BR112016000163B1 - DOUBLE HELIX OLIGO RNA STRUCTURE, NANOPARTICLES, PHARMACEUTICAL COMPOSITION AND FREEZE DRIED FORMULATION - Google Patents

Abstract

GENE-SPECIFIC siRNA RELATED TO RESPIRATORY DISEASE, DOUBLE-HELICEL OLIGO RNA STRUCTURE OLIGO CONTAINING THE siRNA, COMPOSITION CONTAINING THE SAME TO PREVENT OR TREAT RESPIRATORY DISEASE, refers to a gene-specific siRNA related to respiratory diseases, particularly, a gene-specific siRNA with idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease (COPD), and a highly efficient double-stranded RNA oligo structure containing the same, wherein the double-stranded RNA oligo structure has a structure in which hydrophilic materials and hydrophobic are attached at both ends of double helix RNA (siRNA) using a single covalent bond or a ligand-mediated covalent bond to be effectively transferred into a cell, and can be converted into nanoparticles by the hydrophobic interaction of the oligo RNA structure of double helix in a solution. It is desirable that the siRNA contained in the structure of the double-stranded RNA oligo is an siRNA specific to a CTGF, Cyr61, or Plekho1, which are genes related to respiratory diseases, particularly idiopathic pulmonary fibrosis and COPD. Furthermore, the present invention relates to a method for producing the double helix RNA oligo structure and a (...).

Description

Application field

[001] The present invention relates to a respiratory disease-related gene-specific siRNA and a highly efficient structure comprising double-stranded RNA oligo (double-stranded RNA oligo structure) containing the siRNA. The double helix RNA oligo structure has a structure in which a hydrophilic material and a hydrophobic material are conjugated to both ends of double helix RNA (siRNA), using a single covalent bond or a ligand-mediated covalent bond so as to be effectively released into cells, where the structure can be converted into a nanoparticle form by hydrophobic interactions of double-stranded RNA oligo structures in an aqueous solution. The siRNA included in the double-stranded RNA oligo structure is preferably an siRNA specific for CTGF, Cyr61, or Plekho1 (hereinafter referred to as a CTGF, Cyr61, or Plekho1-specific siRNA), which is a gene related to respiratory diseases, particularly, idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease (COPD).

[002] Furthermore, the present invention relates to a method for producing the double-stranded RNA oligo structure and a pharmaceutical composition containing the double-stranded RNA oligo structure for preventing or treating respiratory diseases, particularly idiopathic pulmonary fibrosis. and COPD.

State of the Art

[003] Technologies to inhibit gene expression are important tools in the development of a therapeutic agent and target validation for disease treatment. Among the technologies, since when a role of an interfering RNA (hereinafter referred to as 'RNAi') was found, it was found that the interfering RNA acts on a sequence-specific mRNA in various types of mammalian cells (Silence of the transcripts: RNA interference in medicine. When a long-chain double-stranded RNA is released into a cell, the released double-stranded RNA is converted into a small interfering RNA (hereinafter referred to as 'siRNA') that is processed to 21 to 23 base pairs (bp) by Dicer endonuclease. siRNA is linked to an RNA-induced silencing complex (RISC), whereby a guide (antisense) strand recognizes and cleave a target mRNA to sequence-specifically inhibit the expression of a target gene (NUCLEIC-ACID THERAPEUTICS: BASIC PRINCIPLES AND RECENT APPLICATIONS. Nature Reviews Drug Discovery. 2002. 1, 503-514).

[004] Bertrand et al, found that, compared with an antisense oligonucleotide (ASO) on the same target gene, siRNA has an effect of significantly inhibiting mRNA expression in vitro and in vivo, and the corresponding effect is maintained for a long time (Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem. Biophys. Res. Commun. 2002. 296: 1000-1004). Furthermore, since siRNA is complementary coupled to a target mRNA to sequence-specifically regulate the expression of the target gene, an siRNA mechanism has an advantage in that a target to be applicable can be remarkably enhanced compared to existing antibody-based medicinal products or chemical pharmaceuticals (small molecular drugs) (Progress Towards in vivo Use of siRNAs. MOLECULAR THERAPY. 2006 13(4):664-670).

[005] To develop siRNA as a therapeutic agent even with excellent effect and varied usable range of siRNA, the siRNA needs to be effectively delivered to a target cell with improved stability and more efficient cell release of the siRNA (Harnessing In vivo siRNA Delivery for Drug Discovery and Therapeutic Development. Drug Discov.

[006] In order to solve the problems, research into modifying some nucleotides or structure of siRNA to have a nucleic acid lyase resistance, or using carriers such as viral vectors, liposomes or nanoparticles, etc. to improve in vivo stability have been actively attempted.

[007] Delivery systems using viral vectors such as adenovirus, retrovirus, etc., have high transfection efficacy, and also have high immunogenicity and oncogenicity. Meanwhile, a non-viral delivery system including nanoparticles has a low cell release efficiency compared to the viral delivery system, but has a high stability in vivo and is possible to be released in a target-specific manner. highly improved delivery system such as uptake, internalization, etc. of RNAi oligonucleotide into cells or tissues, and rarely has cytotoxicity and immune stimulation, so that the non-viral delivery system is currently evaluated as a viable delivery method compared to viral delivery systems (Nonviral delivery of synthetic siRNA s in vivo. J Clin Invest. 2007 December 3; 117(12): 3623-3632).

[008] In a method using a nanocarrier in non-viral delivery systems, various polymers such as liposomes, cationic polymer composites, etc., are used to form the nanoparticles, and an siRNA is compatible with the nanoparticles, i.e., nanocarrier, to be released into cells. Among the methods using nanocarriers, a method using polymeric nanoparticles, polymer micelle, lipoplex, or the like is mainly used, in which the lipoplex consists of cationic lipids to interact with anionic lipids of the endosome of a cell, thereby causing a destabilization effect. from the endosome to release the siRNA into a cell (Proc. Natl. Acad. Sci. 15; 93(21):11493-8, 1996).

[009] Furthermore, it is known that chemical materials, etc., are attached to end portions of an siRNA passenger (sense) strand to provide promoted pharmacokinetic characteristics, so that high efficacy can be induced in vivo (Nature 11; 432(7014):173-8, 2004). Here, the stability of the siRNA can vary depending on properties of the chemical materials attached to the ends of the sense (passenger) or antisense (guide) siRNA strand. For example, an siRNA to which a polymer compound such as polyethylene glycol (PEG) is conjugated interacts with an anionic phosphate group of siRNA in the presence of cationic materials to form a complex, thus being a carrier having improved siRNA stability (J. Control Release 129(2):107-16, 2008). In particular, micelle consisting of polymer complexes has an extremely small size, significantly uniform distribution, and is spontaneously formed, thus being easy to manage formulation quality and safe reproducibility, compared to other systems used as a delivery vehicle. of drug, such as microsphere, nanoparticle, etc.

[010] Furthermore, in order to improve the intracellular delivery efficiency of siRNA, the technology of using an siRNA conjugate in which the hydrophilic material that is a biocompatible polymer (e.g., polyethylene glycol (PEG)) is conjugated with siRNA by a single covalent bond or a ligand-mediated covalent bond, to thereby ensure the stability of siRNA and have effective cell membrane permeability, has been developed (see Korean Patent Publication No. 883471). However, chemical modification of siRNA and conjugation with polyethylene glycol (PEG) (PEGylation) still has disadvantages that in vivo stability is slow and release into a target organ is not smooth. In order to solve the disadvantages, a double-stranded RNA oligo structure with a hydrophilic material and a hydrophobic material are linked to oligonucleotides, in particular, double-stranded RNA such as siRNA, was developed, in which the double-stranded RNA oligo structure helix forms a self-assembled nanoparticle called a self-assembled inhibitory RNA micelle (SAMiRNA™) by a hydrophobic interaction of a hydrophobic material (see Korean Patent Publication No. 1224828), SAMiRNA™ technology has an advantage in that homogeneous nanoparticles having a significantly small size are capable of being obtained in comparison with existing release technologies.

[011] As a specific example of SAMiRNA™ technology, PEG (polyethylene glycol) is used as a hydrophilic material, where PEG is a synthetic polymer, which is used to increase the solubility of pharmaceuticals, particularly protein, and to control pharmacokinetics . PEG is a polydisperse material, a polymer in a batch is composed of the sum of different numbers of monomers, a molecular weight is shown on the Gaussian curve, and polydispersity values (Mw/Mn) express the degree of homogeneity of a material. That is, when PEG has a low molecular weight (3 to 5 kDa), the polydisperse value is about 1.01, and when PEG has a high molecular weight (20 kDa), the polydisperse value is about 1. 2, which is high, as well as the molecular weight is higher, the homogeneity of the material is relatively low (F. M. Veronese. Peptide and protein PEGylation: a review of problems and solutions. Biomaterials (2001) 22:405-417). In this sense, when PEG is combined with pharmaceutical products, the polydisperse characteristic of PEG is reflected on the conjugate, so that it is not easy to perform single material verification, and therefore produce materials having a low polydispersity value through PEG synthesis and improvement of purification processes is on an increasing trend, but it still has problems due to the polydisperse characteristic of the material, particularly when PEG is combined with a material having a small molecular weight, it is difficult to confirm whether the combination is easily carried out, etc. (Francesco M. Veronese and Gianfranco Pasut. PEGylation, successful approach to drug delivery. DRUG DISCOVERY TODAY (2005) 10(21):1451-1458).

[012] In this regard, recently, as an improved technology of SAMiRNA™, which are existing self-assembled nanoparticles, a technology of a new form of carrier having a significantly small size compared to existing SAMiRNA™ and remarkably improved polydispersity has been developed by blocking the hydrophilic material of the double helix RNA structure by configuring SAMiRNA™ as a base unit including 1 to 15 uniform monomers having a predetermined molecular weight and a linker as needed, and using the appropriate number of blocked hydrophilic materials as needed.

[013] However, since biopharmaceuticals act specifically to a target gene sequence or protein structure in order to evaluate efficacy and safety in a non-clinical surrogate model, a material acting with the same mechanism as the corresponding biopharmaceuticals in humans is additionally necessary even in the species of the substitute model. Therefore, to avoid the difficulty in additionally finding the material including the same mechanism as in human, a material acting with the same mechanism in both human (treatment target) and mouse (the non-clinical surrogate model) is necessary to be developed.

[014] Idiopathic Pulmonary Fibrosis (hereinafter abbreviated as IPF) is a disease in which chronic inflammatory cells penetrate a wall of an alveolar wall (pulmonary alveolus) to make the lung hard, causing severe structural changes in the lung tissue, so that the Lung function is gradually reduced to induce death. However, an effective treatment for it does not yet exist, and Idiopathic Pulmonary Fibrosis is usually diagnosed when symptoms appear late, and has an extremely poor prognosis as a median survival time is only about three to five years. It is reported that the incidence frequency in foreign countries is about 3-5 people per 100,000, and it is known that the incidence is generally high after the age of 50, and men have an incidence twice as high as women.

[015] The cause of IPF has not been clearly identified yet, and it is only reported that this high frequency is shown in smokers, antidepressants, chronic pulmonary inhalation due to gastroesophageal reflux, metal dust, wood dust, solvent inhalation, etc. , these are considered risk factors related to the occurrence of IPF. However, definitive causal factors cannot be found in most patients.

[016] It is known that IPF is continually worsened without treatment, and about 50% of patients die within 3-5 years, and when a lung is completely hardened by fibrosis as the disease progresses, there is no improvement despite any treatment, and consequently, early treatment is expected to increase efficacy. As the currently used therapeutic agent, a combination therapy method using steroids and azathioprine or cyclophosphamide is known, but it is difficult to say that there are special effects, and the attempts of various fibrosis inhibitors in animal experiments and small group of patients have failed in proving clear effects. In particular, there is no effective treatment in patients with end-stage IPF other than lung transplantation. Therefore, the development of the most effective therapeutic agent against IPF is desperately needed.

[017] However, COPD, one of the representative lung diseases along with asthma, is different from asthma in that it is accompanied by irreversible airway obstruction, and is a respiratory disease that is accompanied by an abnormal inflammatory response of the lung caused by repeated infections, harmful particles, gas entry or smoking, and is not fully reversible, but shows airflow limitation with increasing progression (Pauwels et al, Am J Respir Crit Care Med, 163:12561276, 2001). COPD is a disease caused by pathological changes in the bronchioles and lung parenchyma due to inflammation of the airway and lung parenchyma, and is characterized by obstructive bronchiolitis and emphysema (destruction of the lung parenchyma). Types of COPD include chronic obstructive bronchitis, chronic bronchiolitis, and emphysema. In COPD, the number of neutrophils is increased, and secretion of cytokines such as GM-CSF, TNF-α, IL-8, and MIP-2 is increased. Additionally, inflammation of the airways, a thickened muscle wall, and closure of the bronchi due to increased mucus secretion are also shown. When the bronchus is closed, alveoli are expanded and damaged, so that the exchange capacity of oxygen and carbon dioxide is damaged to increase respiratory failure.

[018] The severity of COPD is emerging around the world, as it is predicted that COPD was ranked 6th in 1990 among the causes of death due to diseases, but will be ranked third in 2020, and is the only disease in which the incidence is highest in 10 diseases. COPD has a high prevalence rate, causes a respiratory disorder, and requires a large amount of direct medical costs necessary for the diagnosis and treatment of COPD, and a significant amount of indirect expenses such as losses due to dyspnea and leave of absence or loss due to premature death, which is a social and economic problem worldwide (Chronic obstructive pulmonary disease (COPD) treatment guidelines 2005. Chronic obstructive airway disease Clinical Research Center. p.30-31).

[019] Existing therapeutic drugs have not been confirmed to reduce the long-term relief in lung function, which is a hallmark of COPD. Therefore, drug treatment in COPD has a primary objective of reducing symptoms or complications. Among therapeutic drugs, a bronchodilator is a typical allopathic COPD drug, and an anti-inflammatory drug or corticosteroid is generally prescribed, but the effect is not significant, the application range is narrow, and there is great concern about side effects. Like other drugs, only the influenza vaccine is known to reduce severe symptoms and death by around 50% in patients with COPD (chronic obstructive pulmonary disease (COPD) treatment guidelines 2005. Chronic obstructive airway disease Clinical Research Center. p.52- 58).

[020] However, many genetic factors are estimated to increase (or decrease) an individual's risk of COPD. One genetic risk factor that has been demonstrated thus far is a genetic α1-antitrypsin deficiency. Smoking significantly increases the risk of COPD, but the occurrence of panlobular emphysema and reduced lung function that rapidly progresses at a young age are shown in both smokers and non-smokers to have significant genetic deficiency. Despite other genes confirmed to be related to the pathogenesis of COPD, there is, however, other than α1-antitrypsin, an attempt to identify biomarkers of the disease to be used for diagnosis through investigation into abnormal cellular, molecular and genetic states that are basically shown in patients with COPD or attempt to find a new treatment method is being progressed (P.J. Barnes and R.A. Stockely. COPD: current therapeutic interventions and future approaches. EUR Respir J. (2005) 25:1084-1106). In particular, research on COPD diagnosis and target selection for treatment through methods such as gene microarray, proteomics, etc., is actively conducted, and analyzes of genetic factors for obtaining COPS sensitivity (susceptibility) and the Causes of smoking-induced worsening of COPD symptoms have mainly been realized (Castaldi et al. The COPD genetic association compendium. Human Molecular Genetics, 2010, Vol. 19, No. 3 526-534).

