Composition for administering a double-stranded oligonucleotide structure using an ultrasonic nebulizer for the prevention or treatment of respiratory viral infections, including COVID-19, pulmonary fibrosis caused by viral infection, or respiratory disease

Abstract

The present invention relates to a composition for administering double-stranded oligonucleotide structures using an ultrasonic nebulizer. This method allows the double-stranded oligonucleotides of the present invention, which form 90 nm neutrally charged self-assembled nanoparticles in aqueous solution, to maintain the same concentration, molecular weight, purity, nanoparticle size, and osmolality as the material in undiluted solution. In addition, the method can maintain target gene suppression ability without cytotoxicity and can specifically deliver drugs to the nasal cavity and lungs, so the present invention can be used to prevent or treat respiratory virus infections, including COVID-19, pulmonary fibrosis caused by viral infection, or respiratory diseases.

Description

[Technical field]

[0001] The present invention relates to a composition for administering a double-stranded oligonucleotide structure using an ultrasonic nebulizer, in particular, to a composition for administering a double-stranded oligonucleotide structure used in the prevention or treatment of respiratory viral infections, including COVID-19, pulmonary fibrosis caused by viral infection, or respiratory diseases, using an ultrasonic nebulizer. [Background technology]

[0002] Drug delivery by inhalation is a drug delivery method that is easy to use, allows drugs to have the desired effect even with a small drug dose, may avoid or minimize systemic side effects by allowing the drug to reach the target organs, especially the bronchi or lungs, directly, and allows the drug to have a rapid effect. Inhalers are classified into metered dose inhalers (MDIs), dry powder inhalers (DPIs), and nebulizers according to the form of the drug. Metered dose inhalers are devices that release a certain amount of drug when used due to the pressure of gas filled in the drug container, while dry powder inhalers are not filled with any gas and are inhaled by the force of inhalation. Nebulizers are devices that use mechanical vibration to atomize the drug into small liquid particles and then deliver the atomized particles into the patient's body through an inhalation tube by air pressure. Nebulizers have the advantage that they can deliver finely atomized drug particles and their effects can reach the bronchi and alveoli well, so they can be used for respiratory viral infections, interstitial pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, bronchiectasis, bronchitis, pneumonia, emphysema, etc. Nebulizers can be classified into compressor nebulizers and ultrasonic nebulizers according to the method of atomization. In compressor nebulizers, a compressor creates atomized particles by passing air through the drug chamber of the nebulizer, and aerosols are generated through the micro-holes of the vibrating mesh. In ultrasonic nebulizers, aerosols are generated by vibrating the drug while ultrasonic waves pass through the drug chamber. Depending on the device, aerosols of a certain size are generated by passing air or microwaves through the micro-holes of the mesh.

[0003] Various attempts have been made to develop siRNA-based drugs into inhalants.However, these inhalants are mainly dry powder formulations, such as hyaluronic acid-coated liposome spray formulations (lyophilized formulations), nanoparticle formulations containing a mixture of albumin and PLGA (polylactic acid-co-glycolic acid), chitosan polymer formulations, cationic lipid or liposome formulations, and nanoparticle formulations containing cell-penetrating proteins (MPG, TAT, CADY, LAH4).The biggest obstacle to developing inhalants for liposome-based siRNA medicines is the phenomenon that liposome stability is impaired after nebulization, such as liposome fragmentation after nebulization. Although various formulation methods have been attempted to improve the stability of liposomes after nebulization, such as mixing liposomes with cholesterol, phospholipids with high phase transition temperatures, or PEGylation of liposomes, there is still a need to improve the stability of the liposomes themselves and the stability of the drug in the liposomes (Mindaugas Rudokas, Med. Princ. Pract. 2016 March; Vol. 25, p.60). In addition, in the case of cationic lipid or liposome formulations, the particles themselves may be toxic, and in the case of conventional RNAi-based drugs, they may cause toxicity by inducing natural immune responses that nonspecifically activate proinflammatory cytokines and interferon responses, resulting in limited delivery efficiency and drug efficacy (Hasan Uludag, front. Bioeng. Biotechnol. 2020 July; Vol. 8, p.916).

[0004] In 1995, Guo and Kemphues reported that not only sense RNA but also antisense RNA was effective in inhibiting gene expression in the nematode C. elegans, and since then, research has been conducted to identify the reason. In 1998, Fire et al. first reported the phenomenon that gene expression is inhibited by specific degradation of the corresponding mRNA when double-stranded RNA (dsRNA) is injected. This phenomenon was named RNA interference (RNAi). RNAi, the process used to inhibit gene expression, can be extremely effective in inhibiting gene expression in a low-cost and simple manner, and the range of applications of this technology has expanded.

[0005] This technique of inhibiting gene expression can regulate the expression of a specific gene, so that it is possible to eliminate a specific gene related to cancer, genetic disease, etc. at the mRNA level, and can be used as an important tool for developing therapeutic drugs for treating diseases and validating targets. As a conventional technique for inhibiting the expression of a target gene, a technique of introducing a transgene into the target gene has been disclosed. This technique includes a method of introducing a transgene in an antisense direction relative to a promoter, and a method of introducing a transgene in a sense direction relative to a promoter.

[0006] Such RNA therapy, which targets RNA, is a method of eliminating the function of a target gene using oligonucleotides against target RNA, and is different from the conventional method in which therapeutic agents such as antibodies and small molecules mainly target proteins. Methods for targeting RNA are broadly classified into two types: RNAi via double-stranded RNA and antisense oligonucleotides (ASOs). Currently, clinical trials are being planned for various diseases by targeting RNA.

[0007] Antisense oligonucleotides (hereinafter referred to as "ASOs") are short synthetic DNAs designed to bind to target genes according to Watson-Crick base pairing, and can specifically inhibit the expression of a particular nucleotide sequence of a gene. Thus, antisense oligonucleotides have been used to study the role of genes and to develop therapeutic drugs that can treat diseases such as cancer at the molecular level. These ASOs have the advantage of being easily produced by setting various targets for inhibiting gene expression, and research has been conducted on the use of ASOs to inhibit the expression of cancer genes and the proliferation of cancer cells. The process of inhibiting the expression of a specific gene by ASOs is accomplished by binding ASOs to complementary mRNA sequences to induce the activity of RNAase H to remove the mRNA, or by interfering with the formation and progression of ribosome complexes for protein translation. In addition, it has also been reported that ASOs bind to genomic DNA to form triple helix structures, thereby inhibiting gene transcription. Although ASO has the potential as described above, in order to use ASO in clinical practice, it is necessary to improve the stability of ASO against nucleases and to efficiently deliver ASO to target tissues or cells so that it specifically binds to the nucleotide sequence of the target gene. In addition, the secondary and tertiary structures of gene mRNA are important factors for the specific binding of ASO, and the region where the formation of the secondary structure of mRNA is weakened is very favorable for ASO to approach. Therefore, efforts have been made to effectively achieve gene-specific inhibition not only in vitro but also in vivo by systematically analyzing the region where the formation of the secondary structure of mRNA is weakened before synthesizing ASO. These ASOs have the advantage of being more stable than siRNA, a type of RNA, and easily soluble in water and saline. To date, three types of ASOs have been approved by the US Food and Drug Administration (FDA) (Jessica, C., J Postdoc Res, 2016, Vol. 4, p.35-50).

[0008] Since the role of RNA interference (hereinafter referred to as "RNAi") was discovered, it has been found that RNAi acts on sequence-specific mRNA in various types of mammalian cells (Barik, S., J Mol. Med., 2005, Vol. 83, p.764-773). When a long double-stranded RNA is delivered into a cell, the delivered double-stranded RNA is converted into small interfering RNA (hereinafter referred to as "siRNA") processed to 21 to 23 base pairs (bp) by Dicer endonuclease. siRNA binds to an RNA-induced silencing complex (RISC) and inhibits the expression of a target gene in a sequence-specific manner through a process in which the guide (antisense) strand recognizes and degrades the target mRNA. The technique of inhibiting gene expression using siRNA is used to observe the changes that occur by inhibiting the expression of a target gene in a target cell, and is effectively used in research to identify the function of the target gene in the target cell. In particular, the inhibition of the function of a target gene in an infectious virus or cancer cell may be effectively used to develop a treatment method for a target disease. In vitro studies and in vivo studies using experimental animals have shown that siRNA can inhibit the expression of target genes.

[0009] Bertrand et al. reported that siRNA has a better inhibitory effect on mRNA expression in vitro and in vivo than antisense oligonucleotides (ASOs) for the same target gene, and the effect lasts longer. In addition, in terms of the mechanism of action, siRNA regulates the expression of target genes in a sequence-specific manner by complementary binding to target mRNA. Therefore, siRNA is superior to conventional antibody-based drugs or chemical drugs (small molecule drugs) in that it can dramatically expand the scope of application of siRNA (MA Behlke, MOLECULAR THERAPY., 2006, Vol. 13, No. 4, p.664-670).

[0010] Although siRNA has excellent efficacy and can be used in a wide range of applications, in order for siRNA to be developed as a therapeutic agent, the in vivo stability and cellular delivery efficiency of siRNA should be improved so that siRNA can be effectively delivered to target cells. In order to improve in vivo stability and solve the problems associated with siRNA's non-specific natural immune stimulation, siRNA research has been actively attempted by modifying some nucleotides or their backbones of siRNA to obtain nuclease resistance, or by using viral vectors, liposomes, or nanoparticles.

[0011] Delivery systems containing viral vectors such as adenovirus or retrovirus have high transfection efficiency, but are highly immunogenic and carcinogenic. On the other hand, non-viral delivery systems containing nanoparticles have lower cell delivery efficiency than viral delivery systems, but have advantages such as high safety in vivo, target-specific delivery, efficient uptake and internalization of RNAi oligonucleotides into cells or tissues, and low cytotoxicity and immune stimulation. Therefore, non-viral delivery systems are currently considered to be more promising delivery methods than viral delivery systems (Akhtar S, J Clin Invest., 2007 December 3, Vol. 117, No. 12, p.3623-3632).

[0012] Among non-viral delivery systems, the method using nanocarriers is a method in which nanoparticles are formed using various polymers such as liposomes and cationic polymer complexes, and siRNA is loaded into such nanoparticles (i.e., nanocarriers) and delivered to cells. Among the methods using nanocarriers, methods using polymer nanoparticles, polymer micelles, lipoplexes, etc. are frequently used. Among them, lipoplexes are composed of cationic lipids and function to interact with anionic lipids in the endosomes of cells to cause destabilization of the endosomes, thereby enabling intracellular delivery of exosomes.

[0013] In addition, it is known that the efficiency of siRNA in vivo can be improved by conjugating a chemical compound or the like to the terminal region of the passenger (sense) strand of siRNA to improve the pharmacokinetic properties (J. Soutschek, Nature 11, 2004, Vol. 432, No. 7014, p.173-8). In this case, the stability of siRNA varies depending on the properties of the chemical compound conjugated to the end of the sense (passenger) strand or antisense (guide) strand of siRNA. For example, siRNA conjugated with a polymer compound such as polyethylene glycol (PEG) provides a carrier with improved siRNA stability by interacting with the anionic phosphate group of siRNA in the presence of a cationic compound to form a complex (SH Kim, J Control Release, 2008, Vol. 129, No. 2, p.107-16). In particular, micelles composed of polymer complexes are very small and have a very uniform size distribution compared to other drug delivery systems such as microspheres or nanoparticles, and are formed spontaneously. Therefore, these micelles are advantageous in that the quality of the micelle formulation can be easily controlled and its reproducibility can be easily ensured.

[0014] In order to improve the intracellular delivery efficiency of siRNA, a technique has been developed to ensure the stability of siRNA and increase the cell membrane permeability of siRNA by using siRNA conjugates obtained by conjugating a hydrophilic compound (e.g., polyethylene glycol (PEG)) that is a biocompatible polymer to siRNA by simple covalent bond or covalent bond via a linker (Korean Patent No. 883471). However, even if siRNA is chemically modified and conjugated (PEGylated) to polyethylene glycol (PEG), siRNA still has the disadvantage of being less stable in vivo and not easily delivered into target organs. To overcome the above disadvantages, a double-stranded oligo-RNA structure has been developed in which a hydrophilic compound and a hydrophobic compound are bound to an oligonucleotide, particularly a double-stranded oligo-RNA such as siRNA. This structure forms a self-assembled nanoparticle named SAMiRNA (Self Assembled Micelle Inhibitory RNA) by the hydrophobic interaction of the hydrophobic compound (Korean Patent No. 1224828). SAMiRNA technology has the advantage over conventional delivery techniques in that it allows obtaining uniform nanoparticles that are very small in size.

[0015] Specifically, in the SAMiRNA technology, PEG (polyethylene glycol) or HEG (hexaethylene glycol) is used as a hydrophilic compound. PEG, a synthetic polymer, is commonly used to enhance the solubility of medical drugs, especially proteins, and to regulate the pharmacokinetics of drugs. PEG is a polydisperse material, and one batch of polymer consists of various numbers of monomers, and thus shows a molecular weight distribution with a Gaussian curve. In addition, the homogeneity of the material is expressed as a polydispersity index (Mw / Mn). In other words, when PEG has a low molecular weight (3-5 kDa), it has a polydispersity index of about 1.01, and when PEG has a high molecular weight (20 kDa), it has a high polydispersity index of about 1.2, indicating that the homogeneity of PEG decreases as its molecular weight increases. Therefore, when PEG is conjugated to a pharmaceutical drug, there is a disadvantage that it is not easy to demonstrate that the polydispersity of PEG is reflected in the conjugate and, as a result, it is a single material. Due to this drawback, the synthesis and purification process of PEG has been improved to produce materials with low polydispersity index. However, when PEG is conjugated to a low molecular weight compound, problems exist with the polydispersity of the compound, such as the fact that it is not easy to confirm whether conjugation has been achieved smoothly (Francesco MV, DRUG DISCOVERY TODAY, 2005, Vol. 10, No. 21, p.1451-1458).

[0016] Therefore, in recent years, SAMiRNA technology (i.e., self-assembled nanoparticles) has been improved by forming the hydrophilic compound of the double-stranded RNA structure (constituting SAMiRNA) in the form of a basic unit block, each of which contains 1 to 15 monomers with a uniform molecular weight and, if necessary, a linker, so that an appropriate number of blocks are used as needed. As a result, a new type of delivery system technology has been developed that is smaller and has a significantly improved polydispersity compared to the conventional SAMiRNA (trademark) (Korean Patent Registration No. 18162349). As is already known, when siRNA is injected, it is rapidly degraded by various enzymes present in the blood, and therefore its delivery efficiency to the target cells or tissues is insufficient. Therefore, the stability and expression inhibition rate variations due to the difference in the target gene have also appeared in the improved SAMiRNA. Therefore, in order to more stably and effectively inhibit the expression of target genes using SAMiRNA composed of improved self-assembled nanoparticles, the inventors attempted to enhance the expression inhibitory effect of SAMiRNA on target genes and the stability of SAMiRNA by utilizing a double-stranded oligonucleotide having an ASO DNA sequence as a guide (sense) strand and an RNA sequence as a passenger (antisense) sequence.

[0017] However, in the delivery of RNAi drugs, conventional RNAi drug delivery methods are mainly based on powder formulations, which, as mentioned above, have the risk of causing toxicity and non-specific immune response due to particles themselves, and only show limited drug delivery efficiency and drug effect.Therefore, the present inventors have attempted to develop a drug delivery method that can overcome the above limitations, and is particularly suitable for SAMiRNA, based on the structural characteristics of SAMiRNA, and have found that when ultrasonic nebulizer is used among various drug delivery means, SAMiRNA can be effectively delivered to the lungs without cytotoxicity, while showing the same concentration, molecular weight, purity, nanoparticle size and osmolality as stock material (SAMiRNA before nebulization), and maintaining its ability to inhibit target genes, and as a result, the present invention has been completed.

[0018] [Patent documents] Korean Patent No. 1224828 Korean Patent No. 1862349 [Non-patent literature] Mindaugas Rudokas, Med. Princ. Pract., 2016 March, Vol. 25, p.60 Hasan Uludag, Front. Bioeng. Biotechnol., 2020 July, Vol. 8, p.916 Jessica, C., J Postdoc Res., 2016, Vol. 4, p.35-50 MA Behlke, MOLECULAR THERAPY., 2006, Vol. 13, No. 4, p.664-670

[0019] Akhtar S, J Clin Invest., 2007 December 3, Vol. 117, No. 12, p.3623-3632 SH Kim, J Control Release, 2008, Vol. 129, No. 2, p.107-16 Francesco MV, DRUG DISCOVERY TODAY, 2005, Vol. 10, No. 21, p.451-1458 Summary of the Invention

[0020] An object of the present invention is to provide a pharmaceutical composition for specifically and effectively delivering a double-stranded oligonucleotide structure suitable for the prevention or treatment of respiratory viral infection, viral pulmonary fibrosis, or respiratory disease to the bronchi and lungs. In order to achieve the above object, the present invention provides a compound having the following structural formula 1: [Structural formula 1] A-X-R-Y-B

[0021] (wherein A represents a hydrophilic compound, B represents a hydrophobic compound, X and Y each independently represent a simple covalent bond or a covalent bond via a linker, and R represents a double-stranded oligonucleotide).

[0022] The present invention provides a pharmaceutical composition for preventing or treating a respiratory viral infection, viral pulmonary fibrosis, or a respiratory disease, comprising a double-stranded oligonucleotide structure having a structure represented by: [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 shows the results of a comparative analysis of the molecular weight, purity, nanoparticle size, and osmolality of SAMiRNA nanoparticles recovered from an ultrasonic nebulizer and a compressor-type nebulizer. [Diagram 2] FIG. 1 shows the results of evaluating the cytotoxicity of SAMiRNA nanoparticles recovered from an ultrasonic nebulizer. [Diagram 3] FIG. 1 shows the results of an analysis of the target gene expression inhibitory activity of SAMiRNA nanoparticles recovered from an ultrasonic nebulizer. [Figure 4]FIG. 1 shows the results of a comparative analysis of Cy5 fluorescence, molecular weight, purity, nanoparticle size, and osmolality of SAMiRNA-Cy5 nanoparticles recovered from an ultrasonic nebulizer and a compressor-type nebulizer. [Figure 5a] FIG. 1 shows the results of analyzing the weights of organs, including lungs, spleen, liver, and kidneys, taken 24 hours after administration of SAMiRNA-Cy5 nanoparticles to hamsters using an ultrasonic nebulizer. [Figure 5b] A figure showing optical images (left), fluorescence images (center) and fluorescence value analysis results for organs such as the lungs, spleen, liver and kidneys taken 24 hours after administration of SAMiRNA-Cy5 nanoparticles to hamsters using an ultrasonic nebulizer. [Figure 6] Confocal images showing effective delivery and distribution of SAMiRNA-Cy5 nanoparticles in lung tissue taken 24 hours after administration of SAMiRNA-Cy5 nanoparticles to hamsters using an ultrasonic nebulizer. [Figure 7a] Experimental scheme showing tissue harvesting at various time points (1, 24, 48, 96, and 168 hours) after administration of SAMiRNA-Cy5 nanoparticles to mice using an ultrasonic nebulizer. [Figure 7b] Optical and fluorescent images of the lungs, spleen, liver, kidneys and heart taken at various time points (1, 24, 48, 96 and 168 hours) after administration of SAMiRNA-Cy5 nanoparticles to mice using an ultrasonic nebulizer. [Figure 8a] Figure 1 shows the quantitative analysis of fluorescence in nasal and lung tissues taken at various time points (1, 24, 48, 96, and 168 hours) after administration of SAMiRNA-Cy5 nanoparticles to mice using an ultrasonic nebulizer. [Figure 8b] FIG. 1 shows the results of PK analysis of lung tissues taken at various time points (1, 24, 48, 96, and 168 hours) after administration of SAMiRNA-Cy5 nanoparticles to mice using an ultrasonic nebulizer. [Figure 9a]Confocal images showing the delivery and distribution of SAMiRNA-Cy5 in lung tissues harvested at various time points (1, 24, 48, 96, and 168 hours) after administration of SAMiRNA-Cy5 nanoparticles to mice using an ultrasonic nebulizer. [Figure 9b] Confocal images showing the delivery and distribution of SAMiRNA-Cy5 in lung tissue taken 1 hour after administration of SAMiRNA-Cy5 nanoparticles to mice using an ultrasonic nebulizer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description of the Invention and Preferred Embodiments Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Generally, the nomenclature used herein is well known and widely used in the art.

[0025] Among various means for delivering the double-stranded oligonucleotide according to the present invention, ultrasonic nebulizer has been found to be the most suitable inhalation drug delivery means without affecting the physical / chemical properties of the structure.Specifically, the physical properties of the double-stranded oligonucleotide structure according to the present invention that has passed through ultrasonic aerosol inhaler have been analyzed, and it has been confirmed that the concentration, molecular weight, purity, nanoparticle size, and osmolality are maintained at the same level as those of stock material (the structure before passing through ultrasonic nebulizer).However, when using a compressor nebulizer, it has been confirmed that the nanoparticle size, molecular weight, and purity of the structure after passing through compressor nebulizer are maintained at the same level as those of stock material, but the concentration and osmolality of the structure are significantly reduced compared to those of stock material.

[0026] At the same time, the cytotoxicity of the structure administered by ultrasonic nebulizer was evaluated using human nasal epithelial cell line RPMI2650 and human lung cancer cell line A549, and it was found that the structure did not show cytotoxicity even at a high concentration of 50 μM. In addition, in order to evaluate the target gene inhibitory activity of each substance, RPMI2650 cells and A549 cells were treated with each substance at various concentrations and then analyzed, and it was confirmed that each substance after passing through the ultrasonic nebulizer showed the same target inhibitory activity as the substance after passing through the ultrasonic nebulizer.

[0027] Finally, to investigate whether the structure of the present invention can be effectively delivered to the lungs in vivo by ultrasonic nebulizer, SAMiRNA was conjugated with Cy5 and administered to hamsters by ultrasonic nebulizer, and then lung tissue was collected and observed. As a result, it was confirmed that the fluorescence of SAMiRNA-Cy5 was found in most cells constituting the lung tissue, and in particular, the strong fluorescence of SAMiRNA-Cy5 was found in the alveoli and bronchi, suggesting that the substance SAMiRNA can be effectively delivered when administered by ultrasonic nebulizer. In other words, it was confirmed that the optimal administration of the double-stranded oligonucleotide structure according to the present invention and the nanoparticles self-assembled from the structure can be achieved by ultrasonic nebulizer. Thus, the present invention provides a compound of the following structural formula 1: [Structural formula 1] A-X-R-Y-B

[0028] (wherein A represents a hydrophilic compound, B represents a hydrophobic compound, X and Y each independently represent a simple covalent bond or a covalent bond via a linker, and R represents a double-stranded oligonucleotide). The present invention relates to a pharmaceutical composition for preventing or treating a respiratory viral infection, viral pulmonary fibrosis, or a respiratory disease, comprising a double-stranded oligonucleotide structure having a structure represented by: In the present invention, the nebulizer may be an ultrasonic nebulizer.

[0029] In the present invention, a double-stranded oligonucleotide can comprise a sense strand and an antisense strand comprising a sequence complementary to the sense strand. In the present invention, each of the sense strand and the antisense strand may consist of 19 to 31 nucleotides, but is not limited thereto.

[0030] In the present invention, the sense or antisense strand may independently be DNA or RNA and may contain sequences in the form of, for example, RNA / RNA, DNA / DNA, or DNA / RNA hybrids. In the present invention, the sense strand or the antisense strand may contain a chemical modification, The chemical modifications include, but are not limited to, the following chemical modifications: a modification in which the OH group at the 2'-position of the sugar structure of one or more nucleotides is replaced with any one selected from the group consisting of a methyl group (-CH3), a methoxy group (-OCH3), an amine group (-NH2), a fluorine (-F), an -O-2-methoxyethyl group, an -O-propyl group, an -O-2-methylthioethyl group, an -O-3-aminopropyl group, an -O-3-dimethylaminopropyl group, an -O-N-methylacetamide group, and an -O-dimethylamidooxyethyl group; A modification in which oxygen in the sugar structure of a nucleotide is replaced by sulfur; A modification in which the bond between nucleotides is any one bond selected from the group consisting of phosphorothioate bond, boranophosphate bond, and methylphosphonate bond; and Modifications made to PNA (peptide nucleic acid), LNA (locked nucleic acid) or UNA (non-locked nucleic acid); The chemical modification may be one or more selected from the group consisting of:

[0031] In the present invention, chemical modifications can contribute to enhancing in vivo stability, i.e., imparting nuclease resistance, and reducing non-specific immune responses. In the present invention, one or more phosphate groups, preferably 1 to 3 phosphate groups, may be attached to, but is not limited to, the 5' end of the antisense strand.

[0032] The double-stranded oligonucleotide according to the present invention is intended to include all substances having general RNAi activity, and as will be apparent to those skilled in the art, examples of target gene-specific double-stranded oligonucleotides include target gene-specific shRNAs, i.e., the oligonucleotides may be siRNAs, shRNAs or miRNAs. A double-stranded oligonucleotide according to the invention may comprise an overhang at the 3' end of one or both strands comprising one or more unpaired nucleotides.

[0033] The double-stranded oligonucleotide according to the present invention is preferably in the form of a DNA-RNA hybrid, siRNA (short interfering RNA), shRNA (short hairpin RNA) or miRNA (microRNA), but is not limited thereto, and may also include a single-stranded miRNA inhibitor that acts as an antagonist to miRNA. More preferably, the pharmaceutical composition according to the present invention comprises a compound represented by the following structural formula 2: [Structural formula 2] [ka]

[0034] (wherein S and AS represent the sense and antisense strands of the double-stranded oligonucleotide, respectively, and A, B, X, and Y are as defined in Structural Formula 1). The structure may include a structure represented by: More preferably, the pharmaceutical composition according to the present invention comprises a compound represented by the following structural formula 3: [Structural formula 3] [ka] This includes structures represented by:

[0035] In another embodiment, the pharmaceutical composition according to the present invention comprises a compound represented by the following structural formula 3': [Structural formula 3'] [ka] This includes structures represented by:

[0036] In the above structural formulas 3 and 3', A, B, X, Y, S and AS are as defined in structural formula 2, and 5' and 3' represent the 5' and 3' ends of the sense strand, respectively. In the present invention, the molecular weight of the hydrophilic compound may be from 200 to 10,000, but is not limited thereto.

[0037] The hydrophilic compound may be selected from the group consisting of, but not limited to, polyethylene glycol (PEG), polyvinylpyrrolidone, and polyoxazoline. In the present invention, the hydrophilic compound is represented by the following structural formula 4 or structural formula 5: [Structural formula 4] (A' m -J) n [Structural formula 5] (J‐A' m ) n

[0038] (In the formula, A' represents a hydrophilic monomer, J represents a linker that connects m hydrophilic monomers together or connects m hydrophilic monomers to an siRNA, m is an integer ranging from 1 to 15, and n is an integer ranging from 1 to 10; The hydrophilic monomer A' is the following compounds (1) to (3): [Compound (1)] [ka] wherein G is selected from the group consisting of CH2, O, S, and NH; [Compound (2)] [ka] [Compound (3)] [ka] and the linker (J) is a compound selected from the group consisting of PO3 - , SO3 and CO2) The compound can have a structure represented by: When the hydrophilic block is represented by the above structural formula 4 or 5, the double-stranded oligonucleotide structure according to the present invention can be represented by the following structural formula 6 or 7: [Structural formula 6] (A' m -J) n -X-R-Y-B [Structural formula 7] (J‐A' m ) n -X-R-Y-B (wherein X, R, Y and B are as defined above in structural formula 1, and A', J, m and n are as defined above in structural formulas 4 and 5). The compound may have a structure represented by:

[0039] As the hydrophilic monomer (A') of the above structural formulas 4 and 5, one selected from nonionic hydrophilic polymers can be used without restriction as long as it meets the purpose of the present invention. Preferably, a monomer selected from compounds (1) to (3) listed in the following Table 1 is used, and more preferably, a monomer of compound (1) is used. In compound (1), G is preferably selected from O, S, and NH.

[0040] In particular, among hydrophilic monomers, the monomer represented by compound (1) is very suitable for producing the structure according to the present invention, because the monomer has the advantages that various functional groups can be introduced into the monomer, the monomer has good in vivo affinity and excellent biocompatibility, and therefore hardly induces an immune response, the monomer can increase the in vivo stability of the double-stranded oligonucleotide contained in the structure represented by structural formula 6 or 7, and the delivery efficiency of the double-stranded oligonucleotide can be increased.

[0041] [Table 1] [Table 1]

[0042] The total molecular weight of the hydrophilic compounds of structural formulae 4 to 7 is preferably within the range of 1,000 to 2,000. Therefore, for example, when compound (1) in structural formulae 6 and 7 is hexaethylene glycol, that is, a compound in which G is O and m is 6, the molecular weight of the hexaethylene glycol spacer is 344, so that the number of repetitions (n) is preferably 3 to 5. In particular, the present invention relates to the compound (A' m -J) n or (J‐A' m ) nThe present invention is characterized in that the repeating unit of the hydrophilic group (hydrophilic block) represented by the formula (I) can be used in an appropriate number (represented by n) as required. The hydrophilic monomer A and the linker J contained in each hydrophilic block may be the same or different between the hydrophilic blocks. The hydrophilic monomer A and the linker J contained in each hydrophilic block may be the same or different between the hydrophilic blocks. In other words, when three hydrophilic blocks are used (n=3), the hydrophilic monomer of the compound (1), the hydrophilic monomer of the compound (2), and the hydrophilic monomer of the compound (3) may be used in the first block, the second block, and the third block, respectively, to indicate that different monomers may be used in all the hydrophilic blocks. As another example, any one of the hydrophilic monomers of the compounds (1) to (3) may be used in all the hydrophilic blocks. Similarly, the same linker may be used in the hydrophilic blocks as a linker mediating the binding of the hydrophilic monomers, or various linkers may be used in the hydrophilic blocks. In addition, the number of hydrophilic monomers, m, may be the same or different between the hydrophilic blocks. In other words, in the first hydrophilic block, three hydrophilic monomers may be connected (m=3), in the second hydrophilic block, five hydrophilic monomers may be connected (m=5), and in the third hydrophilic block, four hydrophilic monomers may be connected (m=4), indicating that different numbers of hydrophilic monomers may be used within the hydrophilic blocks. As another example, the same number of hydrophilic monomers may be used in all hydrophilic blocks.

[0043] In addition, in the present invention, the linker (J) is -PO3 - It is preferred to select from the group consisting of -, -SO3-, and -CO2-, but is not limited thereto. It will be apparent to those skilled in the art that any linker selected in consideration of the hydrophilic monomer used can be used as long as it is compatible with the objectives of the present invention.

[0044] The hydrophobic compounds (B) of the structural formula 3', the structural formula 6 and the structural formula 7 function to form nanoparticles composed of oligonucleotide structures by hydrophobic interaction. The hydrophobic compounds preferably have a molecular weight of 250 to 1,000, and are selected from the group consisting of steroid derivatives, glyceride derivatives, glycerol ethers, polypropylene glycols, C 12 -C 50 The hydrophobic compound may be any hydrophobic compound selected from the group consisting of, but not limited to, unsaturated or saturated hydrocarbons, diacylphosphatidylcholine, fatty acids, phospholipids, lipopolyamines, lipids, tocopherols, and tocotrienols. It will be obvious to those skilled in the art that any hydrophobic compound can be used as long as it is compatible with the purpose of the present invention.

[0045] The steroid derivative may be selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholesterylamine, and the glyceride derivative may be selected from monoglycerides, diglycerides, and triglycerides, etc., wherein the fatty acid of the glyceride is C 12 -C 50 It is preferable that the fatty acid is an unsaturated or saturated fatty acid of the formula:

[0046] In particular, among the hydrophobic compounds, saturated or unsaturated hydrocarbons or cholesterol are preferably used, because they can be easily bound in the step of synthesizing the double-stranded oligonucleotide structure according to the present invention. Most preferably, 24 Hydrophobic hydrocarbons, particularly those containing disulfide bonds, are used.

[0047] The hydrophobic compound can be attached to the end distal from the hydrophilic compound, and can be attached to any site on the sense or antisense strand of the double-stranded oligonucleotide.

[0048] The hydrophilic or hydrophobic compound in the structural formulas 1-3', 6 and 7 according to the present invention is bound to the double-stranded oligonucleotide by a single covalent bond or a covalent bond (X or Y) via a linker. The linker mediating the covalent bond is not particularly limited as long as it is covalently bound to the hydrophilic or hydrophobic compound at the end of the double-stranded oligonucleotide and provides a bond that can be degraded in a specific environment as necessary. Thus, the linker used in the present invention may be any compound that is bound to activate the double-stranded oligonucleotide and / or the hydrophilic (or hydrophobic) compound in the process of producing the double-stranded oligonucleotide structure of the present invention. The covalent bond may be either a non-degradable bond or a degradable bond. Here, examples of the non-degradable bond include, but are not limited to, an amide bond and a phosphate bond, and examples of the degradable bond include, but are not limited to, a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, and an enzymatically degradable bond.

[0049] Additionally, in constructs comprising double-stranded oligonucleotides according to the invention, amine groups or polyhistidine groups may additionally be introduced at the distal ends of hydrophilic compounds attached to the oligonucleotides in the construct.

[0050] This facilitates intracellular uptake and endosomal escape of carriers containing structures comprising the double-stranded oligonucleotides of the present invention, and, as has been reported, the introduction of amine and polyhistidine groups can be used to facilitate intracellular uptake and endosomal escape of carriers such as quantum dots, dendrimers or liposomes.

[0051] Specifically, it is known that the primary amine group introduced at the end, i.e., on the outside, of the carrier is protonated at biological pH and simultaneously interacts with a negatively charged gene to form a conjugate, and that endosomal escape is promoted by the internal tertiary amine that has a buffering effect at low pH after intracellular uptake, thereby protecting the carrier from degradation by lysosomes (Gene Delivery and Expression Inhibition Using Polymer-Based Hybrid Material, Polymer Sci. Technol., Vol. 23, No. 3, p.254-259).

[0052] In addition, histidine, a non-essential amino acid, has an imidazole ring (pKa=6.04) in its side chain (-R) and is therefore known to have the effect of enhancing buffering capacity in endosomes and lysosomes. Therefore, histidine modification can be used in non-viral gene carriers such as liposomes to enhance the efficiency of endosomal escape (Novel histidine-conjugated galactosylated cationic liposomes for efficient hepatocyte selective gene transfer in human hepatoma HepG2 cells. J. Controlled Release Vol. 118, p.262-270). The amine groups or polyhistidine groups can be connected to the hydrophilic compounds or hydrophilic blocks by one or more linkers.

[0053] When an amine group or a polyhistidine group is introduced into the hydrophilic compound of the double-stranded oligonucleotide structure of the present invention represented by the structural formula 1, the structure can be represented by the following structural formula 8: [Structural formula 8] P‐J1‐J2‐A‐X‐R‐Y‐B

[0054] (wherein A, B, R, X and Y are as defined in Structural Formula 1 above, P is an amine group or a polyhistidine group, and J1 and J2 are linkers, each of which is a simple covalent bond, PO3 - , SO3, CO2, C2‐ 12 (which can be independently selected from, but are not limited to, alkyl, alkenyl, and alkynyl)

[0055] As will be apparent to those skilled in the art, any linker selected in view of the hydrophilic compounds used herein can be used as J1 and J2 as long as the linker is compatible with the purpose of the present invention.

[0056] Preferably, when an amine group is introduced, J2 is a simple covalent bond or PO3 - and J1 is preferably, but not limited to, C6 alkyl.

[0057] In addition, when a polyhistidine group is introduced, J2 in formula 8 can be a simple covalent bond or a PO3 - and J1 is preferably, but not limited to, compound (4). [Compound (4)] [ka]

[0058] In addition, when the hydrophilic compound of the double-stranded oligonucleotide structure represented by structural formula 8 is a hydrophilic block represented by structural formula 5 or 6, and an amine group or a polyhistidine group is introduced therein, the double-stranded oligonucleotide structure is represented by the following structural formula 9 or 10: [Structural formula 9] P‐J1‐J2‐(A' m -J) n -X-R-Y-B [Structural formula 10] P‐J1‐J2‐(J‐A' m ) n -X-R-Y-B

[0059] (wherein X, R, Y, B, A', J, m and n are as defined in structural formula 4 or 5 above, and P, J1 and J2 are as defined in structural formula 8 above). The compound may have a structure represented by:

[0060] In particular, the hydrophilic compounds of structural formula 9 and structural formula 10 are preferably bound to the 3' end of the sense strand of the double-stranded oligonucleotide. In this case, structural formula 8 to ... following structural formula 11 to structural formula 13: [Structural formula 11] [ka] [Structural formula 12] [ka] [Structural formula 13] [ka] (In the above formula, X, R, Y, B, A, A', J, m, n, P, J1 and J2 are as defined in the above structural formulas 8 to 10, and 5' and 3' respectively represent the 5' end and 3' end of the sense strand of the target gene-specific double-stranded oligonucleotide.) It can be equivalent to.

[0061] The amine group that can be introduced in the present invention can be a primary, secondary, or tertiary amine group. In particular, it is preferred to use a primary amine group. The introduced amine group can exist as an amine salt. For example, the salt of a primary amine group can be NH3 + It can exist as.

[0062] In addition, the polyhistidine group that can be introduced in the present invention preferably contains 3 to 10 histidines, more preferably 5 to 8 histidines, and most preferably 6 histidines. In addition to histidines, one or more cysteines may be contained.

[0063] On the other hand, when a targeting moiety is provided to a double-stranded oligonucleotide structure containing a target gene-specific oligonucleotide according to the present invention and to a nanoparticle formed from the structure, the targeting moiety promotes efficient delivery of the structure or nanoparticle to a target cell, so that the structure or nanoparticle can be delivered to a target cell even at a relatively low concentration, and therefore can exhibit a strong target gene expression regulating effect.

[0064] Thus, the present invention provides a double-stranded oligo-RNA structure in which a ligand (L), particularly a ligand having the property of specifically binding to a receptor that enhances internalization into a target cell by receptor-mediated endocytosis (RME), is further bound to a structure represented by any of structural formulas 1-3', 6 and 7. For example, a structure in which a ligand is bound to a double-stranded oligo-RNA structure represented by structural formula 1 can be represented by the following structural formula 14: [Structural formula 14] (L i -Z)-A-X-R-Y-B

[0065] (wherein A, B, X and Y are as defined in structural formula 1 above, L is a ligand having the property of specifically binding to a receptor that enhances internalization into a target cell by receptor-mediated endocytosis (RME), and "i" is an integer ranging from 1 to 5, preferably 1 to 3.) It has a structure represented by:

[0066] The ligands in structural formula 14 can be preferably selected from, but are not limited to, target receptor-specific antibodies, aptamers and peptides with RME properties that enhance internalization into target cells; folates (the term "folate" is generally used interchangeably with folic acid, and as used herein, the term "folate" refers to folates in their natural form or activated in the human body); and chemical compounds, such as hexosamines, such as N-acetylgalactosamine (NAG), and sugars or carbohydrates, such as glucose and mannose. In addition, the hydrophilic compound A of the above formula 14 may be used in the form of a hydrophilic block represented by formula 4 or 5.

[0067] In the present invention, the double-stranded oligonucleotide represented by R (or S and AS) in structural formulas 1 to 3', structural formula 6 and structural formula 7 may be any oligonucleotide that specifically inhibits the expression of a gene selected from the group consisting of amphiregulin, RelA / p65 and SARS-CoV-2, but is not limited thereto. Specifically, the double-stranded oligonucleotide represented by R (or S and AS) may be capable of specifically inhibiting the expression of the amphiregulin gene and may include a sequence that targets amphiregulin. The sequence that targets amphiregulin may include, for example, the sequence of SEQ ID NO: 5 and / or 6. