNA hybrid sequence, a double-stranded oligonucleotide structure comprising the double-stranded oligonucleotide, and nanoparticles comprising the double-stranded oligonucleotide structure, and uses thereof for preventing or treating fibrosis or respiratory diseases. [Solution] An amphiregulin-specific double-stranded oligonucleotide is provided, which comprises a sense strand containing any one sequence selected from the group consisting of specific sequences and an anti-sense strand containing a sequence complementary thereto.

Description

[Technical field]

[0001] The present invention relates to a double-stranded oligonucleotide capable of inhibiting amphiregulin expression with high specificity and high efficiency, preferably a double-stranded oligonucleotide comprising a sequence in the form of an RNA / RNA, DNA / DNA, or DNA / RNA hybrid, a double-stranded oligonucleotide structure and nanoparticles comprising said double-stranded oligonucleotide, and use thereof for the prevention or treatment of fibrosis or respiratory diseases. [Background technology]

[0002] In 1995, Guo and Kemphues discovered that sense RNA was as effective as antisense RNA in suppressing gene expression using antisense in C. elegans, and began research to clarify the cause. In 1998, Fire et al. were the first to confirm the phenomenon in which double stranded RNA (dsRNA) was injected and the corresponding mRNA was specifically degraded, suppressing gene expression, and named this phenomenon RNA interference (RNAi). RNAi is a method used to suppress gene expression, and since it is simple and inexpensive, and can clearly achieve gene suppression effects, the fields of application of this technology are becoming more diverse.

[0003] Such a technology for suppressing gene expression can regulate the expression of a specific gene, and can therefore remove target genes at the mRNA level for cancer, genetic diseases, etc., which are believed to be caused by the overexpression of a specific gene, and can be used as an important tool for developing therapeutic agents and validating targets for disease treatment. A technology for introducing a transgene into a target gene has been disclosed as a conventional technology for suppressing the expression of a target gene, and there are two methods: a method for introducing a transgene in the opposite direction (antisense) based on the promoter, and a method for introducing a transgene in the forward direction (sense) based on the promoter.

[0004] Such RNA-targeting RNA therapy uses oligonucleotides against target RNA to eliminate the function of the gene, which is different from the conventional therapeutic agents such as existing antibodies and small molecules that mainly target proteins. There are two main approaches to target RNA: one is RNAi mediated by double-stranded RNA, and the other is antisense oligonucleotides (ASOs). Currently, clinical trials are being conducted to target RNA in various diseases.

[0005] Antisense oligonucleotides (ASOs) are short synthetic DNAs designed to bind to target genes via Watson-Crick base reaction. They can specifically suppress the expression of specific base sequences of genes, and have been used to study gene functions and develop therapeutic agents that can treat diseases such as cancer at the molecular level. ASOs can be easily prepared with various targets for suppressing gene expression, and research has been conducted to utilize them to suppress the expression of oncogenes and the growth of cancer cells. ASOs suppress the expression of specific genes by binding to complementary mRNA sequences and inducing RNase H activity to remove mRNA, or by interfering with the formation and progression of ribosomal complexes for protein translation. It has also been reported that ASOs suppress gene transcription by binding to genomic DNA to form a triple helix structure. Although ASOs have the potential described above, in order to utilize them in clinical practice, they need to be more stable against nucleases and efficiently delivered into target tissues and cells so that they can specifically bind to the base sequences of target genes. In addition, the secondary and tertiary structures of gene mRNA are important factors for the specific binding of ASO, and since the parts of mRNA with less secondary structure are very favorable for ASO to approach, we have systematically analyzed the regions of mRNA with less secondary structure before synthesizing ASO, and have made efforts to effectively achieve gene-specific inhibition not only in vitro but also in vivo. Such ASOs are very stable compared to siRNA, which is a type of RNA, and have the advantage of being well dissolved in water and saline, and currently three ASOs have been approved by the FDA (Federal Drug Administration) (Jessica, C., J Postdoc Res, 4: 35-50, 2016).

[0006] Since the role of interference RNA (RNAi) was discovered, it has been shown to act on sequence-specific mRNA in various mammalian cells (Barik, S., J Mol. Med. (2005) 83: 764-773). When a long RNA double strand is delivered to a cell, it is converted into small interfering RNA (siRNA) processed into 21-23 double strands (base pairs, bp) by an endonuclease called Dicer, and the siRNA binds to RISC (RNA-induced silencing complex), where the guide (antisense) strand recognizes and degrades the target mRNA, thereby inhibiting the expression of the target gene in a sequence-specific manner. The gene expression suppression technique using siRNA suppresses the expression of a target gene in a target cell and observes the changes caused by this, and is useful for research to clarify the function of the target gene in the target cell. In particular, suppressing the function of target genes in infectious viruses or cancer cells would be useful in developing treatments for the relevant diseases, and in vitro studies and in vivo studies using experimental animals have shown that it is possible to suppress the expression of target genes using siRNA.

[0007] According to Bertrand's research team, siRNA for the same target gene has a superior effect of inhibiting mRNA expression in vitro and in vivo compared to antisense oligonucleotide (ASO), and the effect lasts longer. In addition, the mechanism of action of siRNA is to bind complementarily to the target mRNA and regulate the expression of the target gene sequence-specifically, so it has the advantage of being able to dramatically expand the range of applicable subjects compared to existing antibody-based drugs and small molecule drugs (MA Behlke, MOLECULAR THERAPY. 2006 13(4):664-670).

[0008] Despite the excellent effects and wide range of uses of siRNA, in order for siRNA to be developed as a therapeutic agent, the stability of siRNA in the body and the cell delivery efficiency must be improved so that siRNA can be effectively delivered to target cells (FY Xie, Drug Discov. Today. 2006 Jan; 11 (1-2): 67-73). In order to improve the stability in the body and solve the problem of non-specific innate immune stimulation of siRNA, some nucleotides or backbones of siRNA are modified to have resistance to nucleases, or carriers such as viral vectors, liposomes, or nanoparticles are used, and research into this is being actively attempted.

[0009] Delivery systems using viral vectors such as adenovirus and retrovirus have high transfection efficacy but high immunogenicity and oncogenicity. On the other hand, non-viral delivery systems including nanoparticles have lower cell transduction efficiency than viral delivery systems, but are currently considered to be more effective than viral delivery systems because of their high in vivo stability, target-specific delivery, uptake and internalization of the encapsulated RNAi oligonucleotides into cells or tissues, and little cytotoxicity and immune stimulation (Akhtar S, J Clin Invest. 2007 December 3; 117(12): 3623-3632).

[0010] Among non-viral delivery systems, the method using nanocarriers is a method in which nanoparticles are formed using various polymers such as liposomes and cationic polymer complexes, and siRNA is carried by such nanoparticles, i.e., nanocarriers, and delivered to cells. The methods using nanocarriers are mainly used for polymeric nanoparticles, polymer micelles, and lipoplexes, and among them, the method using lipoplexes is composed of cationic lipids, which interact with anionic lipids in the endosomes of cells to induce the destabilization effect of the endosomes and deliver the siRNA into cells.

[0011] It is also known that by linking a chemical to the end of the siRNA passenger (sense) strand, it is possible to give the siRNA improved pharmacokinetic properties and induce high efficiency in vivo (J Soutschek, Nature 11;432(7014):173-8, 2004). In this case, the stability of the siRNA changes depending on the properties of the chemical compound bound to the end of the siRNA sense (passenger) or antisense (guide) strand. For example, siRNA conjugated with a polymer compound such as polyethylene glycol (PEG) interacts with the anionic phosphate group of siRNA in the presence of cationic substances to form a complex, thereby becoming a carrier with improved siRNA stability (SH Kim, J Control Release 129(2):107-16,2008). In particular, micelles made of polymer complexes have a very small size and a very uniform distribution compared to other systems used as drug delivery vehicles, such as microspheres or nanoparticles, and are a structure that forms spontaneously, so they have the advantage of being easy to control the quality and ensure reproducibility of the formulation.

[0012] In order to improve the intracellular delivery efficiency of siRNA, a technology for ensuring siRNA stability and efficient cell membrane permeability was developed using siRNA conjugates in which siRNA is conjugated to a biocompatible polymeric hydrophilic substance (e.g., polyethylene glycol (PEG)) by simple covalent or linker-mediated covalent bonding (Korea Patent No. 883471). However, the chemical modification of siRNA and the conjugation of polyethylene glycol (PEG) alone (PEGylation) still leave issues of low stability in vivo and smooth delivery to target organs. To solve these problems, a double-stranded oligoRNA structure has been developed in which hydrophilic and hydrophobic substances are bound to oligonucleotides, particularly double-stranded oligoRNA such as siRNA. This structure forms self-assembled nanoparticles called SAMiRNATM (self assembled micelle inhibitory RNA) through the hydrophobic interactions of the hydrophobic substances (Korea Patent Registered No. 1224828). SAMiRNATM technology has the advantage of being able to produce homogenous nanoparticles that are very small in size compared to existing delivery technologies.

[0013] In a specific example of SAMiRNATM technology, PEG (polyethylene glycol) or HEG (hexaethylenglycol) is used as a hydrophilic substance. PEG is a synthetic polymer that is often used to increase the solubility and adjust the pharmacokinetics of pharmaceuticals, especially proteins. PEG is a polydisperse substance, and one batch of polymer is composed of a sum of different numbers of monomers, and the molecular weight shows a Gaussian curve shape, and the degree of homogeneity of the substance is expressed by the polydisperse index (Mw / Mn). That is, when PEG has a low molecular weight (3-5kDa), it shows a polydispersity index of about 1.01, and when it has a high molecular weight (20kDa), it shows a high polydispersity index of about 1.2, and the higher the molecular weight, the lower the homogeneity of the substance. Therefore, when PEG is bound to a drug, the polydispersity characteristics of PEG are reflected in the conjugate, making it difficult to verify a single substance. Although there is a trend to produce substances with low polydispersity indexes by improving the synthesis and purification process of PEG, there are problems associated with the polydispersity characteristics of substances, such as the inconvenience of being unable to easily confirm whether PEG has been bound, especially when PEG is bound to a substance with a low molecular weight. (Francesco M.VDRUG DISCOVERY TODAY(2005)10(21):1451-1458).

[0014] Accordingly, a new type of delivery vehicle technology has been developed that is an improved version of the existing self-assembling nanoparticle SAMiRNATM technology, and that has a smaller size and significantly improved polydispersity than the existing SAMiRNATM by blocking the hydrophilic substance of the double-stranded oligonucleotide structure constituting SAMiRNATM with a uniform 1 to 15 monomers having a certain molecular weight and a basic unit including a linker as necessary, and by using an appropriate number of these as necessary. In addition, it is already known that when siRNA is injected into the body, the siRNA is rapidly decomposed by various enzymes present in the blood, and the delivery efficiency to the target cells or tissues is poor, and even the improved SAMiRNATM has variations in stability and expression inhibition rate depending on the target gene. Therefore, in order to more stably and effectively inhibit the expression of the target gene using the improved self-assembling nanoparticle SAMiRNATM, the inventors have attempted to improve the expression inhibition effect and stability on the target gene by applying a double-stranded oligonucleotide in the form of a DNA-RNA hybrid, using a DNA sequence that is an ASO as the sense strand, which is the guide, and an RNA sequence as the antisense strand, which is the passenger.

[0015] Idiopathic Pulmonary Fibrosis (IPF), a type of fibrosis, is a disease in which chronic inflammatory cells invade the walls of the alveoli (lung sacs) causing various changes that harden the lungs, causing major structural changes in the lung tissue, gradually reducing lung function and ultimately leading to death. At present, there is no effective treatment, and once symptoms are felt and a diagnosis is made, the average life expectancy is only about 3 to 5 years, making it a disease with a very poor prognosis. The incidence rate overseas is reported to be about 3 to 5 cases per 100,000 population, and the incidence rate is usually higher after the age of 50, with men being about twice as likely to develop the disease than women.

[0016] The cause of IPF is not yet clear, but it is common in smokers. Antidepressants, chronic pulmonary inhalation due to gastroesophageal reflux, metal dust, wood dust, and solvent inhalation have been reported as risk factors associated with the development of IPF, but no factors with a definite causal relationship have been reported in most patients. The most commonly mentioned factors are activation of Th1 / Th2 reactions and coagulation cascades, regardless of the cause, which secrete fibrotic cytokines, which stimulate fibroblasts to increase ECM (extracellular matrix), resulting in pulmonary fibrosis. Of course, pulmonary fibrosis can occur due to inflammation in the lungs during this process, but recently the opinion that pulmonary fibrosis can occur directly regardless of lung inflammation has become more prevalent. The most recent hypothesis is that pathological pulmonary fibrosis occurs during the wound healing process due to abnormal signaling systems in epithelial-mesenchymal interaction. When epithelial cells are damaged, apoptosis of epithelial cells increases, migration is restricted, differentiation is not regulated, proliferation is inhibited, and soluble factors (TGF, HGF, KGF, angiotensin II, ROS, etc.) are secreted, and apoptosis of mesenchymal cells is inhibited along with ECM, which induces pulmonary fibrosis through increased differentiation of myofibroblasts and ECM infiltration or stimulates epithelial cells again. In other words, lung inflammation does not directly lead to pulmonary fibrosis, but it means that lung inflammation occurs first, and then pulmonary fibrosis occurs in the process of healing as normal tissue, depending on the difference between IPF patients and normal people. IPF can also be induced by the disharmony of Th1 / Th2 cytokines, and while Th1 cytokine responses are related to cell-mediated immunity and restore the damaged site to normal for tissue recovery, Th2 cytokines induce ECM infiltration and fibrosis through the activation and proliferation of fibroblasts.It has been reported that administration of IFN-γ in a bleomycin-based pulmonary fibrosis model can reduce TGF-β and procollagen mRNA and prevent pulmonary fibrosis; however, the exact cause of the disease is unknown. In the future, it will be necessary to clarify the initial causative factors that cause fibrosis and develop substances that can suppress genes and the TGF-β signaling system associated with IPF.

[0017] It is known that IPF will continue to worsen if left untreated, and that more than 50% of patients will die within 3 to 5 years. Moreover, once symptoms have progressed and fibrosis has been completely achieved, no treatment will improve the condition, so it is predicted that early treatment is likely to be effective if treatment is to be performed. Currently, a combination therapy of steroids and azathioprine or cyclophosphamide is known, but it is not particularly effective, and various fibrosis inhibitors have been tried in animal experiments and small-scale patients, but no clear effectiveness has been demonstrated. In particular, for patients with end-stage IPF, there is no effective treatment other than lung transplantation. For this reason, the development of a more efficient IPF treatment is urgently needed.

[0018] Fibrosis is a general term for diseases in which tissues and organs become hardened due to excessive fibrosis of connective tissue for some reason, and all fibrosis processes, regardless of the location, follow the same route as the scar healing process. There is currently no complete cure for fibrosis symptoms, and treatment methods are being developed and researched. Since an effective fibrosis treatment agent can be applied to not only representative fibrosis such as cirrhosis, liver fibrosis, myelofibrosis, myocardial fibrosis, renal fibrosis, and pulmonary fibrosis, but also various diseases accompanied by fibrosis, there is a pressing need to develop an efficient fibrosis treatment agent.

[0019] On the other hand, it is known that amphiregulin binds to epidermal growth factor receptor, activates the epidermal cell receptor pathway (EGFR pathway), and is involved in cell proliferation. It has been disclosed that amphiregulin expression can be inhibited by amphiregulin-specific siRNA, which shows therapeutic effects on certain types of breast cancer. It has also been disclosed that cell invasion in inflammatory breast cancer can be suppressed using shRNA against amphiregulin (Andrea Baillo, J Cell Physiol. 2011 226(10):2691-2701), and that suppression of amphiregulin expression using amphiregulin-specific shRNA suppresses pulmonary artery remodeling in mice exposed to cigarette smoke. It has been disclosed that amphiregulin is associated with airway smooth muscle (ASM) hyperplasia and angiogenesis, in particular promoting airway remodeling in asthmatic patients, and that epidermal growth factor (EGF) and amphiregulin, which are oversecreted in tissue remodeling due to acute asthma, are involved.

[0020] As described above, amphiregulin has been suggested as a possible target for treating respiratory diseases and fibrosis, particularly COPD and IPF. However, technological development of RNAi therapeutic agents against amphiregulin and their delivery technologies is still limited, and there is extremely high market demand for double-stranded oligonucleotide therapeutic agents and their delivery technologies that can specifically and efficiently inhibit amphiregulin expression.

[0021] Therefore, the present inventors selected amphiregulin as a gene associated with fibrosis including IPF, selected double-stranded oligonucleotides targeting amphiregulin, and identified an RNAi therapeutic agent and its delivery vehicle capable of inhibiting amphiregulin expression, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0022] An object of the present invention is to provide a double-stranded oligonucleotide capable of inhibiting amphiregulin expression with high specificity and high efficiency, preferably a double-stranded oligonucleotide comprising an RNA / RNA, DNA / DNA, or DNA / RNA hybrid sequence, a double-stranded oligonucleotide structure comprising the double-stranded oligonucleotide, and nanoparticles comprising the double-stranded oligonucleotide structure.

[0023] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating fibrosis or respiratory diseases, comprising the double-stranded oligonucleotide, a double-stranded oligonucleotide structure containing the same, and / or nanoparticles containing the double-stranded oligonucleotide structure as an active ingredient.

[0024] It is yet another object of the present invention to provide a method for preventing or treating fibrosis or a respiratory disease, comprising the step of administering the pharmaceutical composition for preventing or treating fibrosis or a respiratory disease to an individual in need of such prevention or treatment.

[0025] It is yet another object of the present invention to provide the double-stranded oligonucleotide, a double-stranded oligonucleotide structure comprising the same, and nanoparticles comprising the double-stranded oligonucleotide structure, for use in the prevention or treatment of fibrosis or respiratory diseases.

[0026] It is yet another object of the present invention to provide said pharmaceutical composition for use in the prevention or treatment of fibrotic or respiratory diseases.

[0027] It is yet another object of the present invention to provide uses of the double-stranded oligonucleotide, a double-stranded oligonucleotide structure comprising the same, and nanoparticles comprising the double-stranded oligonucleotide structure for the manufacture of a drug for the prevention or treatment of fibrosis or respiratory disease. [Means for solving the problem]

[0028] In order to achieve the above-mentioned object, the present invention provides a double-stranded oligonucleotide comprising a sense strand containing any one sequence selected from the group consisting of SEQ ID NOs: 1 to 14, more preferably the group consisting of SEQ ID NOs: 10, 11 and 12, and an anti-sense strand containing a sequence complementary thereto.

[0029] The present invention also provides a double-stranded oligonucleotide structure comprising the double-stranded oligonucleotide, and a nanoparticle comprising the double-stranded oligonucleotide structure.

[0030] The present invention also provides a pharmaceutical composition for the prevention or treatment of fibrosis or a respiratory disease, comprising a double-stranded oligonucleotide comprising a sense strand containing any one sequence selected from the group consisting of SEQ ID NOs: 1 to 14, more preferably the group consisting of SEQ ID NOs: 10, 11, and 12, and an anti-sense strand containing a complementary sequence thereto, a double-stranded oligonucleotide structure comprising the double-stranded oligonucleotide, or nanoparticles comprising the double-stranded oligonucleotide structure.

[0031] The present invention also provides a method for preventing or treating fibrosis or a respiratory disease, comprising the step of administering the pharmaceutical composition for preventing or treating fibrosis or a respiratory disease to an individual in need of such prevention or treatment.

[0032] The double-stranded oligonucleotide according to the present invention, which comprises a sense strand comprising any one sequence selected from the group consisting of SEQ ID NOs: 1 to 14, more preferably the group consisting of SEQ ID NOs: 10, 11, and 12, and an anti-sense strand comprising a sequence complementary thereto, a double-stranded oligonucleotide structure comprising the same, and nanoparticles comprising the same can inhibit amphiregulin expression very efficiently, and therefore the double-stranded oligonucleotide according to the present invention, the double-stranded oligonucleotide structure comprising the same, and nanoparticles comprising the same can each be useful in the prevention or treatment of fibrosis or respiratory diseases.

[0033] To achieve the above object, the sequences of SEQ ID NOs: 10, 11 and 12 contained in the preferred double-stranded oligonucleotides provided are as follows: 5'-CACCTACTCTGGGAAGCGT-3' (SEQ ID NO: 10) 5'-ACCTACTCTGGGAAGCGTG-3' (SEQ ID NO: 11) 5'-CTGGGAAGCGTGAACCATT-3' (SEQ ID NO: 12)

[0034] It will be apparent to those skilled in the art that the double-stranded oligonucleotide according to the present invention includes all substances having a general RNAi (RNA interference) effect, and the mRNA-specific double-stranded oligonucleotide encoding the amphiregulin protein includes amphiregulin-specific shRNA, etc. In other words, the oligonucleotide can be characterized as being siRNA, shRNA, or miRNA.

[0035] Furthermore, it will be clear to a person having ordinary skill in the art to which the present invention pertains that amphiregulin-specific siRNAs and antisense oligonucleotides comprising a sense strand containing any one of sequences selected from the group consisting of SEQ ID NOs: 10, 11 and 12, or an antisense strand complementary thereto, in which one or more bases have been substituted, deleted or inserted, are also within the scope of the present invention, as long as specificity for amphiregulin is maintained.

[0036] In the present invention, the sense and antisense strands are characterized by being independently DNA or RNA, and a hybrid form may be used in which the sense strand is DNA and the antisense strand is RNA, or the sense strand is RNA and the antisense strand is DNA.

[0037] In the present invention, the sequences of SEQ ID NOs: 10, 11 and 12 are described as being in the form of DNA, however, when an RNA form is used, the sequences of SEQ ID NOs: 10, 11 and 12 may be used as the corresponding RNA sequences, i.e., sequences in which T is changed to U.

[0038] Furthermore, as long as the specificity for amphiregulin is maintained, the double-stranded oligonucleotide of the present invention includes not only cases in which the sense strand of the sequence is a 100% complementary base sequence to the binding site of the amphiregulin gene, i.e., a perfect match, but also cases in which some of the base sequences do not match, i.e., a mismatch.

[0039] The double-stranded oligonucleotide of the present invention may contain an overhang, which is a structure containing one or more unpaired nucleotides at the 3' end of one or both strands.

[0040] In the present invention, the sense strand or antisense strand can be characterized as being preferably composed of 19 to 31 nucleotides, but is not limited thereto.

[0041] In the present invention, a double-stranded oligonucleotide comprising a sense strand containing any one sequence selected from the group consisting of SEQ ID NOs: 10, 11, and 12 and an antisense strand containing a complementary sequence thereto may be characterized as being specific to amphiregulin, but is not limited thereto.

[0042] In the present invention, the sense strand or antisense strand of the double-stranded oligonucleotide includes various chemical modifications for improving in vivo stability, or for imparting resistance to nucleases and reducing nonspecific immune reactions. The chemical modifications include a modification in which the hydroxyl group (-OH) at the 2' carbon position of the sugar structure in the nucleotide is replaced with any one selected from the group consisting of a methyl group (-CH3), a methoxy group (-OCH3), an amine group (-NH2), fluorine (-F), an O-2-methoxyethyl group, an O-propyl group, an O-2-methylthioethyl group, an O-3-aminopropyl group, an O-3-dimethylaminopropyl group, an O-methylacetamide group, and an O-dimethylamidooxyethyl group; a modification in which the oxygen in the sugar structure in the nucleotide is replaced with a sulfur group; a modification in which the nucleotide bond is any one bond selected from the group consisting of a phosphorothioate bond, a boranophosphate bond, and a methyl phosphonate bond; and a modification in which the nucleotide bond is replaced with any one bond selected from the group consisting of a PNA (peptide nucleic acid). and modification into a DNA-RNA hybrid form; (Ann. Rev. Med. 55, 61-65 2004; US 5,660,985; US 5,958,691; US ​​6,531,584; US 5,808,023; US 6,326,358; US 6,175,001; Bioorg. Med. Chem. Lett. 14: 1139-1143, 2003; RNA, 9: 1034-1048, 2003; Nucleic Acid Res. 31: 589-595, 2003; Nucleic Acids Research, 38 (17) 5761-773, 2010; Nucleic Acids Research, 39(5):1823-1832, 2011), but is not limited to these.

[0043] In the present invention, the double-stranded oligonucleotide is characterized in that one or more phosphate groups, preferably 1 to 3 phosphate groups, are bound to the 5'-end of the antisense strand.

[0044] In another aspect, the present invention relates to a double-stranded oligonucleotide structure comprising the structure of the following structural formula (1), in which A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond mediated by a linker, and R represents a double-stranded oligonucleotide.

[0045] In one preferred embodiment, the double-stranded oligonucleotide structure containing an amphiregulin-specific sequence according to the present invention preferably has a structure as shown in structural formula (1). TIFF0007645849000001.tif6170 In the structural formula (1), A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond mediated by a linker, and R represents an amphiregulin-specific double-stranded oligonucleotide.

[0046] The double-stranded oligonucleotide according to the present invention is preferably in the form of a DNA-RNA hybrid, siRNA (short interfering RNA), shRNA (short hairpin RNA), miRNA (microRNA), etc., but is not limited thereto. A single-stranded miRNA inhibitor that can act as an antagonist against miRNA is also possible.

[0047] Hereinafter, the double-stranded oligonucleotide according to the present invention will be described mainly in terms of RNA, but it will be clear to those of ordinary skill in the art that the invention can also be applied to other double-stranded oligonucleotides having the same properties as the double-stranded oligonucleotide according to the present invention.

[0048] More preferably, the double-stranded oligonucleotide structure comprising the amphiregulin-specific double-stranded oligonucleotide according to the present invention has a structure represented by the following structural formula (2). TIFF0007645849000002.tif13170 In the structural formula (2), A, B, X, and Y are the same as defined in the structural formula (1), S represents the sense strand of the amphiregulin-specific double-stranded oligonucleotide, and AS represents the antisense strand of the amphiregulin-specific double-stranded oligonucleotide.

[0049] More preferably, the double-stranded oligonucleotide structure comprising an amphiregulin-specific double-stranded oligonucleotide has a structure represented by the following structural formula (3) or (4). TIFF0007645849000003.tif30170 In structural formula (3) and structural formula (4), A, B, S, AS, X and Y are the same as defined in structural formula (2), and 5' and 3' represent the 5' end and 3' end of the amphiregulin-specific double-stranded oligonucleotide sense strand.

[0050] The hydrophilic material may be characterized as being selected from the group consisting of, but not limited to, polyethylene glycol (PEG), polyvinylpyrrolidone, and polyoxazoline.

[0051] It will be apparent to those skilled in the art to which the present invention pertains that the double-stranded oligonucleotide constructs comprising the amphiregulin-specific double-stranded oligonucleotides of structural formulas (1) to (4) above can have 1 to 3 phosphate groups bound to the 5' end of the antisense strand, and that shRNA can be used instead of RNA.

[0052] The hydrophilic substances in the structural formulas (1) to (4) are preferably polymeric substances having a molecular weight of 200 to 10,000, and more preferably 1,000 to 2,000. For example, the hydrophilic polymeric substance is preferably a nonionic hydrophilic polymer compound such as polyethylene glycol, polyvinylpyrrolidone, or polyoxazoline, but is not necessarily limited thereto.

[0053] In particular, the hydrophilic substance (A) in structural formulas (1) to (4) can be used in the form of a hydrophilic substance block as shown in structural formula (5) or (6) below. By using an appropriate number of such hydrophilic substance blocks (n in structural formula (5) or (6)) as necessary, problems due to polydispersity that may occur when using general synthetic polymer substances, etc., can be greatly improved. In the structural formula (5) or (6), A' is a hydrophilic substance monomer, J is a linker that links m hydrophilic substance monomers together or m hydrophilic substance monomers and a double-stranded oligonucleotide, m is an integer of 1 to 15, and n is an integer of 1 to 10. m -J) or (J-A' m ) corresponds to the basic unit of the hydrophilic block.

[0054] When having a hydrophilic substance block such as structural formula (5) or structural formula (6), the double-stranded oligonucleotide structure including the amphiregulin-specific double-stranded oligonucleotide of the present invention may have a structure such as structural formula (7) or structural formula (8) below. TIFF0007645849000005.tif12170 In the structural formula (7) and the structural formula (8), X, R, Y and B are the same as defined in the structural formula (1), and A', J, m and n are the same as defined in the structural formula (5) and the structural formula (6).

[0055] In the structural formula (5) and the structural formula (6), the hydrophilic substance monomer (A') may be any of the monomers of a nonionic hydrophilic polymer that meets the object of the present invention, and preferably, a monomer selected from the compounds (1) to (3) shown in Table 1, more preferably, a monomer of the compound (1), in which G is preferably selected from O, S, and NH.

[0056] In particular, among the hydrophilic monomers, the monomer represented by compound (1) is very suitable for producing the structure of the present invention because it has excellent biocompatibility, such as being able to introduce various functional groups, having good affinity in vivo, and inducing a small immune response, as well as the advantage of being able to increase the in vivo stability of the double-stranded oligonucleotide contained in the structure represented by structural formula (7) or structural formula (8) and increasing the delivery efficiency.

[0057] [Table 1]

[0058] It is particularly preferred that the hydrophilic substance in the structural formulae (5) to (8) has a total molecular weight in the range of 1,000 to 2,000. Therefore, for example, in the structural formulae (7) and (8), when hexaethylene glycol according to compound (1), i.e., a substance in which G is O and m is 6, is used, since the molecular weight of the hexaethylene glycol spacer is 344, the number of repetitions (n) is preferably 3 to 5. In particular, in the present invention, if necessary, in the structural formulae (5) and (6), (A' m -J) n Or (J-A' m ) nThe repeating unit of the hydrophilic group represented by n, i.e., the hydrophilic block, can be an appropriate number represented by n. The hydrophilic monomer A and the linker J contained in each hydrophilic block may be independently the same or different between the hydrophilic blocks. That is, when three hydrophilic blocks are used (n=3), a hydrophilic monomer based on compound (1) may be used in the first block, a hydrophilic monomer based on compound (2) may be used in the second block, and a hydrophilic monomer based on compound (3) may be used in the third block, and different hydrophilic monomers may be used for all the hydrophilic blocks, or any one of the hydrophilic monomers based on compounds (1) to (3) may be used for all the hydrophilic blocks. Similarly, the linker mediating the bond between the hydrophilic monomers may be the same for each hydrophilic block, or different linkers may be used for each hydrophilic block. In addition, the number m of hydrophilic substance monomers may be the same or different in each hydrophilic substance block. That is, different numbers of hydrophilic substance monomers may be used, such as three hydrophilic substance monomers linked together (m=3) in the first hydrophilic substance block, five hydrophilic substance monomers linked together (m=5) in the second hydrophilic substance block, and four hydrophilic substance monomers linked together (m=4) in the third hydrophilic substance block, or the same number of hydrophilic substance monomers may be used in all hydrophilic substance blocks.

[0059] In the present invention, the linker (J) is -PO3 - It is preferable that the linker is selected from the group consisting of -, -SO3- and -CO2-, but it is not limited thereto, and it will be apparent to one of ordinary skill in the art that any linker that meets the objectives of the invention can be used depending on the monomer of the hydrophilic substance used, etc.

[0060] The hydrophobic substance (B) in the structural formulas (1) to (4), (7) and (8) plays a role in forming nanoparticles composed of double-stranded oligonucleotide structures represented by the structural formulas (1) to (4), (7) and (8) through hydrophobic interaction. The hydrophobic substance preferably has a molecular weight of 250 to 1,000, and is preferably a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, C 12 ~C 50 The hydrophobic substance may be any one selected from the group consisting of unsaturated or saturated hydrocarbons, diacylphosphatidylcholine, fatty acids, phospholipids, lipopolyamines, lipids, tocopherols, and tocotrienols, but is not limited thereto, and any hydrophobic substance may be used as long as it meets the objectives of the present invention, as will be apparent to a person skilled in the art to which the present invention pertains.

[0061] The steroid derivative may be selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cotestanyl formate and cholesterylamine, and the glyceride derivative may be selected from mono-, di- and tri-glycerides, in which case the fatty acid of the glyceride may be selected from the group consisting of C 12 ~C 50 Preferred are unsaturated or saturated fatty acids of the formula:

[0062] Among the hydrophobic substances, saturated or unsaturated hydrocarbons or cholesterol are particularly preferred since they can be easily bound to the double-stranded oligonucleotide structure of the present invention during synthesis. 24 Carbohydrates, especially those containing disulfide bonds, are most preferred.

[0063] The hydrophobic substance is bound to the distal end opposite to the hydrophilic substance, and may be bound to either the sense strand or the antisense strand of the double-stranded oligonucleotide.

[0064] In the structural formulae (1) to (4), (7) and (8) according to the present invention, the hydrophilic or hydrophobic substance and the amphiregulin-specific double-stranded oligonucleotide are bound by a simple covalent bond or a covalent bond (X or Y) mediated by a linker. The linker mediating the covalent bond is not particularly limited as long as it covalently binds the hydrophilic or hydrophobic substance to the end of the amphiregulin-specific double-stranded oligonucleotide and provides a bond that can be degraded in a specific environment as necessary. Therefore, the linker can be any compound that is bound to activate the amphiregulin-specific double-stranded oligonucleotide and / or the hydrophilic substance (or hydrophobic substance) in the process of producing the double-stranded oligonucleotide structure according to the present invention. The covalent bond may be either a non-degradable bond or a degradable bond. In this case, the non-degradable bond may be an amide bond or a phosphorylated bond, and the degradable bond may be a disulfide bond, an acid degradable bond, an ester bond, an anhydride bond, a biodegradable bond or an enzymatic degradable bond, but is not limited thereto.

[0065] Furthermore, the amphiregulin-specific double-stranded oligonucleotide represented by R (or S and AS) in structural formulas (1) to (4), (7) and (8) can be any double-stranded oligonucleotide that has the property of being able to specifically bind to amphiregulin mRNA, and preferably, in the present invention, is composed of a sense strand containing any one of sequences selected from SEQ ID NOs: 10, 11 and 12 and an antisense strand containing a complementary sequence thereto.

[0066] The present invention also provides that in a double-stranded oligonucleotide structure comprising the amphiregulin-specific double-stranded oligonucleotide of the present invention, an amine group or a polyhistidine group may be further introduced into the terminal site of the hydrophilic substance in the structure opposite to the terminal site bound to the oligonucleotide.

[0067] This is to facilitate intracellular introduction and endosomal escape of a carrier of a double-stranded oligonucleotide structure including the amphiregulin-specific double-stranded oligonucleotide of the present invention. It has already been reported that the introduction of amine groups and the use of polyhistidine groups can be used to facilitate intracellular introduction and endosomal escape of carriers such as quantum dots, dendrimers, and liposomes, and the effects thereof.

[0068] Specifically, the primary amine group modified at the end or outside of the carrier is positively charged at the pH level in vivo and forms a bond with the negatively charged gene through electrostatic interaction. After entering the cell, the internal tertiary amine, which has a buffering effect at the low pH level of the endosome, facilitates escape from the endosome, and is known to be able to protect the carrier from degradation in the lysosome (Gene transfer and expression inhibition using polymer-based hybrid materials. Polymer Sci. Technol., Vol. 23, No. 3, pp. 254-259).

[0069] Histidine, a non-essential amino acid, has an imidazole ring (pKa3 6.04), which has the effect of increasing the buffering capacity in endosomes and lysosomes, and it is known that histidine modification can be used to increase the endosomal escape efficiency in non-viral gene carriers such as liposomes (Novel histidine-conjugated galactosylated cationic liposomes for efficient hepatocyte selective gene transfer in human hepatoma HepG2 cells.J.Controlled Release 118,pp262-270).

[0070] The amine or polyhistidine groups may be linked to a hydrophile or hydrophilic block through one or more linkers.

[0071] When an amine group or a polyhistidine group is introduced into the hydrophilic substance of the double-stranded oligonucleotide structure according to the structural formula (1) of the present invention, it may have a structure as shown in the structural formula (9). In the structural formula (9), A, B, R, and X are the same as those defined in the structural formula (1), P represents an amine group or a polyhistidine group; J1 and J2 are linkers; J1 and J2 are independently a simple covalent bond, PO3-, SO3, CO2, C 2-12 It will be apparent to one of ordinary skill in the art that any linker may be used for J1 and J2 that is suitable for the purposes of the present invention, depending on the hydrophilic material used, including but not limited to alkyl, alkenyl, and alkynyl.

[0072] Preferably, when an amine group is introduced, J2 is a simple covalent bond or PO3-, and J1 is a C6 alkyl, but is not limited thereto.

[0073] When a polyhistidine group is introduced, in the structural formula (9), J2 is preferably a simple covalent bond or PO3-, and J1 is preferably the compound (4), but is not limited thereto. JPEG0007645849000008.jpg32170

[0074] In addition, when the hydrophilic substance of the double-stranded oligonucleotide structure represented by structural formula (9) is a hydrophilic substance block represented by structural formula (5) or structural formula (6) to which an amine group or a polyhistidine group is introduced, it can have a structure such as structural formula (10) or structural formula (11). TIFF0007645849000009.tif14170 In the structural formula (10) and the structural formula (11), X, R, Y, B, A', J, m and n are the same as defined in the structural formula (5) or the structural formula (6), and P, J1 and J2 are the same as defined in the structural formula (9).

[0075] In particular, in structural formula (10) and structural formula (11), it is preferable that the hydrophilic substance is bound to the 3' end of the amphiregulin-specific double-stranded oligonucleotide sense strand, and in this case, structural formula (9) to structural formula (11) can have the following structural formula (12) to structural formula (14). TIFF0007645849000010.tif44170In structural formulas (12) to (14), X, R, Y, B, A, A', J, m, n, P, J1 and J2 are defined as in structural formulas (9) to (11), and 5' and 3' represent the 5' end and 3' end of the amphiregulin-specific double-stranded oligonucleotide sense strand.

[0076] The amine group that can be introduced in the present invention can be a primary to tertiary amine group, and it is particularly preferable to use a primary amine group. The introduced amine group may exist as an amine salt. For example, the salt of the primary amine group can be prepared by reacting NH3 + It can exist in the form of

[0077] Moreover, the polyhistidine group that can be introduced in the present invention preferably contains 3 to 10 histidines, particularly preferably 5 to 8 histidines, and most preferably 6 histidines. In addition to histidines, one or more cysteines may also be contained.

[0078] On the other hand, when a targeting moiety is provided in the double-stranded oligonucleotide structure containing the amphiregulin-specific double-stranded oligonucleotide of the present invention and the nanoparticles formed from the structure, it can efficiently promote delivery to target cells, be delivered to target cells even at a relatively low dosage, exhibit high target gene expression regulation function, and prevent non-specific delivery of the amphiregulin-specific double-stranded oligonucleotide to other organs and cells.

[0079] Accordingly, the present invention provides a double-stranded oligo-RNA structure in which a ligand (L), particularly a ligand having a property of specifically binding to a receptor that promotes internalization into a target cell through receptor-mediated endocytosis (RME), is further bound to the structure represented by structural formula (1) to structural formula (4), structural formula (7), and structural formula (8). For example, the form in which a ligand is bound to the double-stranded oligo-RNA structure represented by structural formula (1) has a structure represented by structural formula (15) below. TIFF0007645849000011.tif8170 In the structural formula (15), A, B, X, and Y are the same as those defined in the structural formula (1), L represents a ligand having a property of specifically binding to a receptor that promotes internalization into a target cell through receptor-mediated endocytosis (RME), and i represents an integer of 1 to 5, preferably an integer of 1 to 3.

[0080] The ligand in structural formula (15) may be selected from, but is not limited to, a target receptor-specific antibody, aptamer, or peptide having RME properties that promote internalization in a target cell-specific manner; or a chemical substance such as folate (generally, folate and folic acid are used interchangeably, and folate in the present invention refers to folate in its natural state or in its activated state in the human body), hexoamines such as N-acetylgalactosamine (NAG), sugars such as glucose, mannose, and carbohydrates.

[0081] Furthermore, the hydrophilic substance A in the structural formula (15) can be used in the form of a hydrophilic substance block represented by structural formula (5) or (6).

[0082] In yet another aspect, the present invention provides a method for producing a double-stranded oligonucleotide construct comprising an amphiregulin-specific double-stranded oligonucleotide.

[0083] The process for producing a double-stranded oligonucleotide structure containing the amphiregulin-specific double-stranded oligonucleotide according to the present invention can be, for example, (1) binding a hydrophilic substance to a solid support; (2) synthesizing a single strand of oligonucleotide on the solid support to which the hydrophilic substance is bound; (3) covalently binding a hydrophobic substance to the 5' end of the single stranded oligonucleotide; (4) synthesizing a single-stranded oligonucleotide having a sequence complementary to that of the single-stranded oligonucleotide; (5) upon completion of the synthesis, isolating and purifying the oligonucleotide-polymer construct and the single stranded oligonucleotide from the solid support; (6) preparing a double-stranded oligonucleotide construct by annealing a single strand of oligonucleotide of complementary sequence to the prepared oligonucleotide-polymer construct.

[0084] The solid support in the present invention is preferably, but not limited to, CPG (Controlled Pore Glass), and may be polystyrene (PS), polymethyl methacrylate (PMMA), silica gel, cellulose paper, etc. In the case of CPG, the diameter is preferably 40 to 180 μm, and the pore size is preferably 500 to 3000 Å. After the step (5), when the preparation is completed, the purified RNA-polymer construct and the single-stranded oligonucleotide are subjected to molecular weight measurement using a MALDI-TOF mass spectrometer to confirm whether the desired oligonucleotide-polymer construct and single-stranded oligonucleotide have been prepared. In the preparation method, the step (4) of synthesizing a single-stranded oligonucleotide having a sequence complementary to the sequence of the single-stranded oligonucleotide synthesized in the step (2) may be performed before the step (1) or during any one of the steps (1) to (5).

[0085] Also possible is a production method characterized in that the single-stranded oligonucleotide having a complementary sequence to the single-stranded oligonucleotide synthesized in step (2) is used in a form in which a phosphate group is bound to the 5' end.

[0086] Meanwhile, the present invention provides a method for producing a double-stranded oligonucleotide structure comprising the amphiregulin-specific double-stranded oligonucleotide of the present invention and a ligand further bound to the double-stranded oligonucleotide structure.

[0087] A method for producing a double-stranded oligonucleotide structure containing a ligand-bound amphiregulin-specific double-stranded oligonucleotide includes, for example, (1) binding a hydrophilic substance to a solid support having functional groups attached thereto; (2) synthesizing a single stranded oligonucleotide based on a solid support having a functional group-hydrophilic substance attached thereto; (3) synthesizing the single-stranded oligonucleotide by covalently binding a hydrophobic substance to the 5'-end; (4) synthesizing a single-stranded oligonucleotide having a sequence complementary to that of the single-stranded oligonucleotide; (5) upon completion of the synthesis, separating the functional group-oligonucleotide-polymer construct and the single stranded oligonucleotide of complementary sequence from the solid support; (6) binding a ligand to the end of the hydrophilic substance using the functional group to prepare a single-stranded ligand-oligonucleotide-polymer construct; (7) preparing a ligand-double-stranded oligonucleotide construct by annealing a single-stranded oligonucleotide having a complementary sequence to the prepared ligand-oligonucleotide-polymer construct; may comprise:

[0088] When the preparation is completed after step (6), the ligand-oligonucleotide-polymer construct and the single stranded oligonucleotide having a complementary sequence can be separated and purified, and the molecular weight can be measured by a MALDI-TOF mass spectrometer to confirm whether the desired ligand-oligonucleotide-polymer construct and complementary oligonucleotide have been prepared. A ligand-double-stranded oligonucleotide construct can be prepared by annealing the prepared ligand-oligonucleotide-polymer construct and the single stranded oligonucleotide having a complementary sequence. In the preparation method, step (4) of synthesizing a single stranded oligonucleotide having a complementary sequence to the single stranded oligonucleotide synthesized in step (3) may be performed as an independent synthesis process before step (1) or during any one of steps (1) to (6).

[0089] In yet another aspect, the present invention relates to a nanoparticle comprising the double-stranded oligonucleotide structure according to the present invention. In the case of the double-stranded oligonucleotide structure according to the present invention, self-assembled nanoparticles are formed by hydrophobic interactions between hydrophobic substances (Korean Patent Publication No. 1224828), and such nanoparticles are not only highly efficient in delivery to the body and highly stable in the body, but also have excellent uniformity in particle size, making QC (Quality Control) easy and simplifying the manufacturing process as a drug.

[0090] In the present invention, the nanoparticles are characterized by being composed of a mixture of double-stranded oligonucleotide structures comprising double-stranded oligonucleotides having different sequences from each other. For example, the nanoparticles may comprise the same type of amphiregulin-specific double-stranded oligonucleotide comprising a sense strand comprising any one of sequences selected from SEQ ID NOs: 10 to 12 and an antisense strand comprising a sequence complementary thereto. In yet another embodiment, the nanoparticles may comprise both heterogeneous amphiregulin-specific double-stranded oligonucleotides comprising a sense strand comprising any one of sequences selected from SEQ ID NOs: 10 to 12 and an antisense strand comprising a sequence complementary thereto, or may comprise both an amphiregulin-specific double-stranded oligonucleotide not disclosed in the present invention.

[0091] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating fibrosis or respiratory diseases, which contains as an active ingredient the double-stranded oligonucleotide, the double-stranded oligonucleotide structure, or the nanoparticles containing the double-stranded oligonucleotide structure according to the present invention.

[0092] The pharmaceutical composition for preventing or treating fibrosis or respiratory diseases according to the present invention inhibits connective tissue remodeling, in particular pulmonary artery remodeling and airway remodeling, and is effective in preventing or treating fibrosis or respiratory diseases.

[0093] In the present invention, the respiratory disease is characterized as chronic obstructive pulmonary disease (COPD), asthma, acute chronic bronchitis, allergic rhinitis, cough suppressant, expectorant, bronchitis, bronchiolitis, pharyngitis, tonsillitis, or laryngitis, and the fibrosis may be characterized as being selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver fibrosis, cirrhosis, myelofibrosis, myocardial fibrosis, renal fibrosis, pulmonary fibrosis, cardiac fibrosis, and radiation-induced fibrosis, but is not limited thereto. In the present invention, the radiation-induced fibrosis is a side effect frequently induced by radiation therapy, which is often used to treat cancer, tumor, etc., and may be used in the same sense as radiation fibrosis syndrome (RFS).

[0094] The composition of the present invention may further contain one or more pharma- ceutically acceptable carriers in addition to the above-described active ingredients for administration. The pharma-ceutically acceptable carrier must be compatible with the active ingredients of the present invention, and may be used as a mixture of one or more of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or any one or more of these components. If necessary, other common additives such as antioxidants, buffers, bacteriostatic agents, etc. may be added. In addition, the composition may be formulated into an injectable dosage form such as an aqueous solution, suspension, or emulsion by further adding a diluent, dispersant, surfactant, binder, or lubricant. In particular, it is preferable to formulate and provide the composition in a lyophilized dosage form. For the preparation of the lyophilized dosage form, a method commonly known in the technical field to which the present invention belongs may be used, and a stabilizer for lyophilization may be added. The formulation can be suitably prepared according to each disease or ingredient by using a suitable method in the art or the method disclosed in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton PA).

[0095] The composition of the present invention can be determined by a person skilled in the art based on the symptoms of the patient and the severity of the disease, and can be formulated in various forms such as powders, tablets, capsules, liquids, injections, ointments, syrups, etc., and can be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles.

[0096] The composition of the present invention can be administered orally or parenterally. The administration route of the composition of the present invention can be, for example, oral, inhalation, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, visceral, sublingual or topical, but is not limited thereto. In particular, administration to the lungs through intrabronchial instillation is also possible for the treatment of respiratory diseases. The dosage of the composition of the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate or disease severity, and can be easily determined by a person skilled in the art. In addition, the composition of the present invention can be formulated into an appropriate dosage form using known techniques for clinical administration.

[0097] In another aspect, the present invention relates to a dosage form in lyophilized form comprising the pharmaceutical composition according to the present invention.

[0098] In another aspect, the present invention relates to a method for preventing or treating fibrosis or a respiratory disease, comprising the step of administering the pharmaceutical composition for preventing or treating fibrosis or a respiratory disease according to the present invention to an individual in need of prevention or treatment of fibrosis or a respiratory disease.

[0099] In the present invention, the respiratory disease may be characterized as chronic obstructive pulmonary disease (COPD), asthma, acute chronic bronchitis, allergic rhinitis, cough and expectorant, bronchitis, bronchiolitis, pharyngitis, tonsillitis, or laryngitis, and the fibrosis may be characterized as, but is not limited to, idiopathic pulmonary fibrosis (IPF), cirrhosis, myelofibrosis, myocardial fibrosis, renal fibrosis, pulmonary fibrosis, cardiac fibrosis, liver fibrosis, or radiation-induced fibrosis.

[0100] In yet another aspect, the present invention relates to the double-stranded oligonucleotide, a double-stranded oligonucleotide structure containing the same, and a nanoparticle containing the double-stranded oligonucleotide structure, for use in the prevention or treatment of fibrosis or respiratory diseases.

[0101] In yet another aspect, the present invention relates to the pharmaceutical composition for use in the prevention or treatment of fibrosis or respiratory diseases.

[0102] In yet another aspect, the present invention relates to use of the double-stranded oligonucleotide, a double-stranded oligonucleotide structure containing the same, and nanoparticles containing the double-stranded oligonucleotide structure for the manufacture of a drug for the prevention or treatment of fibrosis or respiratory diseases. [Brief description of the drawings]

[0103] [Figure 1] The results of a screen of 1,257 SAMiRNAs targeting human amphiregulin are shown. [Diagram 2] The nanoparticle size distribution of double-stranded oligo DNA / RNA hybrids containing the selected amphiregulin-specific double-stranded oligonucleotides is shown. (a) SAMi-AREG#10, (b) SAMi-AREG#11, (c) SAMi-AREG#12 [Diagram 3] This shows the results of the quantitative analysis of amphiregulin mRNA expression levels in Example 4, and is a graph showing the relative expression levels (%) of amphiregulin mRNA when SAMiRNA having each of the sequences of SEQ ID NOs: 1 to 14 of the present invention as a sense strand was treated at different concentrations (200 or 600 nM) with the lung cancer cell line A549. [Figure 4]This shows the results of the quantitative analysis of amphiregulin mRNA expression level in Example 5, in which SAMiRNA having the sequence of sequence number 10 of the present invention as the sense strand was treated at different concentrations (12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 600 nM or 1200 nM) with the lung cancer cell line A549, (a) the relative expression level (%) of amphiregulin mRNA was analyzed, and (b) the IC50 value of SAMiRNA was confirmed. [Diagram 5] FIG. 11 shows the results of quantitative analysis of amphiregulin mRNA expression levels in Example 5, in which SAMiRNA having the sequence of sequence number 11 of the present invention as a sense strand was treated at different concentrations (12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 600 nM or 1200 nM) with the lung cancer cell line A549, (a) the relative expression level (%) of amphiregulin mRNA was analyzed, and (b) the IC50 value of SAMiRNA was confirmed. [Figure 6] FIG. 11 shows the results of quantitative analysis of amphiregulin mRNA expression level in Example 5, in which the lung cancer cell line A549 was treated with different concentrations of SAM iRNA having the sequence of sequence number 12 of the present invention as the sense strand (12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 600 nM or 1200 nM), (a) the relative expression level (%) of amphiregulin mRNA was analyzed, and (b) the IC50 value of SAM iRNA was confirmed. [Figure 7] This shows the results of an innate immune response test analysis for candidate amphiregulin sequences in Example 6. Human peripheral blood mononuclear cells (PBMCs) were treated with 2.5 μM of amphiregulin SAMiRNA having SEQ ID NO: 10 (AR-1), 11 (AR-2), or 12 (AR-3) of the present invention as the sense strand, and the relative increase in mRNA of endogenous immune-related cytokines due to amphiregulin-specific SAMiRNA was analyzed, and in vitro cytotoxicity was evaluated using human peripheral blood mononuclear cells. (a) DNA / RNA hybrid SAMiRNA, (b) RNA / RNA hybrid SAMiRNA [Figure 8] 1 shows the quantitative analysis results of amphiregulin mRNA expression level in Example 7, which is an analysis result comparing the relative expression level (%) of amphiregulin mRNA by double-stranded oligo DNA / RNA hybrid and RNA / RNA hybrid containing the selected amphiregulin-specific SAMiRNA. Graph showing the comparison of the relative expression level (%) of amphiregulin mRNA when the lung cancer cell line A549 was treated with different concentrations (200 nM, 600 nM, or 1200 nM) of SAMiRNA having the sequences of SEQ ID NO: 10 (AR-1), 11 (AR-2), and 12 (AR-3) of the present invention as the sense strand. [Figure 9] The results of screening 237 SAMiRNAs targeting mouse amphiregulin and the effects of nine candidate sequences selected from among them are shown. [Figure 10A] This shows the results of the quantitative analysis of mouse amphiregulin mRNA expression levels in Example 8, and is a graph showing the relative expression levels (%) of amphiregulin mRNA when MLg, a mouse lung fibroblast cell line, was treated with different concentrations (200 or 500 nM) of SAMiRNA having the sequences of SEQ ID NOs: 19, 20, and 21 of the present invention as the sense strand. [Figure 10B] This shows the results of the quantitative analysis of mouse amphiregulin mRNA expression levels in Example 8, and is a graph showing the relative expression levels (%) of amphiregulin mRNA when LA-4, a mouse lung epithelial cell line, was treated with different concentrations (200, 500, or 1000 nM) of SAMiRNA having the sequences of SEQ ID NOs: 19, 20, and 21 of the present invention as the sense strand. [Figure 11] This is a graph showing the results of lung tissue staining analysis and the relative expression levels (%) of target gene and fibrosis marker gene mRNA after SAMiRNA-AREG #20 was administered at 1 mg / kg and 5 mg / kg by microvenous administration to silica-induced mice of Example 9. [Figure 12]13 is a graph showing the results of lung tissue staining analysis and the relative expression levels (%) of target genes and fibrosis marker gene mRNA after SAMiRNA-AREG #20 was administered at 5 mg / kg by microvenous administration to bleomycin-induced mice in Example 10. [Figure 13] This is a graph showing the relative expression levels (%) of target gene, fibrosis marker gene, and inflammatory marker gene mRNA in kidney tissue after SAMiRNA-AREG #20 was administered at 1 mg / kg and 5 mg / kg by microvenous administration to UUO model mice using the UUO surgery of Example 11. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0104] The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of more specifically illustrating the present invention, and the scope of the present invention is not limited by these examples. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

[0105] In the present invention, three specific sequences capable of inhibiting amphiregulin expression were identified, and it was confirmed that these sequences complementarily bind to the mRNA encoding amphiregulin, thereby effectively suppressing the expression of amphiregulin, thereby effectively treating fibrosis and respiratory disease-related diseases.

[0106] Example 1. Algorithm for screening SAMiRNA targeting amphiregulin and selection of candidate sequences

[0107] SAMiRNA based drug high-throughput screening is a method in which a 1-base or 2-base sliding window algorithm is applied to all mRNA to generate all possible candidate sequences, and then homology filtering is performed to remove unnecessary candidate sequences, and the degree of inhibition of gene expression is confirmed for the final selected total SAMiRNA.

[0108] First, the design process for candidate SAMiRNA sequences for amphiregulin was carried out by applying a 1-base or 2-base sliding window algorithm to human amphiregulin mRNA NM_001657.3 (1290 bp), and finally selecting 1,257 candidate SAMiRNA sequences consisting of 19 bases, and then conducting an experiment to determine the degree of amphiregulin inhibition.

[0109] Example 2. Synthesis of double-stranded oligo-RNA constructs

[0110] The double-stranded oligo-RNA structure (SAMiRNA) prepared in the present invention has the following structural formula: The sense strand of the monoSAMiRNA (n=4) double-stranded oligo structure is prepared by sequentially binding three hydrophilic monomers, DMT hexaethylene glycol phosphoramidate, to a support of 3,4,6-triacetyl-1-hexa(ethylene glycol)-N-acetylgalactosamine-CPG (1-Hexa(Ethylene Glycol)-N-Acetyl Galactosamine-CPG) by the above reaction, followed by RNA or DNA synthesis and then further binding to a C 2 -containing disulfide bond, a hydrophobic substance, at the 5'-terminal. 24 (C6-SSC 18) is attached to the 3' end, and C is attached to the 5' end. 24 (C6-SSC 18 We created the sense strand of monoSAMiRNA (n=4) to which 3′-aminobutyric acid (A) was linked.

[0111] After the synthesis was completed, the RNA single strand and oligo (DNA or RNA)-polymer constructs were separated from the CPG by treating with 28% (v / v) ammonia in a water bath at 60°C, and the protecting residues were removed by a deprotection reaction. The RNA single strand and oligo (DNA or RNA)-polymer constructs from which the protecting residues had been removed were treated with N-methylpyrolidone, triethylamine, and triethylaminetrihydrofluoride in a volume ratio of 10:3:4 in a 70°C oven to remove 2'TBDMS (tert-butyldimethylsilyl). From these reaction products, single stranded RNA, oligo (DNA or RNA)-polymer structures, and oligo (DNA or RNA)-polymer structures bound to ligands were separated by high performance liquid chromatography (HPLC), and the molecular weights of these were measured by a MALDI-TOF mass spectrometer (MALDI TOF-MS, SHIMADZU, Japan) to confirm whether they matched the base sequence and oligo-polymer structures to be synthesized. Then, to produce each double-stranded oligo structure, the sense strand and antisense strand were mixed in equal amounts and placed in 1X annealing buffer (30 mM HEPES, 100 mM potassium acetate, 2 mM magnesium acetate, pH 7.0-7.5), reacted in a 90°C thermostatic water bath for 3 minutes, and then further reacted at 37°C to produce the desired SAMiRNA. Annealing of the produced double-stranded oligo RNA structure was confirmed by electrophoresis.

[0112] Example 3. High-throughput screening (HTS) of SAMiRNA nanoparticles that target human amphiregulin and induce RNAi 3.1 Preparation of SAMiRNA nanoparticles

[0113] The 1,257 types of SAMiRNA targeting the amphiregulin sequence synthesized in Example 2 were dissolved in 1X DPBS (Dulbecco's Phosphate Buffered Saline) (WELGENE, KR) and freeze-dried for 5 days using a freeze dryer (LGJ-100F, CN). The freeze-dried nanoparticle powder was dissolved in 1.429 ml of deionized distilled water (Bioneer, KR), homogenized, and used in the experiments for the present invention.

[0114] 3.2 Intracellular processing method of SAMiRNA nanoparticles

[0115] To discover SAMiRNA that suppresses the expression of amphiregulin, we used A549, a human lung cancer cell line. The A549 cell line was cultured in Gibco Biosciences (Gibco) containing 10% fetal bovine serum (Hyclone, US) and 1% penicillin-streptomycin (Hyclone, US). TM The cells were cultured in Ham's F-12K (Kaighn's) medium (Thermo, US) at 37°C and 5% CO2. The A549 cell line was plated in a 96-well plate (Costar, US) at 2 × 10 4 The next day, the SAMiRNA homogenized with deionized distilled water in Example 3.1 was diluted with 1X DPBS and treated with the cells at 500 nM or 1000 nM. SAMiRNA was treated once every 12 hours for a total of four times, and the cells were cultured at 37°C and 5% CO2.

[0116] 3.3 SAMiRNA Screening Using Human Amphiregulin mRNA Expression Inhibition Efficacy Assay

[0117] Total RNA was extracted from the cell line treated with SAMiRNA in Example 3-2, and cDNA was prepared from the total RNA. The mRNA expression level of amphiregulin gene was then quantified using real-time PCR.

[0118] AccuPower® Du Al-HotStart RT-qPCR kit (Bioneer, Korea) was dried and prepared by adding 300 nM AREG forward primer, 300 nM AREG reverse primer, 300 nM AREG probe, 300 nM RPL13A forward primer, 300 nM RPL13A reverse primer, 300 nM RPL13A probe, 400 nM TBP forward primer, 400 nM TBP reverse primer, and 300 nM TBP probe to each well (Table 2, primer and hydrolysis probe sequences used in high-throughput screening (HTS) experiments). The performance of the prepared kit was evaluated by PCR amplification efficiency (Table 3) after creating a calibration curve using total RNA from A549 cells. RT-qPCR was performed at 95°C for 5 minutes, (95°C for 5 seconds, 58°C for 15 seconds) x 45 cycles, and a protocol was used in which the fluorescence value was detected at each cycle.

[0119] The SAMiRNA-treated 96-well plate (Costar, US) was used for the automated procedure from total RNA extraction to RT-qPCR using ExiStation HT TM AccuPower® D, which was manufactured separately and included the HT DNA / RNA extraction kit (Bioneer, Korea) and primers and probes for amphiregulin analysis, was used. Total RNA extraction and one-step RT-qPCR were performed using the Dual-HotStart RT-qPCR Kit (Bioneer, Korea) according to an automated program.

[0120] After qPCR array, the Ct values ​​of the two genes were calculated using the 2(-Delta Delta C(T)) method [Livak KJ, Schmittgen TD. 2001. Analysis The relative amount (%) of amphiregulin mRNA in the experimental group versus the control group was calculated by relative quantitative analysis using [is of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. Dec;25(4):4 02-8].

[0121] [Table 2]

[0122] [Table 3]

[0123] In order to select highly efficient SAMiRNAs, 14 types of SAMiRNAs were selected that had the most efficient reduction in amphiregulin mRNA expression level compared to the control group at a final concentration of 500 nM or 1000 nM, i.e., sequences of SEQ ID NOs: 1 to 14 as the sense strand.

[0124] As shown in Figure 1, 1,257 types of SAMiRNA targeting amphiregulin were finally selected to select 14 types of SAMiRNA that most effectively suppress amphiregulin gene expression. The sequence information of the SAMiRNA is shown in Table 4 below.

[0125] [Table 4]

[0126] Example 4. Screening of SAMiRNA nanoparticles that target human amphiregulin and induce RNAi

[0127] The lung cancer cell line, A549, was treated with SAMiRNA having each of the sequences of SEQ ID NOs: 1 to 14 selected in Example 3 as a sense strand, and the expression pattern of amphiregulin mRNA in the cell line was analyzed.

[0128] 4.1 Intracellular processing method of SAMiRNA nanoparticles

[0129] To identify SAMiRNA that suppresses the expression of amphiregulin, we used A549, a human lung cancer cell line. The A549 cell line was cultured in Gibco Biosciences (Gibco) containing 10% fetal bovine serum (Hyclone, US) and 1% penicillin-streptomycin (Hyclone, US). TM The cells were cultured in Ham's F-12K (Kaighn's) medium (Thermo, US) at 37°C and 5% CO2. The A549 cell line was cultured in the same medium at 8 × 10 4 The next day, the SAMiRNA homogenized with deionized distilled water in Example 3.1 was diluted with 1X DPBS and treated with the cells at 200 nM or 600 nM. SAMiRNA was treated once every 12 hours for a total of four times, and the cells were cultured at 37°C and 5% CO2.

[0130] 4.2 SAMiRNA Screening Using Human Amphiregulin mRNA Expression Inhibition Efficacy Assay

[0131] Total RNA was extracted from the cell line treated with SAMiRNA in Example 4-1, and cDNA was prepared from the total RNA. The mRNA expression level of amphiregulin gene was then quantified using real-time PCR.

[0132] 4-2-1 RNA isolation and cDNA production from SAMiRNA-treated cells

[0133] Total RNA was extracted from the cell line treated with SAMiRNA in Example 4-1 using an RNA extraction kit (AccuPrep Cell total RNA extraction kit, BIONEER, Korea), and the extracted RNA was subjected to RNA reverse transcriptase (AccuPower® RocketScript TM Cycle RT Premix wi cDNA was prepared using AccuPower® RocketScript 1.25 ml of cDNA in a 0.25 ml Eppendorf tube. TM Cycle RT Premix with h 1 μg of extracted RNA was added per tube to oligo(dT)20 (Bioneer, Korea), and the total volume was adjusted to 20 μl by adding DEPC (diethyl ether). Pyrocarbonate-treated distilled water was added. TM The RNA and primers were hybridized at 37°C for 30 seconds using a 96Gradient Thermal Block (BIONEER, Korea), and the two processes of preparing cDNA at 48°C for 4 minutes were repeated 12 times, and the enzyme was inactivated at 95°C for 5 minutes to terminate the amplification reaction.

[0134] 4-2-2 Relative quantitative analysis of human amphiregulin mRNA

[0135] Using the cDNA prepared in Example 4-2-1 as a template, the relative expression rate of amphiregulin mRNA relative to the SAMiRNA control sample was analyzed using real-time qPCR in the SYBR Green format as follows. The cDNA prepared in Example 4-2-1 was diluted 5-fold with distilled water and placed in each well of a 96-well plate. 3 μl of the diluted cDNA for amphiregulin mRNA expression analysis was added to AccuPower® 2X GreenStar TM qPCR Master Mix (BIONE A mixture was prepared by adding 25 μl of PBS (BIONEER, Korea), 19 μl of distilled water, and 3 μl of amphiregulin qPCR primers (SEQ ID NOs: 17 and 18 (Table 5); 10 pmole / μl each, BIONEER, Korea). Meanwhile, to normalize the expression level of amphiregulin mRNA, GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase), a housekeeping gene (hereinafter, HK gene), was used as the standard gene. The 96-well plate containing the mixture was then placed in an Exicycler. TM Real-Time Quantitative The following reactions were performed using rmal Block (BIONEER, Korea): 15 minutes at 95°C to activate the enzyme and eliminate the secondary structure of cDNA, followed by denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, and SYBR Green Scan (SYBR The four steps of the green scan were repeated 42 times, and after a final extension at 72°C for 3 minutes, the temperature was maintained at 55°C for 1 minute, and the melting curve was analyzed from 55°C to 95°C.

[0136] After PCR was completed, the Ct (threshold cycle) value of each target gene was calculated by correcting the Ct value of the target gene by the GAPDH gene, and the difference in ΔCt value was calculated using the experimental group treated with SAMiRNA (SAMiCONT), a control sequence that does not inhibit gene expression, as the control group. The expression level of the target gene in the cells treated with amphiregulin-specific SAMiRNA was relatively quantified using the ΔCt value and the calculation formula 2(-ΔCt)×100.

[0137] To select highly efficient SAMiRNAs, three types of SAMiRNAs were selected that had the most efficient reduction in amphiregulin mRNA expression level compared to the control group at final concentrations of 200 nM or 600 nM, i.e., sequences of SEQ ID NOs: 10, 11, and 12, respectively, as the sense strand.

[0138] As shown in Figure 3, 14 types of SAMiRNAs targeting amphiregulin were finally selected into three types of SAMiRNAs that most effectively suppress amphiregulin gene expression. The sequence information of the SAMiRNAs is shown in Table 6 below.

[0139] [Table 5]

[0140] [Table 6]

[0141] Example 5. Suppression of human amphiregulin expression using SAM iRNA selected from lung cancer cell line (A549)

[0142] The lung cancer cell line A549 was treated with SAMiRNA having the sequences of SEQ ID NOs: 10, 11, and 12 selected in Example 4 as sense strands, and the expression pattern of amphiregulin mRNA in the cell line was analyzed to determine the IC of SAMiRNA. 50 Value confirmed.

[0143] 5.1 Preparation and particle size analysis of SAMiRNA nanoparticles

[0144] Three kinds of SAMiRNAs targeting the amphiregulin sequence synthesized in Example 2 were dissolved in 1X DPBS (WELGENE, KR) and freeze-dried for 5 days using a freeze dryer (LGJ-100F, CN). The freeze-dried nanoparticle powder was dissolved in 2 ml of deionized distilled water (Bioneer, KR) and homogenized for use in the experiments of the present invention. The size and polydispersity index of SAMiRNA were measured using a Zetasizer Nano ZS (Malvern, UK) to analyze the particle size of the prepared SAMiRNA nanoparticles. The size and polydispersity index of the nanoparticles for the selected SAMiRNAs are shown in Table 7 below, and the graph is shown in FIG. 2.

[0145] [Table 7]

[0146] 5.2 Intracellular processing method of SAMiRNA nanoparticles

[0147] To confirm the effect of the selected SAMiRNA in suppressing the expression of amphiregulin, a human lung cancer cell line, A549, was used. The A549 cell line was cultured in Gibco Biosciences (Gibco) containing 10% fetal bovine serum (Hyclone, US) and 1% penicillin-streptomycin (Hyclone, US). TM The cells were cultured in Ham's F-12K (Kaighn's) medium (Thermo, US) at 37°C and 5% CO2. The A549 cell line was cultured in the same medium in a 12-well plate (Costar, US) at 8 × 10 4 The next day, the SAMiRNA homogenized with deionized distilled water in Example 5.1 was diluted with 1X DPBS and treated with the cells at 12.5nM, 25nM, 50nM, 100nM, 200nM, 600nM or 1200nM. SAMiRNA treatment was performed once every 12 hours for a total of four times, and the cells were cultured at 37℃ and 5% CO2.

[0148] 5.3 SAMiRNA IC using human amphiregulin mRNA expression suppression efficacy assay 50 confirmation

[0149] Total RNA was extracted from the cell line treated with SAMiRNA in Example 5-2, and cDNA was prepared from the total RNA. The mRNA expression level of the amphiregulin gene was then relatively quantified using real-time PCR.

[0150] 5-3-1 RNA isolation and cDNA production from SAMiRNA-treated cells

[0151] Total RNA was extracted from the cell line treated with SAMiRNA in Example 5-2 using an RNA extraction kit (AccuPrep Cell total RNA extraction kit, BIONEER, Korea). The extracted RNA was then subjected to RNA reverse transcriptase (AccuPower® RocketScript TM Cycle RT Premix w cDNA was prepared using ith oligo(dT)20 (Bioneer, Korea) in the following manner. TM Cycle RT Premix wi 1 μg of RNA was added to each tube of th oligo(dT)20 (Bioneer, Korea) and diluted with DEPC (diethyl ether) to a total volume of 20 μl. Pyrocarbonate-treated distilled water was added. TM The RNA and primers were hybridized at 37°C for 30 seconds using a 96Gradient Thermal Block (BIONEER, Korea), and the two processes of preparing cDNA at 48°C for 4 minutes were repeated 12 times, and the enzyme was inactivated at 95°C for 5 minutes to terminate the amplification reaction.

[0152] 5-3-2 Relative quantitative analysis of human amphiregulin mRNA

[0153] Using the cDNA prepared in Example 5-3-1 as a template, the relative expression rate of amphiregulin mRNA compared to the SAMiRNA control sample was analyzed using real-time qPCR in the SYBR Green format as follows. The cDNA prepared in Example 5-3-1 was diluted 5-fold with distilled water and placed in each well of a 96-well plate. 3 μl of the diluted cDNA for amphiregulin mRNA expression analysis and AccuPower® 2XGreenSt ar TM A mixture was prepared by adding 25 μl of qPCR MasterMix (BIONEER, Korea), 19 μl of distilled water, and 3 μl of amphiregulin qPCR primer (SEQ ID NOs: 17 and 18 (Table 5); 10 pmole / μl each, BIONEER, Korea). Meanwhile, to normalize the expression level of amphiregulin mRNA, GAP DH (Glyceraldehyde-3-Phosphate Dehydrogenase), a housekeeping gene (hereinafter referred to as HK gene), was used as the standard gene. The 96-well plate containing the mixture was placed in an Exicycler. TM The following reaction was performed using a Real-Time Quantitative Thermal Block (BIONEER, Korea): After 15 minutes at 95°C to activate the enzyme and eliminate secondary structures in the cDNA, the following four processes were repeated 42 times: denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, and SYBR green scan. After a final extension at 72°C for 3 minutes, the temperature was maintained at 55°C for 1 minute, and the melting curve from 55°C to 95°C was analyzed.

[0154] After PCR was completed, the Ct (threshold cycle) value of each target gene was calculated by correcting the Ct value of the target gene by the GAPDH gene, and the difference in ΔCt value was calculated using the experimental group treated with SAMiRNA (SAMiCONT), a control sequence that does not inhibit gene expression, as the control group. The expression level of the target gene in the cells treated with amphiregulin-specific SAMiRNA was relatively quantified using the ΔCt value and the calculation formula 2(-ΔCt)×100.

[0155] As a result, it was confirmed that the amphiregulin-specific SAMiRNAs having the sequences of SEQ ID NOs: 10, 11, and 12 as the sense strands all showed a 50% or greater decrease in amphiregulin mRNA expression even at a low concentration of 100 nM, and thus exhibited a highly efficient effect of inhibiting amphiregulin expression. 50 was confirmed to be 28.75 nM in the case of amphiregulin-specific SAMiRNA having the sequence of SEQ ID NO: 10 as the sense strand, as shown in Figure 4, 26.04 nM in the case of amphiregulin-specific SAMiRNA having the sequence of SEQ ID NO: 11 as the sense strand, as shown in Figure 5, and 12.07 nM in the case of amphiregulin-specific SAMiRNA having the sequence of SEQ ID NO: 12 as the sense strand, as shown in Figure 6. In particular, the amphiregulin-specific SAMiRNA having the sequence of SEQ ID NO: 12 as the sense strand showed a reduction in amphiregulin mRNA expression level of 50% or more even at a low concentration of 25 nM, as shown in Figure 6, demonstrating the most effective effect of inhibiting amphiregulin gene expression among the three selected sequences.

[0156] Example 6. In vitro cytotoxicity evaluation using human peripheral blood mononuclear cells (PBMC)

[0157] To confirm the increase in mRNA levels of endogenous immune-related cytokines by SAMi-hAREG, TM) containing RPMI1640 (Hyclone TM ) ePBMC(R) Cryopreservation medium ved Human PBMCs (human peripheral blood mononuclear cells, Cellular Technology Limited, USA) were plated in a 12-well plate (Costar USA) at 5 × 10 per well. 5 The cells were cultured in a 37°C, 5% CO2 incubator for 1 hour to stabilize the cells, and then the PBMCs were treated with SAMi-CON (DNA / RNA), SAMi-hAREG#10 (DNA / RNA), SAMi-hAREG#11 (DNA / RNA), SAMi-hAREG#12 (DNA / RNA), SAMi-CON (RNA / RNA), SAMi-hAREG#10 (RNA / RNA), SAMi-hAREG#11 (RNA / RNA), and SAMi-hAREG#12 (RNA / RNA) at 2.5 μM, respectively, and cultured in a 37°C, 5% CO2 incubator for 6 hours. As a positive control, 20 μg / ml Concanavalin A (Sigma Aldrich, USA) was used.

[0158] Then, all the cells were harvested, and total RNA was extracted using an RNA extraction kit (RNeasy Mini Kit, Qiagen, Germany) and an RNase-Free DNase set (Qiagen, Germany) according to the protocol provided by the manufacturer.

[0159] The extracted RNA (200 ng) was placed in deionized sterile DW (Bioneer, Korea) and RNA reverse transcriptase (AccuPower® RocketScript TM Cycle RT Premix with ol igo(dT)20, Bioneer, Korea) and this was mixed with a gene amplifier (MyGenie TMA total of 20 μl of cDNA was synthesized using a 96 Gradient Thermal Block (BIONEER, Korea) under the following conditions: (37° C. 30 seconds, 48° C. 4 minutes, 55° C. 30 seconds) x 12 cycles, 95° C., 5 minutes.

[0160] The synthesized cDNA was mixed with qPCR primers for RPL13A, IL1B, IL6, IFNG, TNF, and IL12B genes and then PCR was performed using an Exicycler. TM Amplification was performed using a 96 Real-Time Quantitative Thermal Block (Bioneer, Korea) at 95°C for 5 minutes, followed by 45 cycles of (95°C for 5 seconds, 58°C for 15 seconds).

[0161] After qPCR array, the Ct values ​​of the two genes derived were analyzed by relative quantification using the 2(-Delta Delta C(T)) method [Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. Dec; 25(4): 402-8] to calculate the relative amount (%) of mRNA in the experimental group compared to the control group.

[0162] As a result, as shown in FIG. 7, it was confirmed that amphiregulin-specific SAMiRNA #10, SAMiRNA #11, and SAMiRNA #12 did not induce expression of endogenous immune-related cytokines in human peripheral blood mononuclear cells (human PBMCs).

[0163] Example 7. Comparative analysis of suppression of human amphiregulin expression by DNA / RNA hybrid and RNA / RNA hybrid SAMiRNAs containing the selected sequences of SEQ ID NOs: 10, 11, and 12 as the sense strand

[0164] The lung cancer cell line A549 was treated with double-stranded oligo DNA / RNA hybrids and RNA / RNA hybrids containing amphiregulin-specific SAMiRNAs having the sequences of SEQ ID NOs: 10, 11, and 12, respectively, as the sense strands, selected in Example 4, and the relative expression levels (%) of amphiregulin mRNA in the cell lines were compared and analyzed.

[0165] 7.1 Intracellular processing method of SAMiRNA nanoparticles

[0166] To discover SAMiRNA that suppresses the expression of amphiregulin, we used A549, a human lung cancer cell line. The A549 cell line was cultured in Gibco Biosciences (Gibco) containing 10% fetal bovine serum (Hyclone, US) and 1% penicillin-streptomycin (Hyclone, US). TM The cells were cultured in Ham's F-12K (Kaighn's) medium (Thermo, US) at 37°C and 5% CO2. The A549 cell line was cultured in the same medium at 8 × 10 4 The next day, the SAMiRNA homogenized with deionized distilled water in Example 3.1 was diluted with 1X DPBS and treated with the cells at 200 nM, 600 nM, or 1200 nM. SAMiRNA was treated once every 12 hours for a total of four times, and the cells were cultured at 37°C and 5% CO2.

[0167] 7.2 SAMiRNA Screening Using Human Amphiregulin mRNA Expression Inhibition Efficacy Assay

[0168] Total RNA was extracted from the cell line treated with SAMiRNA in Example 7-1, and cDNA was prepared from the total RNA. The mRNA expression level of amphiregulin gene was then quantified using real-time PCR.

[0169] 7-2-1 RNA isolation and cDNA production from SAMiRNA-treated cells

[0170] Total RNA was extracted from the cell line treated with SAMiRNA in Example 7-1 using an RNA extraction kit (AccuPrep Cell total RNA extraction kit, BIONEER, Korea), and the extracted RNA was subjected to RNA reverse transcriptase (AccuPower® RocketScript TM Cycle RT Premix wi cDNA was prepared using AccuPower￠c RocketScript 1.0 (Bioneer, Korea) in a 0.25 ml Eppendorf tube. TM 1 μg of extracted RNA was added per tube to Cycle RT Premix with oligo(dT)20 (Bioneer, Korea), and distilled water treated with DEPC (diethyl pyrocarbonate) was added to make the total volume 20 μl. TM The RNA and primers were hybridized at 37°C for 30 seconds in a 96 Gradient Thermal Block (BIONEER, Korea), and the two processes of preparing cDNA at 48°C for 4 minutes were repeated 12 times, and the enzyme was inactivated at 95°C for 5 minutes to terminate the amplification reaction.

[0171] 7-2-2 Relative quantitative analysis of human amphiregulin mRNA

[0172] Using the cDNA prepared in Example 7-2-1 as a template, the relative expression rate of amphiregulin mRNA compared to the SAMiRNA control sample was analyzed using real-time qPCR in the SYBR Green format as follows. The cDNA prepared in Example 7-2-1 was diluted 5-fold with distilled water and placed in each well of a 96-well plate. 3 μl of the diluted cDNA for amphiregulin mRNA expression analysis and AccuPower® 2X GreenStar TMA mixture was prepared by adding 25 μl of qPCR MasterMix (BIONEER, Korea), 19 μl of distilled water, and 3 μl of amphiregulin qPCR primer (SEQ ID NOs: 17 and 18 (Table 5); 10 pmole / μl each, BIONEER, Korea). Meanwhile, to normalize the expression level of amphiregulin mRNA, GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase), a housekeeping gene (hereinafter, HK gene), was used as the standard gene. The 96-well plate containing the mixture was placed in an Exicycler. TM The following reaction was performed using a Real-Time Quantitative Thermal Block (BIONEER, Korea): After 15 minutes at 95°C to activate the enzyme and eliminate secondary structures in the cDNA, the following four processes were repeated 42 times: denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, and SYBR green scan. After a final extension at 72°C for 3 minutes, the temperature was maintained at 55°C for 1 minute, and the melting curve from 55°C to 95°C was analyzed.

[0173] After PCR was completed, the Ct (threshold cycle) value of each target gene was calculated by correcting the Ct value of the target gene by the GAPDH gene, and the difference in ΔCt value was calculated using the experimental group treated with SAMiRNA (SAMiCONT), a control sequence that does not inhibit gene expression, as the control group. The expression level of the target gene in the cells treated with amphiregulin-specific SAMiRNA was quantified using the ΔCt value and the calculation formula 2(-ΔCt)×100.

[0174] In order to select highly efficient SAMiRNA from double-stranded oligo DNA / RNA hybrids and RNA / RNA hybrids, we finally selected the sequence that had the most efficient reduction in amphiregulin mRNA expression level compared to the control group at final concentrations of 200 nM, 600 nM, and 1200 nM, i.e., the SAMiRNA, which is a DNA / RNA hybrid having the sequence of sequence number 12 as the sense strand (suppressing gene expression by 90% or more).

[0175] As shown in Figure 8, from the double-stranded oligo DNA / RNA hybrids and RNA / RNA hybrids containing the three selected amphiregulin-specific SAMiRNAs, 12 DNA / RNA hybrid SAMiRNAs that most effectively suppress amphiregulin gene expression were finally selected.

[0176] Example 8. High-throughput screening (HTS) of SAMiRNA nanoparticles that target mouse amphiregulin and induce RNAi

[0177] In the case of siRNA therapeutics, it is not easy to find an optimal sequence that can be commonly applied to different species (strains). In this case, there is a US FDA guideline that requires designing a surrogate sequence (mouse gene-specific siRNA) specific to an animal model (in vivo efficacy test) to analyze the therapeutic effect, and verifying the pharmacological activity and toxicity caused by inhibiting the expression of the gene (presentation by Robert T. Dorsam Ph.D. Pharmacology / Toxicology Reviewer, FDA / CDER).

[0178] Using an existing algorithm-based siRNA design program (our own Turbo-si-designer), we complemented the previously discovered screening and performed SAMiRNA-based siRNA sequence high-throughput screening. 19mer-length siRNAs were scanned in a 1-base sliding window for the entire target gene (the same method as the human amphiregulin target screening), and a total of 1,190 candidate siRNA sequences were generated for the full transcript sequence of the mouse amphiregulin gene (NM_009704.4) that could be used. Homology filtering (blast sequence homology filtering) was performed to remove candidate sequences that may affect the expression of other genes. Finally, 237 types of SAMiRNA were selected and synthesized. The mouse-derived NIH3T3 cell line was treated at a concentration of 1uM under cell culture conditions with 10% FBS, and the in vitro expression inhibitory effect was screened using the primers in Table 8 (primer sequence information for qPCR) (Figure 9).

[0179] Further screening was then carried out on two sequences (SEQ ID NOs: 19 and 20) selected from the NIH3T3 cell line and mouse SAMiRNA-amphiregulin sequence number 21 discovered in a previous milestone study, by treating the MLg cell line, a mouse lung fibroblast cell line, with 10% FBS in cell culture conditions at treatment concentrations of 200 nM and 500 nM, and it was confirmed that sequence number 20 showed the best expression inhibitory effect (Figure 10a).

[0180] Furthermore, mouse lung epithelial cell line LA-4 was treated with two selected sequences (SEQ ID NOs: 19 and 20) and mouse SAMiRNA-amphiregulin sequence 21 discovered in a previous milestone study at concentrations of 200 nM, 500 nM, and 1000 nM under cell culture conditions containing 10% FBS to confirm the additional expression inhibitory effect. As a result, it was confirmed that SEQ ID NO: 20 had the best expression inhibitory effect (Figure 10b).

[0181] As shown in Figure 10, two SAMiRNAs that most effectively suppress amphiregulin gene expression were finally selected from 237 SAMiRNAs targeting mouse amphiregulin. The sequence information of the SA MiRNAs is shown in Table 9 below.

[0182] [Table 8]

[0183] [Table 9]

[0184] Example 9. Efficacy of SAMiRNA-mAREG by microintravenous administration in a mouse model of silica-induced pulmonary fibrosis

[0185] An efficacy analysis was carried out on SAMi-mAREG in an animal model of pulmonary fibrosis induced by silica (Silicon dioxide, SIGMA, Korea). For the experiment, 7-week-old mice were obtained and purified for one week. To induce the model, silica (3 mg) was dissolved and injected intra-organically into the mice. Three days after induction, mice showing no abnormal symptoms were selected and administered 1 mg / kg and 5 mg / kg to the normal group, experimental group administered with saline (PBS), experimental group administered with SAMiRNA-Control, and experimental group (SAMi-mAREG#20), three times at two-day intervals. The mice were sacrificed on the 14th day after model induction.

[0186] 9-1. Gene expression analysis of SAMiRNA in an animal model of pulmonary fibrosis induced by silica

[0187] Lung tissues from sacrificed mice were obtained and crushed using a homogenizer. Total RNA was extracted from the cell lines treated with SAMiRNA in Example 7-1 using an RNA extraction kit (AccuPrep Cell total RNA extraction kit, BIONEER, Korea). The extracted RNA was purified using RNA reverse transcriptase (AccuPower® RocketScript TM cDNA was prepared using Cycle RT Premix with oligo(dT)20 (Bioneer, Korea) as follows. The relative expression rate of total mRNA in each group was analyzed using real-time qPCR with SYBR Green as a template as follows. The prepared cDNA was diluted 5-fold with distilled water in each well of a 96-well plate, and 3 μl of the diluted cDNA was added to AccuPower® 2X GreenStar TM A mixture was prepared by adding 25 μl of qPCR MasterMix (BIONEER, Korea), 19 μl of distilled water, and 3 μl of amphiregulin qPCR primer (SEQ ID NO: 24 and 25 (Table 8); 10 pmole / μl each, BIONEER, Korea). Meanwhile, to normalize the expression levels of amphiregulin, fibronectin, and collagen 3α1 mRNA, RPL13A, a housekeeping gene (hereinafter referred to as HK gene), was used as the standard gene.

[0188] After PCR was completed, the Ct (threshold cycle) values ​​of the target genes obtained were corrected by the RPL13A gene to obtain the Ct values ​​of the target genes, and the ΔCt values ​​were compared between the groups to obtain the difference. The expression levels of amphiregulin, fibronectin, and collagen 3α1 genes were relatively quantified using the ΔCt values ​​and the formula 2(-ΔCt)×100.

[0189] As a result, in the case of amphiregulin, a decreasing effect was confirmed in the groups treated with SAM iRNA-AREG 1 mg / kg and SAM iRNA-AREG 5 mg / kg compared to the group treated with saline in the silica model and the group administered with SAMiRNA-Control in the silica model group. Also, in the case of fibronectin and collagen 3α1, a concentration-dependent decreasing effect was confirmed in the groups administered with SAMiRNA-AREG 1 mg / kg and SAM iRNA-AREG 5 mg / kg compared to the group treated with saline in the silica model and the group administered with SAMiRNA-Control in the silica model group.

[0190] 9-2. Histopathological analysis of SAMiRNA in an animal model of silica-induced pulmonary fibrosis

[0191] Immunohistochemical staining was performed to verify whether SAMiRNA-AREG in the silica model affected the expression of cellular matrix components. Each group of animal models was sacrificed, and paraffin sections were prepared through tissue fixation, washing, dehydration, transparency, infiltration, embedding, and slicing. The paraffin sections were cut thinly using a slicer, and the tissue was attached to a slide. Hematoxylin and eosin (H&E) staining was performed to observe the pathology of the lung tissue, and Masson's trichrome staining was performed to confirm the expression level of collagen 1. Immunohistochemical staining was then performed to analyze the expression level of amphiregulin.

[0192] Hematoxylin and eosin staining revealed that damage occurred in the silica-induced lung tissue compared to the lung tissue of the normal model. However, it was found that the lung tissue of the mice administered SAMiRNA-AREG was relatively less damaged, as in the lung tissue of the normal model. Masson's trichrome staining was then performed to confirm the degree of fibrosis. It was confirmed that the degree of fibrosis expression was reduced in the interstitium between the lung tissues in the SAMiRNA-AREG-administered group compared to the group administered saline and the group administered SAMiRNA-Control in the silica model group. In addition, immunohistochemical staining for amphiregulin revealed that it was highly expressed in the interstitium between cells in the silica-induced animal group. However, it was confirmed that the expression of AREG was reduced in the interstitium between the lung tissues in the group administered SAMiRNA-AREG.

[0193] In conclusion, mouse SAMiRNA-AREG was administered repeatedly to a silica-induced pulmonary fibrosis mouse model three times (10th, 12th, and 14th days) by microintravenous administration at 1 mg / kg and 5 mg / kg, and the lung tissue was examined for the efficacy of inhibiting expression of target genes and fibrosis marker genes, as well as H&E staining and Masson's trichrome staining. The expression of the target gene amphiregulin and the fibrosis marker genes collagen and fibronectin was confirmed to be increased by silica induction, and the expression inhibitory efficacy of SAMiRNA treatment was confirmed in a concentration-dependent manner. In addition, the results of tissue staining confirmed that cellular infiltration and collagen in the lung tissue increased in the silica-induced mice treated with PBS or the control group SAMiRNA-Control, but that cellular infiltration and collagen were significantly reduced in a concentration-dependent manner by the experimental group SAMiRNA-AREG treatment, to a degree comparable to that of the control group treated with DPBS (Figure 11).

[0194] AREG immunohistochemistry staining was then performed on the silica-induced pulmonary fibrosis model. In Figure 11, the level of AREG expression was confirmed using IHC staining in tissues administered 1 mg / kg and 5 mg / kg of SAMiRNA-AREG to a mouse model of silica-induced pulmonary fibrosis. It was confirmed that AREG expression was significantly increased in interstitial sites in models administered silica + PBS and silica + SAMi-Cont compared to normal lung tissue. Meanwhile, confirmation of AREG expression in tissues administered 1 mg / kg and 5 mg / kg of SAMiRNA-AREG confirmed that AREG expression was significantly reduced compared to silica-induced lung tissue (Figure 11).

[0195] Example 10. Efficacy of SAMiRNA-mAREG by microintravenous administration in a mouse model of bleomycin-induced pulmonary fibrosis

[0196] Mouse SAMiRNA-AREG (SAMi-mAREG#20) was administered three times (8th, 1st, and 12th days) at a concentration of 5mg / kg by microintravenous administration to a mouse model of bleomycin-induced pulmonary fibrosis, and Sircol assay analysis was performed. It was confirmed that the amount of collagen protein was reduced by >40% in the experimental group administered with SAMiRNA-AREG compared to the control group, SAM iRNA-Cont. In addition, RNA was extracted from lung tissue of the same experimental group to confirm the efficacy of inhibiting the expression of the target gene amphiregulin and the fibrosis marker gene collagen, and the efficacy of inhibiting the expression was confirmed compared to the control group. In the case of the test substance, the efficacy of the newly discovered sequence number 20 was confirmed to be equal to or greater than that of the existing sequence. H&E staining and Masson's trichrome staining for collagen specific staining were performed on the lung tissue. Histological staining confirmed that the bleomycin-induced mice treated with PBS or the control group, SAMiRNA-Control, had increased cell infiltration into the lung tissue and staining due to collagen accumulation. It was confirmed that cell infiltration and collagen accumulation were significantly reduced by the treatment with SAMiRNA-AREG in the experimental group (FIG. 12). The tissue staining method and the method for confirming the expression of the target gene were the same as in Example 8.

[0197] Example 11. Confirmation of the effect on renal fibrosis induced by UUO (unilateral ureteral obstruction) that occurred in mice administered SAMiRNA-AREG

[0198] This study was carried out to analyze the efficacy of SAMiRNA-AREG (SAMi-mAREG#20) in an animal model of kidney fibrosis induced by UUO surgery. First, to prepare an animal model of kidney fibrosis, mice were anesthetized with Ifran solution (Hana Pharmaceutical, Korea). The skin and peritoneum were incised, and the ureter of the left kidney was tied at two places with 4-0 silk, and two intermediate sites were cut to prevent urinary tract infection. The right side was operated on in the same way, but the ureter was not tied. Similarly, the normal model group was also opened to confirm the ureter of the left kidney, but it was not tied. The peritoneum and skin were sutured to prevent infection. 6 hours after model induction, the first administration of SAMiRNA-AREG was performed at 1 mg / kg and 5 mg / kg. The second administration was performed 24 hours later. The final two administrations were performed, and the animals were sacrificed 24 hours after the last administration. The animal model groups were a total of four groups: a normal group, a UUO model group administered with physiological saline, and a UUO model group administered with 1 mg / kg or 5 mg / kg of SAMiRNA-AREG.

[0199] 11-1. Gene expression analysis of SAMiRNA in renal fibrosis induced by UUO (unilateral ureteral obstruction)

[0200] Lung tissues from sacrificed mice were obtained and crushed using a homogenizer. Total RNA was extracted from the cell lines treated with SAMiRNA in Example 7-1 using an RNA extraction kit (AccuPrep Cell total RNA extraction kit, BIONEER, Korea), and the extracted RNA was purified using RNA reverse transcriptase (AccuPower® RocketScript TM Cycle RT Premix with oli cDNA was prepared using go(dT)20 (Bioneer, Korea) as follows. The relative expression rate of total mRNA in each group was analyzed using real-time qPCR with SYBR Green as a template using the prepared cDNA as follows. The prepared cDNA was diluted 5-fold with distilled water in each well of a 96-well plate, and 3 μl of the diluted cDNA was added to an AccuPower® 2X GreenStar 1000-ml PBS to analyze the expression levels of amphiregulin mRNA and fibrosis markers. TM qPCR Master Mix (BIONEER A mixture was prepared by adding 25 μl of 10 ...

[0201] After PCR was completed, the Ct (threshold cycle) value of each target gene was corrected using the RPL13A gene to obtain the Ct value of the target gene, and the difference in ΔCt value was calculated by comparing each group. The expression levels of transforming growth factor 1, amphiregulin, fibronectin, collagen 1, collagen 3α1, and CCR2 genes were quantified using the ΔCt value and the calculation formula 2(-ΔCt)×100.

[0202] As a result, in the case of amphiregulin, the group administered with saline to the UUO model group increased by more than 60 times compared to the normal group. However, it was confirmed that the gene expression of amphiregulin was reduced by 4 times in the groups treated with SAMiRNA-AREG 1 mg / kg and SAMiRNA-AREG 5 mg / kg. In addition, in the case of fibronectin and transforming growth factor 1, it was confirmed that the groups administered with SAMiRNA-AREG 1 mg / kg and SAMiRNA-AREG 5 mg / kg were reduced in a concentration-dependent manner compared to the UUO model group. In addition, in the case of collagen 3α1, collagen 1, and smooth muscle actin, the group administered with SAMiRNA-AREG 1 mg / kg tended to be reduced compared to the UUO model. On the other hand, the group administered with SAMiRNA-AREG 5 mg / kg showed a superior reduction effect compared to the group administered with SAMiRNA-AREG 1 mg / kg. Furthermore, CCR2 increased by approximately six-fold or more in the UUO model, but a reduction effect was confirmed in the group administered SAMiRNA-AREG.

[0203] Although the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that such specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents. [Industrial Applicability]

[0204] The double-stranded oligonucleotide structure containing the amphiregulin-specific double-stranded oligonucleotide according to the present invention and the pharmaceutical composition containing the same as an active ingredient can highly efficiently suppress the expression of amphiregulin without side effects, and can be highly effective in preventing and treating diseases caused by excessive fibrosis and respiratory diseases.

Claims

1. An amphiregulin-specific double-stranded oligonucleotide construct comprising a DNA sense strand comprising any one sequence selected from the group consisting of SEQ ID NOs: 10, 11, and 12, and an RNA antisense strand comprising a sequence complementary to the DNA sense strand, and comprising a structure represented by the following structural formula (2): In the structural formula (2), S and AS represent the DNA sense strand and the RNA antisense strand, respectively; A means a hydrophilic substance, B is a steroid derivative selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholesterylamine; a glyceride derivative selected from the group consisting of mono-glycerides, di-glycerides, and tri-glycerides; glycerol ether; polypropylene glycol; １２ ~C ５０ "a" refers to a hydrophobic substance selected from the group consisting of unsaturated or saturated hydrocarbons of the formula (I), diacylphosphatidylcholine, fatty acids, phospholipids, lipopolyamines, lipids, tocopherols, and tocotrienols, X and Y each independently represent a simple covalent bond or a covalent bond via a linker.

2. 2. The amphiregulin-specific double-stranded oligonucleotide structure according to claim 1, wherein the sense strand or the antisense strand is composed of 19 to 31 nucleotides.

3. 2. The amphiregulin-specific double-stranded oligonucleotide structure according to claim 1, wherein the sense strand or the antisense strand of the double-stranded oligonucleotide comprises a chemical modification.

4. The chemical modification is to replace a hydroxyl group (-OH) at the 2' carbon position of the sugar structure in a nucleotide with a methyl group (-CH ３ ), a methoxy group (-OCH ３ ), amine group (-NH ２ ), fluorine (-F), O-2-methoxyethyl group, O-propyl group, O-2-methylthioethyl group, O-3-aminopropyl group, O-3-dimethylaminopropyl group, O-N-methylacetamide group, and O-dimethylamidooxyethyl; a modification in which oxygen in the sugar structure in a nucleotide is replaced with sulfur; a modification in which the nucleotide bond is a phosphorothioate bond, a boranophosphate bond, and a methyl phosphonate bond; and a modification in which the nucleotide bond is a peptide nucleic acid (PNA), a locked nucleic acid (LNA), or an unlocked nucleic acid (UNA), 4. The amphiregulin-specific double-stranded oligonucleotide structure according to claim 3, characterized in that the amphiregulin-specific double-stranded oligonucleotide structure is at least one selected from the group consisting of:

5. 2. The amphiregulin-specific double-stranded oligonucleotide structure according to claim 1, wherein one or more phosphate groups are bound to the 5' end of the antisense strand of the double-stranded oligonucleotide.

6. The amphiregulin-specific double-stranded oligonucleotide structure according to claim 1, comprising the structure of the following structural formula (3) or structural formula (4): In the structural formulas (3) and (4), A, B, X, Y, S and AS are the same as defined in claim 1, and 5' and 3' refer to the 5' and 3' ends of the double-stranded oligonucleotide sense strand.

7. The amphiregulin-specific double-stranded oligonucleotide structure according to claim 1, wherein the hydrophilic substance has a structure represented by the following structural formula (5) or structural formula (6): In the structural formula (5) or (6), A' is a hydrophilic substance monomer, J is a linker that links m hydrophilic substance monomers together or m hydrophilic substance monomers and a double-stranded oligonucleotide, m is an integer of 1 to 15, and n is an integer of 1 to 10. The hydrophilic substance monomer A' is any one compound selected from the following compounds (1) to (3), and the linker (J) is -PO ３ － -, -SO ３ - and -CO ２ - selected from the group consisting of; In the compound (1), G is selected from the group consisting of O, S, and NH;

8. 2. The amphiregulin-specific double-stranded oligonucleotide structure according to claim 1, wherein the hydrophilic substance has a molecular weight of 200 to 10,000.

9. 2. The amphiregulin-specific double-stranded oligonucleotide structure according to claim 1, wherein the molecular weight of the hydrophobic substance is 250 to 1,000.

10. 2. The amphiregulin-specific double-stranded oligonucleotide structure according to claim 1, wherein the covalent bonds represented by X and Y are non-degradable or degradable bonds.

11. 11. The amphiregulin-specific double-stranded oligonucleotide structure according to claim 10, wherein the non-degradable bond is an amide bond or a phosphate bond.

12. The amphiregulin-specific double-stranded oligonucleotide structure according to claim 10, wherein the degradable bond is any one selected from the group consisting of a disulfide bond, an acid degradable bond, an ester bond, an anhydride bond, a biodegradable bond and an enzymatically degradable bond.

13. A nanoparticle comprising the double-stranded oligonucleotide structure according to any one of claims 1 to 12.

14. The nanoparticle according to claim 13, wherein the nanoparticle is composed of a mixture of double-stranded oligonucleotide structures including double-stranded oligonucleotides having different sequences.

15. A pharmaceutical composition for preventing or treating fibrosis or respiratory diseases, comprising the double-stranded oligonucleotide structure according to any one of claims 1 to 12 as an active ingredient.

16. A pharmaceutical composition for preventing or treating fibrosis or respiratory diseases, comprising the nanoparticles according to claim 13 as an active ingredient.

17. The pharmaceutical composition for preventing or treating fibrosis or respiratory diseases according to claim 15, wherein the respiratory disease is any one selected from the group consisting of chronic obstructive pulmonary disease (COPD), asthma, acute and chronic bronchitis, allergic rhinitis, antitussive and expectorant bronchitis, bronchitis, bronchiolitis, pharyngitis, tonsillitis and laryngitis.

18. The pharmaceutical composition for preventing or treating fibrosis or respiratory diseases according to claim 15, wherein the fibrosis is any one selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver fibrosis, liver cirrhosis, myelofibrosis, myocardial fibrosis, renal fibrosis, pulmonary fibrosis, cardiac fibrosis and radiation-induced fibrosis.

19. The pharmaceutical composition for preventing or treating fibrosis or respiratory diseases according to claim 16, wherein the respiratory disease is any one selected from the group consisting of chronic obstructive pulmonary disease (COPD), asthma, acute and chronic bronchitis, allergic rhinitis, antitussive and expectorant bronchitis, bronchitis, bronchiolitis, pharyngitis, tonsillitis and laryngitis.

20. 17. The pharmaceutical composition for preventing or treating fibrosis or respiratory diseases according to claim 16, wherein the fibrosis is any one selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver fibrosis, liver cirrhosis, myelofibrosis, myocardial fibrosis, renal fibrosis, pulmonary fibrosis, cardiac fibrosis and radiation-induced fibrosis.

21. A pharmaceutical composition for the prevention or treatment of fibrosis or respiratory diseases according to claim 15 in lyophilized form.

22. A pharmaceutical composition for the prevention or treatment of fibrosis or respiratory diseases according to claim 16 in lyophilized form.

23. The hydrophilic substance is hexaethylene glycol-(-PO ３ － -Hexaethylene glycol) ３ and the hydrophobic substance is C ２４ (C ６ -S-S-C １８ 2. The amphiregulin-specific double-stranded oligonucleotide structure according to claim 1, wherein

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