[021] CTGF (connective tissue growth factor; CCN2), which is one of the matricellular proteins included in the CCN family, is known to be a secretory cytokine involved in various biological processes, such as cell adhesion, migration, proliferation and angiogenesis, wound repair, etc. Overexpression of CTGF is considered to be the main cause of symptoms such as scleroderma, fibrotic diseases, and scar formation (Brigstock DR. Connective tissue growth factor (CCN2, CTGF) and organ fibrosis: lessons from transgenic animals. J Cell Commun Signal ( 2010) 4 (1): 1-4). In particular, in relation to fibrosis, CTGF is known to cause sustained fibrosis in conjunction with TGF-β (Transforming growth factor-e) or to promote ECM (extracellular matrix) production under a condition in which the formation of fibers is caused by, and recently, is known to be able to treat ocular disorders or muscular dystrophy caused by abnormal expression of CTGF by treating samples or materials that prevent the expression of CTGF or inhibit the action of it, but relevance with a respiratory disease is not has been suggested (U.S. Patent No. 7,622,454 and U.S. Patent Application Publication No. 20120164151).

[022] CYR61 (cysteine-rich angiogenic inducer 61) is an extracellular matrix (ECM)-related signaling protein included in the CCN family, which is known to control diverse cellular activities such as cell adhesion, migration, proliferation, differentiation, apoptosis, etc. . Purified CYR61 promotes endothelial cell binding and spreading in a manner similar to fibronectin, and has no mitogenic activity, but acts to reinforce a mitogenic effect of a fibroblast growth factor (MARIA L. KIREEVA et al. Cyr61, a Product of a Growth Factor-Inducible Immediate-Early Gene, Promotes Cell Proliferation, Migration, and Adhesion. MOLECULAR AND CELLULAR BIOLOGY, Apr. 1996, p.

[023] It is reported that Plekho1 (a member of the pleckstrin homology domain O family 1) is present in a plasma membrane or nucleus, acts as a non-enzymatic regulator of the protein kinase CK2α1 (Casein kinase 2, alpha 1), and is involved in apoptosis through the inhibition of AP-1 action through the C-terminal part produced when being broken down by Caspase 3 (Denis G. Bosc et al. Identification and Characterization of CKIP-1, a Novel Pleckstrin Homology Domaincontaining Protein That Interacts with Protein Kinase CK2. The Journal of Biological Chemistry (2000) 275, 14295-14306; Lingqiang Zhang et al. The EMBO Journal (2005) 24, 766-778).

[024] Patent application US2008108583, entitled Treatment or prevention of oto-pathologies by inhibition of pro-apoptotic genes refers to one or more inhibitors, in particular siRNAs, which negatively regulate the expression of human pro-apoptotic genes. The invention also relates to a pharmaceutical composition comprising the compound, or a vector capable of expressing the compound, and a pharmaceutically acceptable carrier. The present invention also contemplates a method of treating or preventing the incidence of hearing impairment (or impairment of balance), particularly hearing impairment associated with cell death of inner ear hair cells or outer ear hair cells, comprising administering to the patient the composition at a therapeutically effective dose.

[025] Publication WO2011119887, entitled RNA Interference in Dermal and Fibrotic Indications refers to RNAi constructs with improved tissue and cell uptake characteristics and methods of using these compounds in dermal and fibrotic applications. It describes the efficient in vivo delivery of sd-rxRNA molecules to the skin and the use of such molecules to silence genes. This class of RNAi molecules has superior efficacy in vitro and in vivo than previously described RNAi molecules. The molecules associated with the invention have broad potential as therapeutics for disorders or conditions associated with compromised skin and fibrosis. Aspects of the invention relate to double-stranded ribonucleic acids (dsRNAs), including a sense strand and an antisense strand, wherein the antisense strand is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences listed therein. document, and in which the dsRNA is an sd-rxRNA.

[026] Application US2013096288, entitled siRNA Conjugate and Preparation Method Thereof and its equivalent from the same family KR20100123214 disclose an siRNA-polymer conjugate and a method for preparing the same, and more specifically, a hybrid conjugate formed by the covalent bond of the siRNA , and a polymeric compound to improve the in vivo stability of siRNA, and a method for preparing the conjugated hybrid. The conjugate of the invention can improve the in vivo stability of siRNA, thereby achieving efficient delivery of therapeutic siRNA into cells and exhibiting siRNA activity even at a relatively low concentration. Therefore, the conjugate can advantageously be used not only as an siRNA treatment tool for cancers and other infectious diseases, but also as a new type of siRNA delivery system.

[027] Application US2008015114, entitled siRNA Targeting Connective Tissue Growth Factor (CTGF) concerns efficient sequence-specific gene silencing, made possible through the use of siRNA technology. By selecting specific siRNAs by rational design, one can maximize the generation of an effective gene silencing reagent as well as methods for silencing genes. Methods, compositions and kits generated through the rational design of siRNAs are disclosed, including those targeting CTGF.

[028] Patent CN101287835, entitled siRNA-Hydrophilic Polymer Conjugates for Intracellular Delivery of siRNA and Method Thereof discloses hybrid conjugates formed by covalently linking siRNA (small interfering RNA) molecules to hydrophilic polymers to improve the stability of siRNA molecules effective for in vivo gene therapy and complex polyelectrolyte micelles formed by ionic interactions between conjugates and multifunctional cationic compounds. Hydrophilic siRNA-polymer conjugates and polyelectrolyte complex micelles derived therefrom can be advantageously used to improve the stability of siRNA molecules in vivo. Consequently, the delivery of siRNA molecules for therapeutic applications into cells can be facilitated, and the siRNA is still active even if a reduced dose of the siRNA is used.

[029] Publication WO2013089522, entitled Novel Oligonucleotide Conjugates and Use Thereof, discloses a double-stranded oligo-RNA structure, in which target-specific ligands are linked to the double-stranded oligo-RNA structure, where high-weight compounds are covalently linked molecular, which can be advantageously used in the treatment of diseases, specifically, cancer. The present invention also provides a method for preparing the RNA structure and a technology for delivering target-specific double-stranded RNA oligo-RNA through said structure. . Nanoparticles constituted by the double-stranded oligo-RNA structure to which ligands are attached can efficiently deliver double-stranded oligo-RNA to a target and therefore can exhibit an activity of the double-stranded oligo-RNA structure in a cell. target, even with administration of relatively low concentration double-stranded oligo-RNA. Nanoparticles can prevent the delivery of nonspecific double-stranded oligo-RNA to other organs and cells, and therefore can be advantageously used not only as a tool for double-stranded oligo-RNA for the treatment of various diseases, specifically cancer, but also as a new type of double-stranded oligo-RNA delivery system. The present invention also relates to hybrid conjugates in which a hydrophilic material and a hydrophobic material are connected to both ends of the antisense oligonucleotide (ASO) through a covalent bond, so as to obtain in vivo stability of the ASO, and to a method to prepare the hybrid conjugates, and nanoparticles made up of the conjugates. The conjugates of the invention can improve the in vivo stability of ASO and therefore can efficiently deliver ASO for treatment.

[030] The article Chemical Modification and Clinical Application of siRNA, by Sun Liping et al., (Chemistry of Life 25 (4): Pages 339 - 342), discusses the appropriate chemical modification to allow the application of siRNA in vivo. Several commonly used chemical modification methods include phosphate backbone modification, ribose modification, and base modification. The technology of transformation from in vitro to in vivo is discussed, enabling clinical applications in the treatment of viral, tumor and genetic diseases.

[031] The article Effects of CTGF gene silencing on the proliferation and myofibroblast differentiation of human lung fibroblasts, by Liu Xiaojing, (Sheng Wu Yi Xue Gong Cheng Xue Za Zhi 25 (2): 407 — 412), presents a plasmid that expresses the CTGF small interfering RNA (siRNA) (CTGF-siRNA) that was constructed and stably transfected into the human lung fibroblast cell line, MRC-5. Stable clones with CTGF gene silencing (CTGF-siRNA/MRC-5) were successfully established by G418 screening and further confirmed by real-time quantitative PCR and Western blot. Cell proliferation was investigated by growth curve analysis, and the cell doubling time of CTGF-siRNA/MRC-5 cells was markedly longer than that of control cells (P < 0.05). Compared with control cells, the expression of alpha-smooth muscle actin (alpha-SMA), the marker of myofibroblast differentiation, was decreased in CTGF-siRNA/MRC-5 cells. Furthermore, the deposition of extracellular matrix (ECM) proteins (such as type I collagen and fibronectin) in CTGF-siRNA/MRC-5 cells was also decreased. The resulting data suggest that CTGF may play an important role in the differentiation of lung fibroblast into myofibroblast, and that siRNA targeting the CTGF gene may provide a new strategy for gene therapy of pulmonary fibrosis.

[032] As described above, the prevalence of respiratory diseases, particularly idiopathic pulmonary fibrosis and COPD has increased, but therapeutic agents being able to basically prevent and treat idiopathic pulmonary fibrosis and COPD do not yet exist. Therefore, the demand for the therapeutic agent for idiopathic pulmonary fibrosis and COPD showing high level of prevention and treatment effects without side effects is significantly widely present in the market.

GENERAL DESCRIPTION OF THE INVENTION

[033] An object of the present invention is to provide a new siRNA specific for CTGF, Cyr61 or Plekho1 (hereinafter referred to as an siRNA specific for CTGF, Cyr61 or Plekho1), capable of inhibiting their expression at a significantly high efficiency, a double-stranded RNA oligo structure containing the siRNA, and a method for producing the double-stranded RNA oligo structure.

[034] Another object of the present invention is to provide a pharmaceutical composition including the siRNA specific for CTGF, Cyr61 or Plekho1 or the double-stranded RNA oligo structure containing the siRNA as an effective component, for the prevention or treatment of respiratory diseases, particularly , idiopathic pulmonary fibrosis and COPD.

[035] Another object of the present invention is to provide a method for preventing or treating respiratory diseases, particularly idiopathic pulmonary fibrosis and COPD using siRNA specific for CTGF, Cyr61 or Plekho1 or the double-stranded RNA oligo structure containing the siRNA.

BRIEF DESCRIPTION OF FIGURES

[036] FIG. 1 is a schematic diagram of a nanoparticle formed from a double helix oligo polymer structure in accordance with the present invention.

[037] FIG. 2 is a graph showing an amount of inhibition of target gene expression confirmed after transformation of a human fibroblast cell line with siRNA having sequence ID SEQ NOs: 1 to 10, 101 to 110, and 201 to 210 as an sense tape according to the present invention.

[038] A: Graph showing the amount of CTGF expression for treatment with 5 or 20 nM siRNA having sequence ID SEQ NOs: 1 to 10 as a sense strand.

[039] B: Graph showing the amount of Cyr61 expression for treatment with 5 or 20 nM siRNA having sequence ID SEQ NOs: 101 to 110 as a sense strand.

[040] C: Graph showing the amount of Plekho1 expression for treatment with 5 or 20 nM siRNA having sequence ID SEQ NOs: 201 to 210 as a sense strand.

[041] FIG. 3 is a graph showing an amount of inhibition of target gene expression confirmed after transformation of a human fibroblast lineage cell with siRNA having sequence ID SEQ NO: 1, 3, 4, 8, 9, 10, 102, 104, 105, 106, 107, 108, 109, 204, 206, 207, 208, 209 or 210 as the sense tape according to the present invention.

[042] A: Graph showing the amount of CTGF expression according to treatment of 0.2 or 1 nM siRNA having sequence ID SEQ NO: 1, 3, 4, 8, 9 or 10 as a sense strand.

[043] B: Graph showing the amount of Cyr61 expression according to treatment of 0.2 or 1 nM siRNA having sequence ID SEQ NO: 102, 104, 105, 106, 107, 108 or 109 as a sense strand .

[044] C: Graph showing the amount of Plekho1 expression according to treatment of 0.2 or 1 nM siRNA having sequence ID SEQ NO: 204, 206, 207, 208, 209, or 210 as a sense strand.

[045] FIG. 4 is a graph showing an amount of inhibition of target gene expression confirmed after transformation of a human lung cancer cell line with siRNA having sequence ID SEQ NO: 35, 42, 59, 602, 603, 604, 124, 153, 166, 187, 197, 212, 218, 221 or 223 as a sense tape according to the present invention.

[046] A: Graph showing the amount of CTGF expression according to treatment of 0.04, 0.2 or 1 nM siRNA having sequence ID SEQ NO: 35, 42, 59, 602, 603 or 604 as a sense tape.

[047] B: Graph showing the amount of Cyr61 expression according to treatment of 0.5, 1 or 5 nM siRNA having sequence ID SEQ NO: 124, 153, 166, 187 or 197 as a sense strand.

[048] C: Graph showing the amount of Plekho1 expression according to treatment with 0.5, 1 or 5 nM siRNA having sequence ID SEQ NO: 212, 218, 221 or 223 as a sense strand.

[049] FIG. 5 is a graph showing an amount of inhibition of target gene expression confirmed after transformation of a human lung cancer cell line with SAMiRNA having sequence SEQ ID NO: 42, 59, or 602 as a sense strand accordingly. with the present invention.

[050] FIG. 6 is a graph showing an amount of inhibition of target gene expression confirmed after transformation of a mouse fibroblast cell line with siRNA having sequence ID SEQ NOs: 301 to 330, 401 to 430, 501 to 530 as a sense tape according to the present invention.

[051] A: Graph showing the amount of CTGF expression according to 20 nM siRNA treatment having sequence ID SEQ NOs: 301 to 330 as a sense strand.

[052] B: Graph showing the amount of Cyr61 expression according to 20 nM siRNA treatment having sequence ID SEQ NOs: 401 to 430 as a sense strand.

[053] C: Graph showing the amount of Plekho1 expression according to treatment with 20 nM siRNA having sequence ID SEQ NOs: 501 to 530 as a sense strand.

[054] FIG. 7 is a graph showing an amount of inhibition of target gene expression confirmed after transformation of a mouse fibroblast cell line with siRNA having sequence ID SEQ NO: 404 to 406, 408 to 410, 414 to 418, 420 to 422, 424, 427, 429, 430, 503 to 509, 514 to 517, 519, 521 to 526 or 528 as a sense tape according to the present invention.

[055] A: Graph showing the amount of Cyr61 expression according to 5 nM siRNA treatment having sequence ID SEQ NOs: 404 to 406, 408 to 410, 414 to 418, 420 to 422, 424, 427, 429 , or 430 as a sense tape.

[056] B: Graph showing the amount of Plekho1 expression according to treatment of 5 nM siRNA having sequence ID SEQ NO: 503 to 509, 514 to 517, 519, 521 to 526 or 528 as a sense strand.

[057] FIG. 8 is a graph showing an amount of inhibition of target gene expression confirmed after transformation of a mouse fibroblast cell line with siRNA having sequence SEQ ID NO: 301, 303, 307, 323, 410, 422, 424 , 507, 515 or 525 as a sense tape according to the present invention.

[058] A: Graph showing the amount of CTGF expression according to treatment of 0.2, 1 or 5 nM siRNA having sequence ID SEQ NO: 301, 303, 307 or 323 as a sense strand.

[059] B: Graph showing the amount of Cyr61 expression according to treatment of 0.2, 1 or 5 nM siRNA having sequence ID SEQ NO: 410, 422 or 424 as a sense strand.

[060] C: Graph showing the amount of Plekho1 expression according to treatment of 0.2, 1 or 5 nM siRNA having sequence ID SEQ NO: 507, 515 or 525 as a sense strand.

[061] FIG. 9 is a graph showing an amount of inhibition of target gene expression confirmed after transforming a mouse fibroblast cell line with siRNA having a SEQ ID NO: 4, 5, 6, 8, 9, 102, 104, 105, 107, 108, 109, 202, 204, 206 to 209, 307, 424 or 525 as the sense tape according to the present invention.

[062] A: Graph showing the amount of CTGF expression according to treatment of 5 nM siRNA having sequence ID SEQ NO: 4, 5, 6, 8, 9 or 307 as a sense strand.

[063] B: Graph showing the amount of Cyr61 expression according to 5 nM siRNA treatment having sequence ID SEQ NO: 102, 104, 105, 107, 108, 109 or 424 as a sense strand.

[064] C: Graph showing the amount of Plekho1 expression according to treatment of 5 nM siRNA having sequence ID SEQ NO: 202, 204, 206 to 209 or 525 as a sense strand.

[065] FIG. 10 is a graph showing an amount of inhibition of target gene expression confirmed after transformation of a mouse fibroblast cell line with siRNA having sequence ID SEQ NO: 6, 8, 102, 104, 105, 204, 207 , 208, 307, 424 or 525 as the sense tape according to the present invention.

[066] A: Graph showing the amount of CTGF expression according to treatment with 5 or 20 nM siRNA having sequence ID SEQ NO: 6, 8 or 307 as a sense strand.

[067] B: Graph showing the amount of Cyr61 expression according to treatment with 5 or 20 nM siRNA having sequence ID SEQ NO: 102, 104, 105 or 424 as a sense strand.

[068] C: Graph showing the amount of Plekho1 expression according to treatment with 5 or 20 nM siRNA having sequence ID SEQ NO: 204, 207, 208 or 525 as a sense strand.

BEST WAY TO CARRY OUT THE INVENTION

[069] To achieve the above objectives, the present invention provides an siRNA specific for CTGF, Cyr61 or Plekho1 (gene related to respiratory diseases) consisting of the first oligonucleotide which is a sense strand including any sequence selected from the group consisting of ID SEQ NOs: 1 to 600 and 602 to 604 and a second oligonucleotide which is an antisense strand including a complementary sequence thereto.

[070] The siRNA in the present invention includes all materials having a general RNAi action (RNA interference), and therefore, it is obvious to one skilled in the art that siRNA specific for CTGF, Cyr61 or Plekho1 includes shRNA specific for CTGF, Cyr61 or Plekho1, etc.

[071] SEQ ID NOs: 1 to 100, or 602 to 604 are siRNA sense strand sequences specific for CTGF (Homo sapiens), SEQ ID NOs: 101 to 200 are siRNA sense strand sequences specific to Cyr61 (Homo sapiens ), SEQ ID NOs: 201 to 300 are siRNA sense strand sequences specific for Plekho1 (Homo sapiens), SEQ ID NOs: 301 to 400 are siRNA sense strand sequences specific to CTGF (Mus musculus), SEQ ID NOs: 401 to 500 are siRNA sense strand sequences specific for Cyr61 (Mus musculus), and SEQ ID NOs: 501 to 600 are siRNA sense strand sequences specific to Plekho1 (Mus musculus).

[072] The siRNA according to the present invention is preferably siRNA specific for CTGF including any sequence selected from the group consisting of ID SEQ NOs: 1 to 10, 35, 42, 59, 602, 603, 604, 301 to 303 , 305 to 307, 309, 317, 323 and 329, like a sense tape,

[073] Cyr61-specific siRNA including any sequence selected from the group consisting of ID SEQ NOs: 101 to 110, 124, 153, 166, 187, 197, 409, 410, 415, 417, 418, 420, 422, 424 , 427 and 429, like a sense tape, or

[074] siRNA specific for Plekho1 including any sequence selected from the group consisting of ID SEQ NOs: 201 to 210, 212, 218, 221, 223, 504 to 507, 514, 515 and 522 to 525, as a sense strand.

[075] More preferably, the siRNA according to the present invention is CTGF-specific siRNA including any sequence selected from the group consisting of ID SEQ NOs: 4, 5, 8, 9, 35, 42, 59, 601, 602 , 604, 301, 303, 307 and 323, like a sense tape,

[076] siRNA specific for Cyr61 including any sequence selected from the group consisting of ID SEQ NOs: 102, 104, 107, 108, 124, 153, 166, 187, 197, 410, 422 and 424, as a sense strand, or

[077] siRNA specific for Plekho1 including any sequence selected from the group consisting of ID SEQ NOs: 206 to 209, 212, 218, 221, 223, 507, 515 and 525, as a sense strand.

[078] More preferably, the siRNA according to the present invention is CTGF-specific siRNA including any sequence selected from the group consisting of SEQ ID NO: 42, 59, 602 and 323, as a sense strand,

[079] siRNA specific for Cyr61 including any sequence selected from the group consisting of SEQ ID NO: 124, 153, 187, 197 and 424, such as a sense strand, or siRNA specific for Plekho1 including any sequence selected from the group consisting in ID SEQ NO: 212, 218, 221, 223 and 525, as a sense tape.

[080] In particular, it was confirmed that some of the siRNA specific for human CTGF, Cyr61 or Plekho1 according to the present invention are capable of simultaneously inhibiting the expression of mouse CTGF, Cyr61 or Plekho1.

[081] The siRNA capable of simultaneously inhibiting the expression of human and mouse CTGF, Cyr61 or Plekho1 preferably includes a sense strand of siRNA specific for CTGF according to SEQ ID NO: 6 or 8, a sense strand of siRNA specific for Cyr61 according to SEQ ID NO: 102, 104 or 105, or a sense strand of Plekho1-specific siRNA according to SEQ ID NO: 204, 207 or 208.

[082] More preferably, the siRNA includes a CTGF-specific siRNA sense strand according to SEQ ID NO: 6, a Cyr61-specific siRNA sense strand according to SEQ ID NO: 102, or a specific siRNA sense strand for Plekho1 according to SEQ ID NO: 207.

[083] The sense strand or antisense strand of the siRNA according to the present invention preferably has 19 to 31 nucleotides, and the siRNA includes a sense strand including any sequence selected from SEQ ID NOs: 1 to 604 and an antisense strand complementary to it.

[084] Since the siRNA specific for CTGF, Cyr61 or Plekho1 according to the present invention has a base sequence designed to be capable of being complementary linked to the mRNA encoding the corresponding gene, expression of the corresponding gene is capable of be effectively inhibited. Furthermore, the siRNA specific for CTGF, Cyr61 or Plekho1 according to the present invention may include overhang which is a structure including one or two or more unpaired nucleotides at the 3' end of the siRNA, and may include various modifications of the siRNA to provide lyase resistance to the nucleic acid, and to decrease non-specific immune response to improve in vivo stability. The modification of the first oligonucleotide or the second oligonucleotide configuring the siRNA may be one or more combinations selected from the modification in which the -OH group at the 2' carbon in a sugar backbone in one or more nucleotides is replaced with -CH3(methyl ), -OCH3 (methoxy), -NH2, -F(fluorine), -O-2-methoxyethyl, -O-propyl, -O- 2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -O-N-methylacetamido or -O-dimethylamidooxyethyl; modification in which oxygen in a sugar backbone in nucleotides is replaced with sulfur; and modification of phosphorothioate or boranophophate, methyl phosphonate linkages of nucleotide linkages, or may be modification to peptide nucleic acid (PNA), modification to locked nucleic acid (LNA), or modification to unlocked nucleic acid (UNA) (Ann . Rev. Med. 55, 61-65 2004; 14:1139-1143, 2003; Nucleic Acids Research, 38(17) 5761-5773, 2010; 5) 1823-1832, 2011).

[085] The siRNA specific for CTGF, Cyr61 and/or Plekho1 according to the present invention can inhibit the expression of the corresponding gene, and can notably inhibit the expression of the corresponding protein.

[086] The present invention also provides a conjugate in which a hydrophilic material and a hydrophobic material are conjugated to both ends of siRNA, for effective in vivo release to improve the stability of siRNA specific to genes related to respiratory diseases, particularly, the siRNA specific for CTGF, Cyr61 or Plekho1.

[087] In the siRNA conjugate in which a hydrophilic material and a hydrophobic material are linked to the siRNA, a self-assembled nanoparticle is formed by a hydrophobic interaction of the hydrophobic material (see Korean Patent Publication No. 1224828), in which the self-assembled nanoparticle has advantages in internal release efficiency and in vivo stability are significantly excellent, and the uniformity of a particle size is excellent, so that quality control (QC) is easily carried out, according to which a process for producing drugs is easy.

[088] In a preferred embodiment, a double-stranded oligo RNA structure containing the siRNA specific for CTGF, Cyr61 or Plekho1 according to the present invention preferably has a structure represented by the following Structural Formula (1): Structural Formula 1 A-X-R-Y-B

[089] In Structural Formula (1), A is a hydrophilic material, B is a hydrophobic material, X and Y are each independently a single covalent bond or a ligand-mediated covalent bond, and R is siRNA specific for CTGF, Cyr61 or Plekho1.

[090] More preferably, a double-stranded oligo RNA structure containing the siRNA specific for CTGF, Cyr61 or Plekho1 according to the present invention has a structure represented by the following Structural Formula (2): Structural Formula 2 A-X-S-Y-B AS

[091] In Structural Formula (2), A, B, X and Y are the same as those defined in Structural Formula (1), S is a sense strand of the siRNA specific for CTGF, Cyr61 or Plekho1, and AS is an antisense strand of siRNA specific for CTGF, Cyr61 or Plekho1.

[092] More preferably, a double-stranded RNA oligo structure containing the siRNA specific for CTGF, Cyr61 or Plekho1 according to the present invention has a structure represented by the following Structural Formula (3) or (4): Structural Formula 3 A-X -5' S 3'-Y-B AS Structural Formula 4 A-X-3' S 5'-Y-B AS

[093] In Structural Formulas (3) and (4), A, B, S, AS, X and Y are the same as those defined in Structural Formula (1) and 5' and 3' mean 5' end and 3' end of the sense strand of siRNA specific for CTGF, Cyr61 or Plekho1.

[094] It is obvious to one skilled in the art that in Structural Formulas (1) to (4), one to three phosphate groups can be attached to the 5' end of the antisense strand of the double-stranded oligo RNA structure containing the specific siRNA. for CTGF, Cyr61 or Plekho1, and shRNA is able to be used instead of using siRNA.

[095] The hydrophilic material in Structural Formulas (1) to (4) is preferably a cationic or nonionic polymer material having a molecular weight of 200 to 10,000, more preferably, a nonionic polymer material having a molecular weight of 1,000 to 2,000. For example, non-ionic hydrophilic polymer compounds such as polyethylene glycol, polyvinyl pyrrolidone, polyoxazoline, etc., are preferably used as the hydrophilic polymer material, but the present invention is not necessarily limited thereto.

[096] In particular, the hydrophilic material (A) in Structural Formulas (1) to (4) can be used in a block form of hydrophilic material represented by the following Structural Formula (5) or (6), and using a number appropriate (n in Structural Formula (5) or (6)) of blocks of hydrophilic material as required, problems arising from polydispersity that may occur in case of using a general synthetic polymer material, etc., may be largely improved. Structural Formula 5 (A’m-J)n Structural Formula 6 (J-A’m)n

[097] In Structural Formula (5), A' is a monomer of hydrophilic material, J is a ligand to link monomers of hydrophilic material (the sum is m) between them or a ligand to link monomers of hydrophilic material (the sum is m) for siRNA, m is an integer from 1 to 15, n is an integer from 1 to 10, and the repeat unit represented by (A'm-J) or (J-A'm) corresponds to a unit base block of hydrophilic material.

[098] When the block of hydrophilic material is represented by Structural Formula (5) or (6), the double-stranded RNA oligo structure containing the siRNA specific for CTGF, Cyr61 or Plekho1 according to the present invention has a structure represented by the following Structural Formula (7) or (8): Structural Formula 7 (A'm-J)n-X-R-Y-B Structural Formula 8 (J-A'm)n-X-R-Y-B

[099] In Structural Formulas (7) and (8), X, R, Y and B are the same as those defined in Structural Formula (1) and A', J, m and n are the same as those defined in Structural Formulas (5) and (6).

[0100] In Structural Formulas (5) and (6), the hydrophilic material monomer A' is usable without limitation as long as it meets the objectives of the present invention among the non-ionic hydrophilic polymer monomers. The hydrophilic material monomer A' is preferably a monomer selected from the following compounds (1) to (3) shown in Table 1, more preferably a monomer of the compound (1), and G in the compound (1) may preferably be selected from CH2, O, S and NH.

[0101] In particular, among the monomers of hydrophilic material, the monomer of Compound (1) can have several functional groups introduced into them and good affinity in vivo, and induce a small immune response, thus having excellent biocompatibility, and, in addition , can increase the in vivo stability of the oligonucleotide included in the structure of Structural Formula (7) or (8) and can increase the release efficiency, which is considerably suitable for producing the structure according to the present invention. [Table 1] Structure of the hydrophilic material monomer in the present invention

[0102] In particular, the hydrophilic material in structural formulas 5 to 8 preferably has a total molecular weight of 1,000 to 2,000. Therefore, for example, when hexaethylene glycol represented by Compound (1), that is, G is O, and m is 6, is used in Structural Formulas (7) and (8), a molecular weight of hexaethylene glycol spacer is 344, so that the repeat number (n) is preferred to be from 3 to 5. In particular, the repeating unit of the hydrophilic group, i.e. the block of hydrophilic material, represented by (A'm-J) or ( J-A'm)n in Structural Formulas (5) and (6), is capable of being used in an appropriate number represented by n, as needed. A which is the monomer of hydrophilic material and J which is the ligand included in each block of hydrophilic material may be independently equal to each other or may be different from each other. That is, when 3 blocks of hydrophilic material are used (n = 3), the hydrophilic material monomer of compound (1) is used in the first block, the hydrophilic material monomer of compound (2) is used in the second block, the hydrophilic material monomer of compound (3) is used in the third block, etc. That is, different hydrophilic material monomers for all hydrophilic material blocks can be used, and any hydrophilic material monomer selected from the hydrophilic material monomers of compounds (1) to (3) can also be used equally in all blocks of hydrophilic material. Likewise, ligands mediating the combination of hydrophilic material monomer can also be the same and different from each other for all blocks of hydrophilic material. Furthermore, m, which is the number of monomers of hydrophilic material, can be equal to each other or different from each other among all blocks of hydrophilic material. That is, 3 monomers of hydrophilic material (m=3) are linked to the first block of hydrophilic material, 5 monomers of hydrophilic material (m=5) are linked to the second block of hydrophilic material, 4 monomers of hydrophilic material (m=4 ) are attached to the third block of hydrophilic material, etc. That is, monomers of hydrophilic material each having different numbers or each having the same number can be used in blocks of hydrophilic material.

[0103] Furthermore, in the present invention, the ligand (J) is preferably selected from the group consisting of PO3-, SO3 and CO2, but the present invention is not limited to them. It is obvious to one skilled in the art that any binder can be used depending on the monomer of hydrophilic material used, etc., as long as it satisfies the objectives of the present invention.

[0104] The hydrophobic material (B) in Structural Formulas (1) to (4), (7) and (8) serves to form a nanoparticle formed from the oligonucleotide structures according to Structural Formulas (1) to (4), (7) and (8) through hydrophobic interaction. The hydrophobic material preferably has a molecular weight of 250 to 1,000 and may include a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, C12 to C50 saturated or unsaturated hydrocarbon, diacylphosphatidylcholine, fatty acids, phospholipids, lipopolyamine, etc. ., but the present invention is not limited thereto. It is obvious to one skilled in the art that any hydrophobic material can be used, as long as it satisfies the objectives of the present invention.

[0105] The steroid derivative can be selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesterol formate, cholestanyl formate, and cholesteryl amine, and the glyceride derivative can be selected from mono, di and triglycerides, etc. wherein the fatty acid of the glyceride is preferably saturated or unsaturated fatty acid C12 to C50.

[0106] In particular, among hydrophobic materials, saturated or unsaturated hydrocarbons or cholesterol is preferred since it is capable of easily being linked in a synthetic step of the oligonucleotide structure according to the present invention, and C24 hydrocarbon, particularly, tetradocosane , including a disulfide bond is more preferred.

[0107] The hydrophobic material is attached to the distal end of the hydrophilic material, and it does not matter whether the hydrophobic material is attached to any position of the sense strand or antisense strand of the siRNA.

[0108] The hydrophilic material or the hydrophobic material in Structural Formulas (1) to (4), (7) and (8) according to the present invention is linked to the siRNA specific for CTGF, Cyr61 or Plekho1 by a single covalent bond or a ligand-mediated covalent bond (X or Y). Furthermore, the linker mediating the covalent linkage is covalently linked to the hydrophilic material or the hydrophobic material at the end of the siRNA specific for CTGF, Cyr61 or Plekho1, and is not particularly limited as long as the linkage that is possible to be cleaved in an environment specific information is provided as needed. Therefore, any compound for binding to activate the siRNA specific for CTGF, Cyr61 or Plekho1 and/or the hydrophilic material (or the hydrophobic material) in producing the double helix RNA oligo structure according to the present invention can be used as the binder. The covalent bond can be either a non-degradable bond or a degradable bond. Here, examples of the non-degradable bond may include an amide bond or a phosphorylation bond, and examples of the degradable bond may include a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond or an enzymatically degradable bond and the like, but the present invention is not limited to these.

[0109] Furthermore, any siRNA specific for CTGF, Cyr61 or Plekho1 represented by R (or S and AS) in Structural Formulas (1) to (4), (7), and (8) is usable without limitation, provided that the siRNA is capable of being specifically bound to CTGF, Cyr61 or Plekho1. Preferably, the siRNA specific for CTGF, Cyr61 or Plekho1 consists of a sense strand including any sequence selected from SEQ ID NOs: 1 to 600 and 602 to 604 and an antisense strand including a complementary sequence thereto.

[0110] In particular, the siRNA included in Structural Formulas (1) to (4), (7) and (8) according to the present invention is preferably CTGF-specific siRNA including any sequence selected from the group consisting of ID SEQ NOs: 1 to 10, 35, 42, 59, 602 to 604 or 301 to 303, 305 to 307, 309, 317, 323 and 329, as a sense tape,

[0111] siRNA specific for Cyr61 including any sequence selected from the group consisting of ID SEQ NOs: 101 to 110, 124, 153, 166, 187, 197, 409, 410, 415, 417, 418, 420, 422, 424 , 427 and 429, like a sense tape, or

[0112] siRNA specific for Plekho1 including any sequence selected from the group consisting of ID SEQ NOs: 201 to 210, 212, 218, 221, 223, 504 to 507, 514, 515 and 522 to 525, as a sense strand.

[0113] More preferably, the siRNA according to the present invention is CTGF-specific siRNA including any sequence selected from the group consisting of ID SEQ NOs: 4, 5, 8, 9, 35, 42, 59, 602, 603 , 604, 301, 303, 307 and 323, like a sense tape,

[0114] siRNA specific for Cyr61 including any sequence selected from the group consisting of ID SEQ NOs: 102, 104, 107, 108, 124, 153, 166, 187, 197, 410, 422 and 424, as a sense strand, or

[0115] siRNA specific for Plekho1 including any sequence selected from the group consisting of ID SEQ NOs: 206 to 209, 212, 218, 221, 223, 507, 515 and 525, as a sense strand.

[0116] More preferably, the siRNA according to the present invention is siRNA specific for CTGF including any sequence of SEQ ID NO: 42, 59, 602 or 323, as a sense strand,

[0117] siRNA specific for Cry61 including any sequence of SEQ ID NO: 124, 153, 187, 197 or 424, as a sense strand, or

[0118] siRNA specific for Plekho1 including any sequence of SEQ ID NO: 212, 218, 221, 223 or 525, as a sense strand.

[0119] In addition, siRNA including sense strand of siRNA specific for human and mouse CTGF according to SEQ ID NO: 6 or 8, siRNA including sense strand of siRNA specific for human and mouse Cyr61 according to SEQ ID NO: 102, 104 or 105, and siRNA including sense strand of siRNA specific for human and mouse Plekho1 according to SEQ ID NO: 204, 207 or 208, are particularly preferred, which is because the siRNA having the sequence as the sense strand has an effect of simultaneously inhibiting the expression of human or mouse CTGF, Cyr61 or Plekho1, such that siRNA including sense strand siRNA specific for human and mouse CTGF according to SEQ ID NO: 6, siRNA including sense strand siRNA specific for human and mouse Cyr61 according to SEQ ID NO: 102, and siRNA including sense strand siRNA specific for human and mouse Plekho1 according to SEQ ID NO: 207 are most preferred.

[0120] In the double-stranded RNA oligo structure containing the siRNA for CTGF, Cyr61 or Plekho1 according to the present invention, an amine group or a polyhistidine group can be additionally introduced into a portion of the distal end linked with the siRNA of the hydrophilic material in the structure.

[0121] This makes it easier to carry out the intercellular introduction and escape from the endosome of the double-stranded oligo RNA structure containing the siRNA specific for CTGF, Cyr61 or Plekho1 according to the present invention, and the possibility of introducing an amino group and using a polyhistidine group to easily realize the intercellular introduction and endosome escape of transporters such as Quantum dot, Dendrimers, liposomes, etc., and the effect thereof, has been reported.

[0122] Specifically, it is known that the primary amine group expressed at the end or outside of the transporter is protonated at pH in vivo to form a conjugate with a negatively charged gene by an electrostatic interaction, and escape from the endosome is easily accomplished due to internal tertiary amines having a buffering effect at the low pH of the endosome after being introduced into the cells, thus being able to protect the transporter from lysosomal breakdown (gene release and inhibition of expression using a polymer-based hybrid material . Polymer Sci. Technol., Vol. 23, No.3, pp254-259).

[0123] Furthermore, it is known that histidine, which is one of the non-essential amino acids, has an imidazole ring (pKa3 6.04) at a residue (-R) to increase the buffering capacity in the endosome and lysosome, so that histidine expression can be used to increase endosome escape efficiency in non-viral gene transporters including liposomes (Novel histidine-conjugated galactosylated cationic liposomes for efficient hepatocyte selective gene transfer in human hepatoma HepG2 cells. J. Controlled Release 118, pp262-270).

[0124] The amine group or the polyhistidine group can be linked to the hydrophilic material or the hydrophilic material block through at least one linker.

[0125] When the amine group or polyhistidine group is introduced into the hydrophilic material of the double helix RNA oligo structure represented by Structural Formula (1), the double helix RNA oligo structure can have a structure represented by Structural Formula (9 ) below: Structural Formula 9 P-J1-J2-A-X-R-Y-B

[0126] In Structural Formula (9), A, B, R, X and Y are the same as those defined in Structural Formula (1),

[0127] P is an amine group or a polyhistidine group.

[0128] J1 and J2 are ligands and can each be independently selected from a simple covalent bond, PO3-, SO3, CO2, C2-12 alkyl, alkenyl, and alkynyl, but the present invention is not limited to them. It is obvious to one skilled in the art that any ligand can be used with J1 and J2, as long as it satisfies the objectives of the present invention depending on the hydrophilic material used.

[0129] Preferably, when the amine group is introduced, J2 is preferably a single covalent bond or PO3- and J1 is preferably C6 alkyl, but the present invention is not limited to the same.

[0130] Furthermore, when the polyhistidine group is introduced, in Structural Formula (9), J2 is preferably a single covalent bond or PO3-, and J1 is preferably the following Compound (4), but the present invention is not limited to the same.Compound 4

[0131] Furthermore, when the hydrophilic material of the double helix RNA oligo structure represented by Structural Formula (9) is a block of hydrophilic material represented by Structural Formula (5) or (6), and the amine group or the polyhistidine is introduced, the double helix RNA oligo structure can be represented by Structural Formula (10) or (11): Structural Formula 10 P-J1-J2-(A'm-J)n-X-R-Y-B Structural Formula 11 P-J1- J2-(J-A'm)n-X-R-Y-B

[0132] In Structural Formulas (10) and (11), X, R, Y, B, A', J, m and n are the same as those defined in Structural Formula (5) or (6), and P, J1 and J2 are the same as those defined in Structural Formula (9).

[0133] In particular, in Structural Formulas (10) and (11), the hydrophilic material is preferentially linked to the 3' end of the sense strand of the siRNA specific for CTGF, Cyr61 or Plekho1, and in this case, Structural Formulas (9) a (11) can be represented by the following Structural Formulas (12) to (14): Structural Formula 12 P-J1-J2-A-X-3'S 5'-Y-B AS Structural Formula 13 P-J1-J2-(A'm-J )n-X-3'S 5'-Y-B AS Structural Formula 14 P-J1-J2-(J-A'm)n-X-3'S 5'-Y-B AS

[0134] In Structural Formulas (12) and (14), X, R, Y, B, A, A' J, m, n, P, J1 and J2 are the same as those defined in Structural Formulas (9) to ( 11), and 5' and 3' mean the 5' end and 3' end of the sense strand of the siRNA specific for CTGF, Cyr61 or Plekho1.

[0135] The amine group that can be introduced in the present invention can be primary to tertiary amine groups, and the primary amine group is particularly preferred. The introduced amine group may be present as an amine salt. For example, the salt of the primary amine group may be present in an NH3+ form.

[0136] Furthermore, the polyhistidine group that can be introduced in the present invention preferably includes from 3 to 10 histidines, more preferably, 5 to 8 histidines and most preferably, 6 histidines. In addition to histidine, at least one cysteine can be additionally included.

[0137] However, when a target moiety is provided in the double-stranded RNA oligo structure containing the siRNA specific for CTGF, Cyr61 or Plekho1 according to the present invention and nanoparticles formed therefrom, delivery to the target cell is effectively promoted to achieve release to the target cell, even at a relatively low dosage concentration, thus showing a high control function for target gene expression, and thereby preventing non-specific release of siRNA specific for CTGF, Cyr61 or Plekho1 in other organs and cells.

[0138] In this sense, the present invention provides a double-stranded RNA oligo structure in which a ligand (L), particularly a ligand specifically bound to a receptor that promotes internalization of the target cell through receptor-mediated endocytosis (RME ), is additionally attached to the structure according to Structural Formulas (1) to (4), (7) and (8). For example, the form in which the linker is attached to the double-stranded RNA oligo structure according to Structural Formula (1), has a structure represented by the following Structural Formula (15): Structural Formula 15 (Li-Z)- A-X-R-Y-B

[0139] In Structural Formula (15), A, B, X and Y are the same as those defined in Structural Formula (1), L is a ligand specifically bound to a receptor that promotes internalization of the target cell through receivers (RME), and i represents an integer from 1 to 5, preferably an integer from 1 to 3.

[0140] The ligand in Structural Formula (15) can be preferably chosen from the group consisting of a target receptor-specific antibody or aptamer, peptide that has RME properties to promote cell internalization in a target cell-specific manner; or folate (generally, folate and folic acid are used interchangeably, and folate in the present invention means folate in a natural state or in an activated state in a human body), chemicals such as sugar, carbohydrates, etc., including hexoamines such as N-acetyl galactosamine (NAG), etc., glucose, mannose, but the present invention is not limited to the same.

[0141] Furthermore, the hydrophilic material A in Structural Formula (15) can be used in the form of the block of hydrophilic material represented by Structural Formulas (5) and (6).

[0142] The present invention also provides a method for producing the double-stranded RNA oligo structure containing siRNA specific for CTGF, Cyr61 or Plekho1.

[0143] The method for producing the double-stranded RNA oligo structure containing the siRNA specific for CTGF, Cyr61 or Plekho1 according to the present invention may include the following steps:

[0144] (1) attaching a hydrophilic material based on a solid support;

[0145] (2) synthesize a single strand of RNA based on the solid support containing the hydrophilic material linked to it;

[0146] (3) covalently attach a hydrophobic material to the 5' end of the single-stranded RNA;

[0147] (4) synthesizing a single-stranded RNA having a sequence complementary to a sequence of the single-stranded RNA;

[0148] (5) separating and purifying an RNA-polymer structure and the single-stranded RNA from the solid support when the synthesis of the single-stranded RNA is completed; It is

[0149] (6) produce the double helix RNA oligo structure by annealing the RNA-polymer structure and a single strand of RNA having a sequence complementary to it.

[0150] The solid support in the present invention is preferably a controlled pore glass (CPG), but the present invention is not limited to the same, but can be polystyrene, cellulose paper, silica gel, etc. In CPG, a diameter is preferably 40 to 180 μm, and a pore size is preferably 500 to 3000Å.

[0151] After step (5) above, when production is completed, it can be confirmed whether the purified RNA-polymer structure and the purified single-stranded RNA are produced as the desired RNA-polymer structure and single-stranded RNA. Desired RNA measuring molecular weights by MALDI-TOF mass spectrometer. In the production method, Step (4) which is a step of synthesizing single-stranded RNA having a sequence complementary to a sequence of single-stranded RNA synthesized in Step (2) may be performed before Step (1) or may be performed during any stage of one of Steps (1) to (5).

[0152] Furthermore, the single-stranded RNA having a complementary sequence to the single-stranded RNA synthesized in Step (2) may contain a phosphate group attached to the 5' end thereof.

[0153] However, the present invention also provides a method for producing the double-stranded RNA oligo structure in which a linker is additionally linked to the double-stranded RNA oligo structure containing the siRNA specific for CTGF, Cyr61 or Plekho1.

[0154] The method for producing the double-stranded RNA oligo structure containing the siRNA specific for CTGF, Cyr61 or Plekho1 bound to the ligand according to the present invention may include the following steps:

[0155] (1) attaching a hydrophilic material to a solid support containing a functional group attached thereto;

[0156] (2) synthesize a single strand of RNA based on the solid support containing a functional group-hydrophilic material attached to it;

[0157] (3) covalently attach a hydrophilic material to the 5' end of the RNA single strand;

[0158] (4) synthesizing a single-stranded RNA having a sequence complementary to a sequence of the single-stranded RNA;

[0159] (5) separating a functional group-RNA-polymer structure and the single-stranded RNA having the complementary sequence from the solid support when the synthesis of the single-stranded RNA is completed;

[0160] (6) produce a single-stranded linker-RNA-polymer structure by attaching a linker to one end of the hydrophilic material using the functional group; It is

[0161] (7) produce the double helix RNA linker-oligo structure by annealing the linker-RNA-polymer structure and a single strand of RNA having a sequence complementary to it.

[0162] After step (6) above, when production is completed, whether the double helix RNA linker-oligo structure and the desired single-stranded RNA having a sequence complementary to it will be prepared or not can be confirmed by separating and purifying the linker-RNA-polymer structure and the single-stranded RNA having a sequence complementary to it, and measuring the molecular weights by MALDI-TOF mass spectrometer. The double helix RNA linker-oligo structure can be produced by annealing the RNA linker-polymer structure and a single strand of RNA having a sequence complementary to it. In the production method, Step (4), which is a step of synthesizing single-stranded RNA having a sequence complementary to a sequence of single-stranded RNA synthesized in Step (3), is an independent synthesis process, and can be carried out before Step (1) or may be carried out during any step of one of Steps (1) to (6).

[0163] The present invention also provides a nanoparticle including double-stranded oligo RNA structure containing siRNA specific for CTGF, Cyr61 or Plekho1.

[0164] As described above, the double-stranded RNA oligo structure containing the siRNA specific for CTGF, Cyr61 or Plekho1 is an amphipathic structure containing both hydrophobic materials and hydrophilic materials, in which the hydrophilic materials have affinity through an interaction such as a hydrogen bond, etc., with water molecules present in the body to face outward, and the hydrophobic materials face inward through a hydrophobic interaction between them, thus forming a thermodynamically stable nanoparticle. That is, hydrophobic materials are positioned in the center of the nanoparticle, and hydrophilic materials are positioned toward the outside of the siRNA specific for CTGF, Cyr61, or Plekho1 to form nanoparticles protecting the siRNA specific for CTGF, Cyr61, or Plekho1. These formed nanoparticles enhance the intracellular release of siRNA specific for CTGF, Cyr61, and/or Plekho1 and improve the effect of siRNA.

[0165] The nanoparticles according to the present invention can be formed from just the double-stranded RNA oligo structure containing siRNAs each having the same sequence as each other, or can be formed from the double-stranded RNA oligo structure containing siRNAs , each having a different sequence, wherein it is interpreted in the present invention that siRNAs each having a different sequence include siRNA having different target genes, for example, siRNA specific for CTGF, Cyr61 or Plekho1, or siRNA having the same target gene specificity. target, but having a different sequence.

[0166] Furthermore, the double-stranded RNA oligo structure containing other respiratory disease-related gene-specific siRNA in addition to siRNA specific for CTGF, Cyr61 or Plekho1 can also be included in the nanoparticles according to the present invention.

[0167] Furthermore, the present invention provides a composition for preventing or treating respiratory diseases, particularly idiopathic pulmonary fibrosis and COPD, including: the siRNA specific for CTGF, Cyr61 or Plekho1, the double-stranded RNA oligo structure containing the siRNA , and/or the nanoparticles formed from the double helix RNA oligo structure.

[0168] The composition including the siRNA specific for CTGF, Cyr61 or Plekho1 according to the present invention, the double-stranded RNA oligo structure containing the siRNA, and/or the nanoparticle formed from the double-stranded RNA oligo structure as components effective, inhibits pulmonary artery remodeling and airway remodeling, so that the siRNA specific for CTGF, Cyr61 or Plekho1 or the composition containing the siRNA has the effect of preventing or treating respiratory diseases.

[0169] In particular, the composition for preventing or treating respiratory diseases, including the double-stranded RNA oligo structure according to the present invention may include:

[0170] a double-stranded oligo RNA structure including a CTGF-specific siRNA that includes a sense strand including any one sequence selected from the group consisting of ID SEQ NOs: 1 to 100 or 602 to 604 and 301 to 400, preferably, any sequence selected from the group consisting of ID SEQ NOs: 1 to 10, 35, 42, 59, 602 to 604, 301 to 303, 305 to 307, 309, 317, 323 and 329, more preferably, any sequence selected from the group consisting of SEQ ID NOs: 4, 5, 8, 9, 35, 42, 59, 602 to 604, 301, 303, 307 and 323, more preferably, any sequence of SEQ ID NO: 42, 59, 602 or 323, and an antisense strand including a complementary sequence thereto;

[0171] a double-stranded oligo RNA structure including an siRNA specific for Cyr61 that includes a sense strand including any one sequence selected from the group consisting of ID SEQ NOs: 101 to 200, and 401 to 500, preferably any sequence selected from the group consisting of ID SEQ NOs: 101 to 110, 124, 153, 166, 187, 197, 409, 410, 415, 417, 418, 420, 422, 424, 427 and 429, more preferably, any sequence selected from the group consisting of SEQ ID NOs: 102, 104, 107, 108, 124, 153, 166, 187, 197, 410, 422 and 424, more preferably, any sequence of SEQ ID NO: 124, 153, 187 , 197 or 424, and an antisense strand including a complementary sequence thereto; or

[0172] a double-stranded oligo RNA structure including an siRNA specific for Plekho1 that includes a sense strand including any one sequence selected from the group consisting of ID SEQ NOs: 201 to 300, and 501 to 600, preferably any one sequence selected from the group consisting of ID SEQ NOs: 201 to 210, 212, 218, 221, 223, 504 to 507, 514, 515 and 522 to 525, more preferably, any sequence selected from the group consisting of ID SEQ NOs: 206 to 209, 212, 218, 221, 223, 507, 515, and 525, more preferably, any sequence of SEQ ID NO: 212, 218, 221, 223 or 525, and an antisense strand including a sequence complementary to the same.

[0173] Furthermore, the composition for preventing or treating respiratory diseases, including the double-stranded RNA oligo structure according to the present invention may include: a double-stranded RNA oligo structure including a CTGF-specific siRNA that includes a sense strand of siRNA specific for human and mouse CTGF according to the sequence of SEQ ID NO: 6 or 8, preferably, SEQ ID NO: 6, and an antisense strand including a complementary sequence thereto;

[0174] a double-stranded oligo RNA structure including an siRNA specific for Cyr61 that includes a sense strand of siRNA specific for human and mouse Cyr61 according to the sequence of ID SEQ NO: 102, 104 and 105, preferably ID SEQ NO: 102 and an antisense strand including a complementary sequence thereto; or

[0175] a double-stranded oligo RNA structure including a siRNA specific for Plekho1 that includes a sense strand of siRNA specific for human and mouse Plekho1 according to the sequence of ID SEQ NO: 204, 207 and 208, preferably ID SEQ NO: 207 and an antisense strand including a complementary sequence thereto.

[0176] Additionally, a double-stranded RNA oligo structure containing the CTGF-specific siRNA, a double-stranded RNA oligo structure containing the Cyr61-specific siRNA, and/or a double-stranded RNA oligo structure containing the siRNA specific for Plekho1 can be mixed and included in the composition, and additionally, siRNA specific for another respiratory disease-related gene other than CTGF, Cry61 or Plekho1 or a double-stranded RNA oligo structure containing the other siRNA specific for respiratory disease-related gene may also be included in the composition according to the present invention.

[0177] At the time of use of a composition for preventing or treating respiratory diseases additionally including the double-stranded RNA oligo structure containing the other gene-specific siRNA related to respiratory diseases together with the siRNA specific for CTGF, Cyr61, and/or or Plekho1, or the double-stranded RNA oligo structure containing the siRNA specific for CTGF, Cyr61, and/or Plekho1, a synergistic effect like that of a combination therapy can be obtained.

[0178] Examples of respiratory diseases capable of being prevented or treated by the composition of the present invention include idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease (COPD), acute or chronic bronchitis, allergic rhinitis, cough and phlegm, lower respiratory tract infection acute, bronchitis, and bronchiolitis, acute upper respiratory tract infection, pharyngitis, tonsillitis, laryngitis, etc., but the present invention is not limited to the same.

[0179] Furthermore, nanoparticles included in the composition for preventing or treating respiratory diseases, including nanoparticles formed from the double-stranded RNA oligo structure according to the present invention may purely consist of just any one structure selected from the oligo structure. double-stranded RNA containing the siRNA specific for CTGF, Cyr61, or Plekho1, or can be configured in a form in which two or more types of double-stranded RNA oligo structures including the siRNA specific for CTGF, Cyr61, or Plekho1 are mixed together. yes.

[0180] The composition of the present invention can be prepared by additionally including at least one pharmaceutically acceptable carrier in addition to the effective components. The pharmaceutically acceptable carrier is required to be compatible with the effective components of the present invention, and can be used by mixing one or more components selected from saline, sterile water, Ringer's solution, buffered saline, dextrose solution, of maltodextrin, glycerol and ethanol, and other conventional additives such as antioxidant, buffer, fungistat and the like, can be added thereto as necessary. Furthermore, the composition can be prepared as an injection formulation, such as an aqueous solution, suspension, emulsion and the like, by adding diluent, dispersant, surfactant, binder and lubricant thereto. In particular, it is preferred to provide the prepared composition as a lyophilized formulation. To prepare the lyophilized formulation, any method that is generally known in the technical field of the present invention can be used, in which a stabilizer for lyophilization can be added thereto. Furthermore, appropriate methods in the art or a method disclosed in Remington's pharmaceutical Science, Mack Publishing Company, or Easton PA may preferably be used for formulation depending on each disease or component.

[0181] The method of dosing and administering the effective components, etc., included in the pharmaceutical composition of the present invention can be determined based on the patient's general symptoms and severity of the disease and by those skilled in the art. Furthermore, the composition may be formulated with various types such as powder, tablet, capsule, solution, injection, ointment, syrup, and the like and may be provided as a unit dose or a multiple dose container, e.g., a sealed ampoule. , bottle and the like.

[0182] The pharmaceutical composition of the present invention can be administered orally or parenterally. Examples of a route of administration of the pharmaceutical composition according to the present invention may include oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, sublingual or topical administration, but the present invention is not limited to it. Particularly, the route of administration may also include administration into the lung through drip infusion into respiratory organs for treating respiratory diseases. The administration amount of the composition may have various ranges depending on a patient's weight, age, gender, health condition, diet, administration time, method, excretion rate, disease severity, and the like, and can be easily determined. by a general expert in the art. Furthermore, the composition of the present invention can be formulated into an appropriate pharmaceutical form using known technologies for clinical administration.

[0183] Furthermore, the present invention provides a method for preventing or treating respiratory diseases, particularly idiopathic pulmonary fibrosis and COPD, including: administering the double-stranded RNA oligo structure according to the present invention and the nanoparticles including the structure of double-stranded RNA oligo to a patient requiring this treatment.

EXAMPLES

[0184] Below, the present invention will be described in detail with reference to the following Examples. These examples are only to exemplify the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not construed to be limited to these examples.

[0185] Example 1. Design of the CTGF, Cyr61 or Plekho1 target sequence and siRNA production

[0186] 604 types of target sequences (sense strands) capable of being linked to the mRNA sequence of the CTGF gene (Homo sapiens) (NM_001901), mRNA sequence of the Cyr61 gene (Homo sapiens) (NM_001554), mRNA sequence of the gene Plekho1 (Homo sapiens) (NM_016274), CTGF gene (Mus musculus) mRNA sequence (NM_010217), Cyr61 gene (Mus musculus) mRNA sequence (NM_010516), or Plekho1 gene (Mus musculus) mRNA sequence (NM_023320 ) were designed, and antisense stranded siRNAs having sequences complementary to the target sequences were produced.

[0187] First, a gene design program (Turbo si-Designer) developed by Bioneer Co., was used to design the target sequence to which the siRNA is capable of being ligated from corresponding gene mRNA sequences. The siRNA for respiratory disease-related genes according to the present invention has a double-stranded structure including a sense strand consisting of 19 nucleotides and an antisense strand complementary thereto. Furthermore, siCONT (having sequence of SEQ ID NO: 601 as a sense strand) which is siRNA having a sequence in which the expression of any gene is not inhibited, was produced. siRNA was produced by phosphodiester linkage forming an RNA structure using β-cyanoethyl phosphoramidite (Nucleic Acids Research, 12:4539-4557, 1984). Specifically, a reaction product including RNA having a desired length was obtained by repeating a series of unblocking, coupling, oxidation and protection processes, on a solid support to which the nucleotide is attached, using RNA synthesizer (384 Synthesizer, BIONEER , Korea). RNA from the reaction product was separated and purified by HPLC LC918 (Japan Analytical Industry, Japan) equipped with a Daisogel C18 column (Daiso, Japan), and it was confirmed whether or not the purified RNA meets the target sequence by MALDI-TOF mass spectrometer (Shimadzu, Japan). Then, the desired double-stranded siRNA (SEQ ID NOs: 1 to 604) was produced by ligating the sense strand of RNA to the antisense strand of RNA.

[0188] Example 2. Production of double-stranded oligo RNA (PEG-SAMiRNA) structure.

[0189] The double-stranded oligo RNA (PEG-SAMiRNA) structure produced in the present invention has a structure represented by the following Structural Formula (16): Structural Formula 16 C24-5'-S-3'-PEG AS

[0190] In Structural Formula (16), S is a sense strand of siRNA; AS is an antisense strand of siRNA; PEG is a hydrophilic material, that is, polyethylene glycol; C24 is a hydrophobic tetradocosane material including a disulfide bond; and 5' and 3' mean double-stranded oligo RNA end directions.

[0191] The siRNA sense strand of Structural Formula (16) was produced by synthesizing a double helix RNA oligo structure-hydrophilic material of a sense strand in which polyethylene glycol is attached to the 3' end by the method described above in which phosphodiester is alloy forming an RNA structure are linked using β-cyanoethyl phosphoramidite, based on 3' polyethylene glycol (PEG, Mn=2,000)-CPG produced in Example 1 of Korean Patent Open Publication No. 10-2012-0119212, as support, and tetradocosane ligand including a disulfide bond to the 5' end, thereby producing a sense strand of a desired RNA-polymer structure. For an antisense strand in which annealing is performed with the sense strand, the antisense strand having a sequence complementary to the sense strand was produced by the reaction described above.

[0192] When the synthesis was completed, the single-stranded RNA and the RNA-polymer structure synthesized by treatment of 28% (v/v) ammonia in a water bath at 60°C were separated from CPG and protection fractions were removed from it by a deprotection reaction, respectively. The single-stranded RNA and the RNA-polymer backbone from which the protective moieties were removed were treated with N-methylpyrrolidone, triethylamine and triethylaminetrihydrofluoride in a volume ratio of 10:3:4 in an oven at 70°C to remove 2' TBDMS(tert-butyldimethylsilyl).

[0193] RNA from the reaction product was separated and purified by HPLC LC918 (Japan Analytical Industry, Japan) equipped with a Daisogel C18 column (Daiso, Japan), and it was confirmed whether or not the purified RNA meets the target sequence by mass spectrometer MALDI-TOF (Shimadzu, Japan). Then, to produce each double-stranded RNA oligo structure, the sense strand and antisense strand each having the same amount were mixed together, placed in 1X annealing buffer (30mM HEPES, 100mM potassium acetate, 2mM potassium acetate). magnesium, pH 7.0 ~ 7.5), and reacted in a constant temperature water bath at 90°C for 3 minutes, and reacted again at 37°C, thus producing each double-stranded RNA oligo structure including siRNA having sequence ID SEQ NO: 42, 59, 602, 124, 153, 187, 197, 212, 218, 221, 223, 323, 424, 525 or 601 as the sense strand (hereinafter referred to as SAMiRNALP-hCTGF, SAMiRNALP-hCyr, SAMiRNALP-hPlek, SAMiRNALP-mCTGF, SAMiRNALP-mCyr, and SAMiRNALP-mPlek SAMiRNALP-CONT, respectively). It was confirmed that the double-stranded RNA oligo structure produced was annealed by electrophoresis.

[0194] Example 3. Production of improved double-stranded oligo RNA structure (Mono-HEG-SAMiRNA).

[0195] The improved double-stranded oligo RNA structure produced in the present invention is obtained using [PO3--hexaethylene glycol]4 (hereinafter referred to as 'Mono-HEG-SAMiRNA', see Structural Formula (17)) which is the building block of hydrophilic material instead of using PEG which is the hydrophilic material, and the following Structural Formula (17). Structural Formula 17 C24-5’-S-3’-[(Hexa Ethylene Glycol)-PO3-]4 AS

[0196] In Structural Formula (17), S is a sense strand of siRNA; AS is an antisense siRNA strand; [Hexa Ethylene Glycol]4 is a hydrophilic material monomer; C24 is a hydrophobic tetradocosane material including a disulfide bond; and 5' and 3' mean sense strand end directions of double helix RNA oligo.

[0197] The structure of Mono-HEG SAMiRNA according to Structural Formula (17) can be represented by the following Structural Formula (18):

[0198] RNA from the reaction product was separated and purified by HPLC LC918 (Japan Analytical Industry, Japan) equipped with a Daisogel C18 column (Daiso, Japan), and it was confirmed whether or not the purified RNA meets the target sequence by mass spectrometer MALDI-TOF (Shimadzu, Japan). Then, to produce each double-stranded RNA oligo structure, the sense strand and antisense strand each having the same amount were mixed together, placed in 1X annealing buffer (30 mM HEPES, 100 mM potassium acetate, 2 mM acetate magnesium, pH 7.0 ~ 7.5), and reacted in a constant temperature water bath at 90°C for 3 minutes, and reacted again at 37°C, thus producing each double-stranded RNA oligo structure helix including siRNA having sequence of SEQ ID NO: 42, 59, 602, 124, 153, 187, 197, 212, 218, 221, 223, 323, 424, 525 or 601 as the sense strand (hereinafter referred to as Mono-HEG -SAMiRNALP-hCTGF, Mono-HEG-SAMiRNALP-hCyr, Mono-HEG-SAMiRNALP-hPlek, Mono-HEG-SAMiRNALP-mCTGF, Mono-HEG-SAMiRNALP-mCyr, Mono-HEG-SAMiRNALP-mPlek, and Mono-HEG- SAMiRNALP- CONT, respectively). It was confirmed that the double-stranded RNA oligo structure produced was annealed by electrophoresis.

[0199] Example 4. Production of nanoparticles formed from improved double helix oligo RNA structure (Mono-HEG-SAMiRNA) and measurement of their size

[0200] The double helix RNA oligo structure (Mono-HEG-SAMiRNA) produced by Example 3 forms a nanoparticle, that is, micelle by a hydrophobic interaction between the hydrophobic materials linked to the end of the double helix RNA oligo (FIG. 1).

[0201] It was confirmed that the corresponding Mono-SAMiRNA-HEG nanoparticles (SAMiRNA) were formed by analyzing the PDI (polydispersity index) of the nanoparticles formed from Mono-HEG-SAMiRNA-hCTGF, Mono-HEG-SAMiRNA-hCyr , Mono-HEG-SAMiRNA-hPlek, Mono-HEG-SAMiRNA-mCTGF, Mono-HEG-SAMiRNA- mCyr, Mono-HEG-SAMiRNA-mPlek and Mono-HEG-SAMiRNA-CONT.

[0202] Example 4-1. Nanoparticle production

[0203] Mono-HEG-SAMiRNA-hCTGF was dissolved in 1.5 mL DPBS (Dulbecco's Phosphate Buffered Saline Solution) at a concentration of 50 μg/mL, the mixture obtained was lyophilized at -75°C and 5mTorr for 48 hours to produce nanoparticle powder, and the nanoparticle powder was dissolved in DPBS which is a solvent for producing homogenized nanoparticles. Nanoparticles formed from Mono-HEG-SAMiRNA-hCyr, Mono-HEG-SAMiRNA-hPlek, Mono-HEG-SAMiRNA-mCTGF, Mono-HEG-SAMiRNA-mCyr, Mono-HEG-SAMiRNA-mPlek, and Mono-HEG-SAMiRNA -CONT were produced by the same method.

[0204] Example 4-2. Measurement of size and polydispersity index (PDI) of nanoparticles

[0205] A nanoparticle size was measured by measuring the zeta potential. The size of the homogenized nanoparticles produced by Example 4-1 was measured by measuring the zeta potential (Nano-ZS, MALVERN, England), under conditions in which a refractive index of the material is 1.459, an absorption index is 0.001, a temperature of a solvent: DPBS is 25°C and the corresponding viscosity and refractive index are 1.0200 and 1.335, respectively. Once the measurement was carried out by a size measurement including repeating 15 times and then repeating 6 times.

[0206] As the PDI value is reduced, the corresponding particles become uniformly distributed, and thus, it could be appreciated that the nanoparticles of the present invention have a significantly uniform size.

[0207] Example 5. Confirmation of inhibition of target gene expression using human target gene-specific siRNA in human fibroblast cell line (MRC-5).

[0208] Human fibroblast (MRC-5) which is a fibroblast cell line was transformed by siRNAs having sequences of SEQ ID NOs: 1 to 10, 101 to 110, 201 to 210 and 601 produced by Example 1 as the strand sense, and the expression aspect of the target gene was analyzed in a transformed fibroblast cell line (MRC-5).

[0209] Example 5-1. Human fibroblast cell line culture

[0210] A human fibroblast cell line (MRC-5) obtained from the Korean Cell line bank (KCLB) was cultured in RPMI-1640 culture medium (GIBCO/Invitrogen, USA, 10% (v/v) fetal serum bovine, penicillin 100 units/ml and 100 μg/mL streptomycin) under conditions of 37°C and 5%(v/v) CO2.

[0211] Example 5-2. Transfection of targeted siRNA into human fibroblast cell line

[0212] 1.8x105 fibroblast cell lines (MRC-5) cultured by Example 5-1 were cultivated in RPMI 1640 medium for 18 hours in the 6-well plate under condition of 37°C and 5% (v/v) CO2, and the medium was removed, and 500 μl of Opti-MEM medium (GIBCO, USA) for each well was dispensed.

[0213] Meanwhile, 3.5 μl of Lipofectamine™ RNAi Max (Invitrogen, USA) was mixed with 246.5 μl of Opti-MEM medium to prepare a mixed solution, and the mixed solution was reacted at room temperature for 5 minutes . siRNA solutions each having a final concentration of 5 or 20 nM were prepared by adding 5 or 20 μl of siRNAs (1 pmole/μl) having sequences from SEQ ID NOs: 1 to 10, 101 to 110, 201 to 210, and 601 by Example 1, as sense tape, in 230 μl of Opti-MEM medium. The Lipofectamine™ RNAi Max mixture and siRNA solution were mixed and reacted at room temperature for 15 minutes, thereby preparing a solution for transfection.

[0214] Then, 500 μl of each transfection solution was dispensed into each well of the tumor cell line containing Opti-MEM dispensed here, and cultured for 6 hours, and the Opti-MEM medium was removed. Here, 1 ml of RPMI 1640 culture medium was dispensed into it and cultivated under the condition of 37°C and 5%(v/v) CO2 for 24 hours.

[0215] Example 5-3. Quantitative analysis of target gene mRNA

[0216] cDNA was produced by extracting total RNA from the cell line transfected by Example 5-2, and an amount of target gene mRNA expression was subjected to relative quantification by real-time PCR.

[0217] Example 5-3-1. Separation of RNA from the transfected cell and production of cDNA

[0218] Total RNA was extracted from the transfected cell line of Example 5-2 by the RNA extraction kit (AccuPrep Cell total RNA extraction kit, BIONEER, Korea), and the extracted RNA was used to produce cDNA by transcriptase reverse RNA (AccuPower CycleScript RT Premix/dT20, Bioneer, Korea) according to the following method. Specifically, 1 μg of the extracted RNA per tube was added to AccuPower CycleScript RT Premix/dT20(Bioneer, Korea) contained in 0.25 μl Eppendorf tube, and distilled water treated with DEPC (diethyl pyrocarbonate) was added in order to have a total volume of 20 μl. A process of hybridizing RNA and primers at 30°C for 1 minute by a gene amplifier (MyGenie™ 96 Gradient Thermal Block, BIONEER, Korea) and a process for producing cDNA at 52°C for 4 minutes were repeated 6 times, and then, an amplification reaction was completed by inactivating the enzyme at 90°C for 5 minutes.

[0219] Example 5-3-2. Relative quantitative analysis of target gene mRNA

[0220] A relative amount of respiratory disease-related gene mRNA was quantified by the following method through real-time PCR with the cDNA produced by Example 5-3-1 as a template. cDNA produced by Example 5-3-1 was diluted 5-fold with distilled water in each well of the 96-well plate. Then, 3 μl of the diluted cDNA, 25 μl of 2 x GreenStar™ PCR Master Mix (BIONEER, Korea), 19 μl of distilled water, and 3μ^' of qPCR primers (Table 2; F and R each having 10 pmole /μl; BIONEER, Korea) were added to them to prepare each mixed solution. Meanwhile, RPL13A (ribosomal protein L13a) which is a housekeeping gene (hereinafter referred to as HK gene) was determined as a standard gene to normalize the expression amount of target gene mRNA. The following reaction was performed in a 96-well plate containing Exicycler™96 Real-Time Quantitative Thermal Block mixture (BIONEER, Korea). After the reaction at 95°C for 15 minutes to activate the enzyme and remove a secondary structure from the cDNA, four processes, including a denaturation process at 94°C for 30 seconds, an annealing process at 58°C for 30 seconds, an extension process at 72°C for 30 seconds, and a SYBR green scanning process were repeated 42 times, and final extension was performed at 72°C for 3 minutes. Then, the temperature was maintained at 55°C for 1 minute, and a melting curve was analyzed from 55°C to 95°C. After the PCR was completed, for the Ct value (threshold cycle) for each of the target gene obtained, Ct values of the target gene corrected by the GAPDH gene were calculated, and then the difference (ΔCt) was obtained using a test group treated with siRNA (SEQ ID NO: 601, siCONT) having a control sequence preventing inhibition of gene expression, as a control group.

[0221] The amount of expression of the target gene of the cell treated with the siRNA specific for CTGF (Homo sapiens) (having sequence ID SEQ NOs: 1 to 10 as the sense strand) was relatively quantified using the ΔCt value and calculation formula 2 (-ΔCt)x 100 (FIG. 2A). Furthermore, the amount of target gene expression of the cell treated with the Cyr61 (Homo sapiens)-specific siRNA (having sequence ID SEQ NOs: 101 to 110 as the sense strand) was relatively quantified (FIG. 2B), and the The amount of expression of the target gene of the cell treated with the siRNA specific for Plekho1 (Homo sapiens) (having sequence ID SEQ NOs: 201 to 210 as the sense strand) was relatively quantified (FIG. 2C). As a result, it can be confirmed that the various types of siRNA of the present invention showed high level of inhibition of target gene expression. Furthermore, in order to select siRNA with high efficiency, siRNAs having ID sequences SEQ NOs: 1, 3, 4, 8, 9, 10, 102, 104, 105, 106, 107, 108, 109, 204, 206, 207, 208, 209 and 210, in which the amount of mRNA expression for each gene at a concentration of 5 nM is significantly decreased, as sense strand, were selected.

[0222] Table 2 qPCR primer sequences (m, Mus musculus; h, Homo sapiens. F, forward primer; R - reverse primer)

[0223] Example 6. Selection of human target gene-specific siRNA with high efficiency in human fibroblast cell line (MRC-5)

[0224] The human fibroblast cell line (MRC-5) was transformed using siRNAs having sequences from SEQ ID NOs: 1, 3, 4, 8, 9, 10, 102, 104, 105, 106, 107, 108, 109, 204, 206, 207, 208, 209, 210 and 601 selected by Example 5-3-2, as the sense strand, and the target gene expression aspect was analyzed in the transformed fibroblast cell line (MRC-5 ) to select siRNA with high efficiency.

[0225] Example 6-1. Human fibroblast cell line culture

[0226] The human fibroblast cell line (MRC-5) obtained from the Korean Cell lineage bank (KCLB, Korea) was cultured under the same condition as Example 5-1.

[0227] Example 6-2. Transfection of targeted siRNA into human fibroblast cell line

[0228] 1.8x105 fibroblast cell lines (MRC-5) cultivated by Example 6-1 were cultivated in RPMI 1640 medium for 18 hours in the 6-well plate under condition of 37°C and 5% (v/v) CO2, and the medium was removed, and 500 μl of Opti-MEM medium (GIBCO, USA) for each well was dispensed.

[0229] Meanwhile, 3.5 μl of Lipofectamine™ RNAi Max (Invitrogen, USA) was mixed with 246.5 μl of Opti-MEM medium to prepare a mixed solution, and the mixed solution was reacted at room temperature for 5 minutes . siRNA solutions each having a final concentration of 0.2 or 1 nM were prepared by adding 0.2 or 1 μl of siRNAs (1 pmole/μl) having sequences of SEQ ID NOs: 1, 3, 4, 8, 9, 10, 102, 104, 105, 106, 107, 108, 109, 204, 206, 207, 208, 209, 210 and 601 by Example 1, as the sense tape, at 230 μl of Opti-MEM medium. The Lipofectamine™ RNAi Max mixture and siRNA solution were mixed and reacted at room temperature for 15 minutes, thereby preparing a solution for transfection.

[0230] Then, 500 μl of each transfection solution was dispensed into each well of the tumor cell line containing Opti-MEM dispensed here, and cultured for 6 hours, and the Opti-MEM medium was removed. Here, 1 ml of RPMI 1640 culture medium was dispensed into it and cultivated under the condition of 37°C and 5%(v/v) CO2 for 24 hours.

[0231] Example 6-3. Quantitative analysis of target gene mRNA

[0232] cDNA was produced by extracting total RNA from the cell line transfected by Example 6-2 through the same method as Example 4-3, and an amount of mRNA expression of the target gene was subjected to relative quantification by time-lapse PCR real. The amount of inhibition of target gene expression according to low-concentration siRNA treatment was observed to clearly confirm each siRNA efficacy, and it was confirmed that siRNAs having SEQ ID sequence NOs: 8, 107 and 206 of the sense strand showed a relatively high level of inhibition for target gene expression even at significantly lower concentrations (FIG. 3).

[0233] Example 7. Confirmation of inhibition of target gene expression using human target gene-specific siRNA in human lung cancer cell line (A549).

[0234] The human lung cancer cell line (A549) which is a lung tumor cell line was transformed by siRNAs having sequences of SEQ ID NOs: 35, 42, 59, 602, 603, 604, 124, 153, 166, 187, 197, 212, 218, 221, 223 and 601 produced by Example 1 as the sense strand, and the expression aspect of the target gene was analyzed in the transformed lung cancer cell line (A549).

[0235] Example 7-1. Human lung cancer cell line culture

[0236] A human lung cancer cell line (A549) obtained from American Type Culture Collection (ATCC) was cultured in DMEM culture medium (GIBCO/Invitrogen, USA, 10% (v/v) fetal bovine serum, penicillin 100 units/ml and 100 μg/mL streptomycin) under conditions of 37°C and 5%(v/v) CO2.

[0237] Example 7-2. Transfection of targeted siRNA into human lung cancer cell line

[0238] 1.2x105 lung cancer cell lines (A549) cultured by Example 7-1 were cultivated in DMEM 1640 medium for 18 hours in the 6-well plate under condition of 37°C and 5% (v/v) CO2, and the medium was removed, and 500 μl of Opti-MEM medium (GIBCO, USA) for each well was dispensed.

[0239] Meanwhile, 3.5 μl of Lipofectamine™ RNAi Max (Invitrogen, USA) was mixed with 246.5 μl of Opti-MEM medium to prepare a mixed solution, and the mixed solution was reacted at room temperature for 5 minutes . siRNA solutions each having a final concentration of 5nM, 1nM, 0.5nM, 0.2nM or 0.04nM were prepared by adding 1 μl of siRNAs (1 pmole/μl) having sequences of SEQ ID NOs: 35, 42 , 59, 602, 603, 604, 124, 153, 166, 187, 197, 212, 218, 221 and 223 by Example 1, as sense tape, at 230 μl of Opti-MEM medium. The Lipofectamine™ RNAi Max mixture and siRNA solution were mixed and reacted at room temperature for 15 minutes, thereby preparing a solution for transfection.

[0240] Then, 500 μl of each transfection solution was dispensed into each well of the tumor cell line containing Opti-MEM dispensed here, and cultured for 6 hours, and the Opti-MEM medium was removed. Here, 1 ml of RPMI 1640 culture medium was dispensed into it and cultured under the condition of 37°C and 5%(v/v) CO2 for 24 hours.

[0241] Example 7-3. Quantitative analysis of target gene mRNA

[0242] cDNA was produced by extracting total RNA from the cell line transfected by Example 7-2, and an amount of target gene mRNA expression was subjected to relative quantification by real-time PCR.

[0243] Example 7-3-1. Separation of RNA from the transfected cell and production of cDNA

[0244] Total RNA was extracted from the transfected cell line of Example 5-2 by the RNA extraction kit (AccuPrep Cell total RNA extraction kit, BIONEER, Korea), and the extracted RNA was used to produce cDNA by transcriptase reverse RNA (AccuPower CycleScript RT Premix/dT20, Bioneer, Korea) according to the following method. Specifically, 1 μg of the extracted RNA per tube was added to AccuPower CycleScript RT Premix/dT20(Bioneer, Korea) contained in 0.25 μl Eppendorf tube, and distilled water treated with DEPC (diethyl pyrocarbonate) was added in order to have a total volume of 20 μl. A process of hybridizing RNA and primers at 30°C for 1 minute by a gene amplifier (MyGenie™ 96 Gradient Thermal Block, BIONEER, Korea) and a process for producing cDNA at 52°C for 4 minutes were repeated 6 times, and then, an amplification reaction was completed by inactivating the enzyme at 90°C for 5 minutes.

[0245] Example 7-3-2. Relative quantitative analysis of target gene mRNA

[0246] A relative amount of respiratory disease-related gene mRNA was quantified by the following method via real-time PCR with the cDNA produced by Example 7-3-1 as a template. cDNA produced by Example 6-3-1 was diluted 5-fold with distilled water in each well of the 96-well plate. Then, 3 μl of the diluted cDNA, 25 μl of 2 x GreenStar™ PCR Master Mix (BIONEER, Korea), 19 μl of distilled water, and 3 μl of qPCR primers (Table 2; F and R each having 10 pmole/ μl; BIONEER, Korea) were added to them to prepare each mixed solution. Meanwhile, RPL13A (ribosomal protein L13a) which is a housekeeping gene (hereinafter referred to as HK gene) was determined as a standard gene to normalize the expression amount of target gene mRNA. The following reaction was performed in a 96-well plate containing Exicycler™96 Real-Time Quantitative Thermal Block mixture (BIONEER, Korea). After the reaction at 95°C for 15 minutes to activate the enzyme and remove a secondary structure from the cDNA, four processes, including a denaturation process at 94°C for 30 seconds, an annealing process at 58°C for 30 seconds, an extension process at 72°C for 30 seconds, and a SYBR green scanning process were repeated 42 times, and final extension was performed at 72°C for 3 minutes. Then, the temperature was maintained at 55°C for 1 minute, and a melting curve was analyzed from 55°C to 95°C. After the PCR was completed, for the Ct value (threshold cycle) for each of the target gene obtained, Ct values of the target gene corrected by the GAPDH gene were calculated, and then the difference (ΔCt) was obtained using a test group treated with siRNA (SEQ ID NO: 601, siCONT) having a control sequence preventing inhibition of gene expression, as a control group.

[0247] The amount of expression of the target gene of the cell treated with the siRNA specific for CTGF (Homo sapiens) (having sequence ID SEQ NOs: 132, 42, 59, 602, 603, 604 as the sense strand) was quantified relatively using the ΔCt value and calculation formula 2 (-ΔCt) x 100 (FIG. 4A). Furthermore, the amount of target gene expression from the cell treated with the Cyr61 (Homo sapiens)-specific siRNA (having sequence ID SEQ NOs: 124, 153, 166, 187, and 197 as the sense strand) was relatively quantified ( FIG. 4B), and the amount of target gene expression of the cell treated with the siRNA specific for Plekho1 (Homo sapiens) (having sequence ID SEQ NOs: 212, 218, 221, and 223 as the sense strand) was relatively quantified. (FIG. 4C).

[0248] As a result, it can be confirmed that the various types of siRNA of the present invention showed high level of inhibition of target gene expression. Furthermore, in order to select siRNA with high efficiency, siRNAs having sequences ID SEQ NOs: 42, 59, 602, 124, 153, 187, 197, 212, 218, 221 and 223, wherein the amount of mRNA expression for each gene at 5 nM concentration is significantly decreased, such as sense strand, were selected.

[0249] Example 8. Inhibition of target gene expression in human lung cancer cell line (A549) by nanoparticles (SAMiRNA), formed from double helix oligo polymer structure

[0250] Human lung cancer cell line (A549) was transformed using nanoparticles formed from SAMiRNA LP including siRNA having sequences of SEQ ID NOs: 42, 59, 602, 124, 153, 187, 197, 212, 218, 221 and 223 selected by Example 7-3-2 as the sense strand, and the expression aspect of the target gene was analyzed in the transformed lung cancer cell line (A549).

[0251] Example 8-1. Human lung cancer cell line culture

[0252] Human lung cancer cell line (A549) obtained from American Type Culture Collection (ATCC) was cultured under the same condition as Example 7-1.

[0253] Example 8-2. Transfection of targeted SAMiRNA into human lung cancer cell line

[0254] 1.2x105 lung cancer cell lines (A549) cultured by Example 8-1 were cultivated in RPMI 1640 medium for 18 hours in a 12-well plate under condition of 37°C and 5% (v/v) CO2, and the medium was removed, and the same amount of Opti-MEM medium (GIBCO, USA) for each well was dispensed. 100 μl of Opti-MEM medium and SAMiRNALP and monoSAMiRNALP produced by Example 4-2 were added to DPBS at a concentration of 50 μg, the obtained mixture was lyophilized at -75°C and 5mTorr condition for 48 hours by the same method as in Example 5-1 to produce homogenized nanoparticles. Next, each well of the tumor cell line into which Opti-MEM is dispensed was treated with a transfection solution at a concentration of 200 nM, and cultured at 37°C and 5%(v/v) CO2 to the total of 48 hours.

[0255] Example 8-3. Relative quantitative analysis of target gene mRNA

[0256] cDNA was produced by extracting total RNA from the cell line transfected by Example 8-2 through the same method as Example 6-3, and an amount of mRNA expression of the target gene was subjected to relative quantification by time-lapse PCR real. The amount of inhibition of target gene expression according to low-concentration siRNA treatment was observed to clearly confirm each siRNA efficacy, and it was confirmed that siRNAs having SEQ ID sequence NOs: 42, 59 and 602 of the sense strand showed a relatively high level of inhibition for target gene expression even at significantly lower concentrations (FIG. 5).

[0257] Example 9. Confirmation of inhibition of target gene expression using mouse target gene-specific siRNA in mouse fibroblast cell line (NIH3T3).

[0258] Mouse fibroblast (NIH3T3) which is a fibroblast cell line was transformed by siRNAs having sequences of SEQ ID NOs: 301 to 330, 401 to 430, 501 to 530 and 601 produced by Example 1 as the sense strand , and the expression aspect of the target gene was analyzed in transformed fibroblast cell line (NIH3T3).

[0259] Example 9-1. Mouse fibroblast cell line culture

[0260] A mouse fibroblast cell line (NIH3T3) obtained from American Type Culture Collection (ATCC) was cultured in RPMI-1640 culture medium (GIBCO/Invitrogen, USA, 10% (v/v) fetal bovine serum, penicillin 100 units/ml and 100 μg/mL streptomycin) under conditions of 37°C and 5%(v/v) CO2.

[0261] Example 9-2. Transfection of targeted siRNA into mouse fibroblast cell line

[0262] 1x105 fibroblast cell lines (NIH3T3) cultivated by Example 9-1 were cultivated in RPMI 1640 medium for 18 hours in the 12-well plate under condition of 37°C and 5% (v/v) CO2, and the Medium was removed, and 500 μl of Opti-MEM medium (GIBCO, USA) to each well was dispensed.

[0263] Meanwhile, 1.5 μl of Lipofectamine™ RNAi Max (Invitrogen, USA) was mixed with 248.5 μl of Opti-MEM medium to prepare a mixed solution, and the mixed solution was reacted at room temperature for 5 minutes . 5 or 20 μl of siRNAs (1 pmole/μl) having sequences of ID SEQ NOs: 301 to 330, 401 to 430, 501 to 530 and 601 produced by Example 1, as sense strand, were added to 230 μl of Opti-medium. MEM, thus preparing siRNA solutions each having a final concentration of 5 or 20 nM. The Lipofectamine™ RNAi Max mixture and siRNA solution were mixed and reacted at room temperature for 20 minutes, thereby preparing a solution for transfection.

[0264] Then, 500 μl of each transfection solution was dispensed into each well of the tumor cell line containing Opti-MEM dispensed here, and cultured for 6 hours, and the Opti-MEM medium was removed. Here, 1 ml of RPMI 1640 culture medium was dispensed into it and cultivated under the condition of 37°C and 5%(v/v) CO2 for 24 hours.

[0265] Example 9-3. Quantitative analysis of target gene mRNA

[0266] cDNA was produced by extracting total RNA from the cell line transfected by Example 9-2 through the same method as Example 5-3, and an amount of mRNA expression of the target gene was subjected to relative quantification by time-lapse PCR real.

[0267] The amount of expression of the target gene of the cell treated with the siRNA specific for CTGF (Mus musculus) (having sequence ID SEQ NOs: 301 to 330 as the sense strand) was relatively quantified (FIG. 6A). Furthermore, the amount of expression of the target gene of the cell treated with the Cyr61 (Mus musculus)-specific siRNA (having sequence ID SEQ NOs: 401 to 430 as the sense strand) was relatively quantified (FIG. 6B), and the The amount of expression of the target gene of the cell treated with the siRNA specific for Plekho1 (Mus musculus) (having sequence ID SEQ NOs: 501 to 530 as the sense strand) was relatively quantified (FIG. 6C).

[0268] As a result, it can be confirmed that the various types of siRNA of the present invention showed high level of inhibition of target gene expression. Furthermore, in order to select siRNA with high efficiency, siRNAs having sequences of ID SEQ NOs: 301, 302, 303, 305, 306, 307, 309, 317, 323 or 329 in which amount of mRNA expression for CTGF (Mus musculus) at a concentration of 20 nM is significantly decreased, like the sense strand, siRNA having sequence ID SEQ NO: 409, 410, 415, 417, 418, 420, 422, 424, 427 or 429 in which amount of mRNA expression for Cyr61 (Mus musculus) at a concentration of 20 nM is significantly decreased, as the sense strand, were selected, and siRNA having sequence ID SEQ NO: 504, 505, 506, 507, 514, 515, 522, 523 , 524 or 525 in which the amount of mRNA expression for Plekho1 (Mus musculus) at a concentration of 20 nM is significantly decreased, as sense strand, was selected.

[0269] Furthermore, in order to select siRNA with more preferential efficiency, siRNAs having sequences of SEQ ID NO: 301, 303, 307 or 323 in which the amount of mRNA expression for CTGF (Mus musculus) at a concentration of 5 nM is significantly decreased, such as the sense strand, siRNA having sequence ID SEQ NO: 410, 422, or 424 were selected in which the amount of mRNA expression for Cyr61 (Mus musculus) at a concentration of 5 nM is significantly decreased, as the sense strand, were selected, and siRNA having sequence ID SEQ NO: 507, 515, or 525 in which the amount of mRNA expression for Plekho1 (Mus musculus) at a concentration of 5 nM is significantly decreased, as the sense strand, was selected (FIG. 7).

[0270] Example 10. Selection of mouse target gene-specific siRNA with high efficiency in mouse fibroblast cell line (NIH3T3)

[0271] The target gene expression aspect was analyzed in mouse fibroblast cell line (NIH3T3) using siRNAs having sequences of SEQ ID NOs: 301, 303, 307, 323, 410, 422, 424, 507, 515, 525 and 601 selected by Example 9-3, as the sense strand, to select siRNA with high efficiency.

[0272] Example 10-1. Mouse fibroblast cell line culture

[0273] Mouse fibroblast cell line (NIH3T3) obtained from American Type Culture Collection (ATCC) was cultured under the same condition as Example 9-1.

[0274] Example 10-2. Transfection of targeted siRNA into mouse fibroblast cell line

[0275] 1x105 fibroblast cell lines (NIH3T3) cultivated by Example 10-1 were cultivated in RPMI 1640 medium for 18 hours in the 12-well plate under condition of 37°C and 5% (v/v) CO2, and the Medium was removed, and 500 μl of Opti-MEM medium (GIBCO, USA) to each well was dispensed.

[0276] Meanwhile, 1.5 μl of Lipofectamine™ RNAi Max (Invitrogen, USA) was mixed with 248.5 μl of Opti-MEM medium to prepare a mixed solution, and the mixed solution was reacted at room temperature for 5 minutes . 0.2, 1 or 5 μl of siRNAs (1 pmole/μl) having sequences of ID SEQ NOs: 301, 303, 307, 323, 410, 422, 424, 507, 515, 525 and 601 produced by Example 1, as sense strand, were added to 230 μl of Opti-MEM medium, thus preparing siRNA solutions each having a final concentration of 0.2, 1 or 5 nM. The Lipofectamine™ RNAi Max mixture and siRNA solution were mixed and reacted at room temperature for 20 minutes, thereby preparing a solution for transfection.

[0277] Then, 500 μl of each transfection solution was dispensed into each well of the tumor cell line containing Opti-MEM dispensed here, and cultured for 6 hours, and the Opti-MEM medium was removed. Here, 1 ml of RPMI 1640 culture medium was dispensed into it and cultured under the condition of 37°C and 5%(v/v) CO2 for 24 hours.

[0278] Example 10-3. Quantitative analysis of target gene mRNA

[0279] cDNA was produced by extracting total RNA from the cell line transfected by Example 10-2 through the same method as Example 5-3, and an amount of mRNA expression of the target gene was subjected to relative quantification by time-lapse PCR real. The amount of target gene expression of the cell treated with the CTGF (Mus musculus)-specific siRNA (having sequence ID SEQ NOs: 301, 303, 307, and 323 as the sense strand) was relatively quantified (FIG. 8A). Furthermore, the amount of target gene expression of the cell treated with the Cyr61 (Mus musculus)-specific siRNA (having sequence ID SEQ NOs: 410, 422 and 424, as the sense strand) was relatively quantified (FIG. 8B). , and the amount of expression of the target gene of the cell treated with the siRNA specific for Plekho1 (Mus musculus) (having sequence ID SEQ NOs: 507, 515, and 525 as the sense strand) was relatively quantified (FIG. 8C).

[0280] As a result, it was confirmed that each target gene-specific siRNA inhibits the expression of the target gene in a concentration-dependent manner, and it was confirmed that siRNAs having sequences of ID SEQ NOs: 307, 424 and 525 of the sense strand showed a level relatively high inhibition for target gene expression even at significantly lower concentrations, to select siRNA with high efficiency.

[0281] Example 11. Confirmation of inhibition of target gene expression using human target gene-specific siRNA in mouse fibroblast cell line (NIH3T3).

[0282] Since biopharmaceuticals have species-specific sites of action such as protein structure or gene sequence, the identity of the treatment drug is significantly important to ensure efficiency in the biopharmaceutical development of new drugs. The gene sequence homology between the human target gene-specific siRNA and the mouse target gene-specific siRNA designed in Example 1 was analyzed to select siRNA sequences that can confirm the effect of inhibiting target gene expression on mouse fibroblast cell.

[0283] The selected siRNA sequences are siRNAs having sequences of ID SEQ NOs: 4, 5, 6, 8, 9, 102, 104, 105, 107, 108, 109, 202, 204, 206, 207, 208 and 209 , such as the sense strand, which are the human target gene-specific siRNAs produced by Example 1, siRNAs having sequences of SEQ ID NOs: 307, 424 and 525, such as the sense strand, which are the mouse target gene-specific siRNAs , and siRNA having sequence ID SEQ NO: 601, as sense strand, which is a control group. The target gene expression aspect was analyzed in mouse fibroblast cell line (NIH3T3) using these selected siRNAs, and their efficacy was confirmed in the mouse cell of siRNA designed based on the human gene.

[0284] Example 11-1. Mouse fibroblast cell line culture

[0285] Mouse fibroblast cell line (NIH3T3) obtained from American Type Culture Collection (ATCC) was cultured under the same condition as Example 9-1.

[0286] Example 11-2. Transfection of targeted siRNA into mouse fibroblast cell line

[0287] 1.8x105 fibroblast cell lines (NIH3T3) cultivated by Example 11-1 were cultivated in RPMI 1640 medium for 18 hours in the 6-well plate under condition of 37°C and 5% (v/v) CO2, and the medium was removed, and 500 μl of Opti-MEM medium (GIBCO, USA) to each well was dispensed.

[0288] Meanwhile, 3.5 μl of Lipofectamine™ RNAi Max (Invitrogen, USA) was mixed with 246.5 μl of Opti-MEM medium to prepare a mixed solution, and the mixed solution was reacted at room temperature for 5 minutes . siRNA solutions each having a final concentration of 5 or 20 nM were prepared by adding 5 or 20 μl of siRNAs (1 pmole/μl) having sequences of SEQ ID NOs: 4, 5, 6, 8, 9, 102, 104, 105, 107, 108, 109, 202, 204, 206, 207, 208, 209, 307, 424, 525 and 601 produced by Example 1, as sense tape, at 230% of Opti-MEM medium. The Lipofectamine™ RNAi Max mixture and siRNA solution were mixed and reacted at room temperature for 15 minutes, thereby preparing a solution for transfection.

[0289] Then, 500 μl of each transfection solution was dispensed into each well of the tumor cell line containing Opti-MEM dispensed here, and cultured for 6 hours, and the Opti-MEM medium was removed. Here, 1 ml of RPMI 1640 culture medium was dispensed into it and cultivated under the condition of 37°C and 5%(v/v) CO2 for 24 hours.

[0290] Example 11-3. Quantitative analysis of target gene mRNA

[0291] cDNA was produced by extracting total RNA from the cell line transfected by Example 11-2 through the same method as Example 5-3, and an amount of target gene mRNA expression was subjected to relative quantification by time-lapse PCR real. The amount of target gene expression of the cell treated with the siRNA specific for CTGF (Mus musculus) (SEQ ID NO: 307) or treated with the siRNA specific for CTGF (Homo sapiens) (SEQ ID NO: 4, 5, 6, 8 or 9) was relatively quantified (FIG. 9A). The amount of target gene expression from the cell treated with Cyr61-specific siRNA (Mus musculus) (having a sequence of SEQ ID NO: 424 as the sense strand) or treated with Cyr61-specific siRNA (Homo sapiens) (having a sequence of SEQ ID NO: 102, 104, 105, 107, 108 or 109 as the sense strand) was relatively quantified (FIG. 9B), and the amount of target gene expression of the cell treated with the siRNA specific for Plekho1 (Mus musculus ) (having a sequence of SEQ ID NO: 525 as the sense strand) or treated with siRNA specific for Plekho1 (Homo sapiens) (having a sequence of SEQ ID NO: 202, 204, 206, 207, 208 or 209 as the sense strand ) was relatively quantified (FIG. 9C).

[0292] As a result, it was confirmed that each human target gene-specific siRNA inhibits the expression of the target gene according to sequence homology, and it was confirmed that siRNAs having sequences of ID SEQ NOs: 6, 8, 102, 104 , 105, 204, 207 and 208 as the sense strand showed a relatively high level of inhibition for target gene expression at 20nM and among these, IC50(inhibition concentration 50%) was less than 20nM in siRNAs having SEQ ID sequences NOs: 6, 102 and 207 even in mouse cell lines. Therefore, it was confirmed that the relatively high level of inhibition for target gene expression was maintained even at low concentration, that is, these siRNAs had high efficiency (FIG. 10).

ADVANTAGEOUS EFFECTS

[0293] The siRNA specific for CTGF, Cyr61 or Plekho1 according to the present invention, the double-stranded RNA oligo structure containing the siRNA, and the pharmaceutical composition containing the double-stranded RNA oligo structure for treatment, are capable of inhibit the expression of CTGF, Cyr61 or Plekho1 at a high efficiency without side effects to provide treatment effects for respiratory diseases, particularly, idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease (COPD), which can be significantly usefully used in the treatment of respiratory diseases , in which there is no appropriate therapeutic agent at this time, particularly idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease (COPD).

[0294] From the foregoing, it will be understood by those skilled in the art to which the present invention belongs that the present invention can be carried out in other concrete embodiments without altering the technical spirit or essential characteristic thereof. In this regard, it should be understood that the above-mentioned examples are illustrative in all respects, but not limited. The scope of the present invention is to be construed to include the meaning and scope of the appended claims, and all changes and modified forms which are derived from the equivalent concept thereof, rather than the detailed description.

Claims (12)

1. Double helix RNA oligo structure, characterized by the fact that it is represented by the following Structural Formula (1): Structural Formula 1 AXRYB wherein A is a hydrophilic material, B is a hydrophobic material, X and Y are each independently a simple covalent bond or a ligand-mediated covalent bond, and R is CTGF-specific siRNA, and where the hydrophilic material has its structure represented by the following Structural Formula (5) or Structural Formula (6): Structural Formula 5 (A'm -J)n Structural formula 6 (J-A'm)n where A' is a monomer of hydrophilic material, J is a linker to connect between m monomers of hydrophilic material, or a linker to connect between m monomers of hydrophilic materials with siRNA, m is an integer from 1 to 15, n is an integer from 1 to 10, the hydrophilic material monomer A' being a compound selected from the group consisting of compounds (1) to (3), and the ligand J being selected from the group consisting of PO3-, SO3 and CO2. 2. Structure, according to claim 1, characterized by the fact that the structure comprising the double helix RNA oligo represented by the following Structural Formula (2): Structural Formula 2 A-X-S-Y-B AS where S is the sense strand of the siRNA, according to of claim 1, and AS is an antisense tape, according to claim 1, A, B, X and Y are the same as being defined in claim 1. 3. Structure, according to claim 2, characterized by the fact that the structure comprising the double helix RNA oligo represented by the following Structural Formula (3) or Structural Formula (4): Structural Formula 3 A-X-5' S 3'- Y-B AS Structural formula 4 A-X-3' S 5'-Y-B AS wherein A, B, X, Y S and AS are the same as defined in claim 2, and 5' and 3' mean 5' end and 3' end of the siRNA sense strand. 4. Structure according to any one of claims 1 characterized by the fact that the hydrophilic material has a molecular weight of 200 to 10,000, said hydrophilic material being any one selected from the group consisting of polyethylene glycol, polyvinyl pyrrolidone and polyoxazoline. 5. Structure according to claim 1, characterized by the fact that the CTGF-specific siRNA is siRNA comprising a selected sense strand selected from the group consisting of ID SEQ NOs: 1 to 100 and 301 to 400 and an anti-sense strand. complementary sense to said sense tape. 6. Structure according to any one of claims 1 to 4, characterized by the fact that the hydrophobic material has a molecular weight of 250 to 1,000, being selected from the group consisting of a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, saturated or unsaturated C12 to C50 hydrocarbon, diacylphosphatidylcholine, fatty acid, phospholipid and lipopolyamine, where steroid derivative is selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholesteryl amine and wherein the glyceride derivative is selected from the group consisting of mono-, di- and tri-glyceride. 7. Structure according to any one of claims 1 to 6, characterized by the fact that the covalent bond represented by X or Y is a non-degradable bond or a degradable bond, where the non-degradable bond is an amide bond or a bond of phosphorylation and where the degradable bond is a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond or an enzymatically degradable bond. 8. Structure according to any one of claims 1 to 7, further characterized by the fact that it further comprises a ligand linked to the hydrophilic material, which is specifically linked to a receptor that promotes internalization of the target cell with receptor-mediated endocytosis (RME), where the ligand is selected from the group consisting of a target receptor-specific antibody, aptamer, peptide, N-acetyl galactosamine (NAG), glucose, and mannose. 9. Structure according to any one of claims 1 to 14, characterized by the fact that it further comprises an amine or polyhistidine group introduced at the distal end linked with the siRNA in the hydrophilic material, where the amine or polyhistidine group is linked to the hydrophilic material or hydrophilic block with more than one ligand, the amine group being selected from the group consisting of tertiary amine groups and where the polyhistidine comprises 3 to 10 histidines. 10. Nanoparticles characterized by the fact that it comprises the structure according to any one of claims 1 to 9, where the structure comprising the double-stranded RNA oligo containing siRNAs with different sequences is mixed into the nanoparticles. 11. Pharmaceutical composition, characterized by the fact that it comprises the structure according to any one of claims 1 to 9, or the nanoparticles, according to claim 10, as an active ingredient, where the composition is intended for the prevention or treatment of respiratory diseases said to be respiratory diseases being selected from the group consisting of asthma, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute or chronic bronchitis, allergic rhinitis, cough and phlegm, acute lower respiratory tract infection, bronchitis, bronchiolitis, acute upper respiratory tract infection, pharyngitis, tonsillitis and laryngitis. 12. Lyophilized formulation characterized by the fact that it comprises the pharmaceutical composition, according to claim 11.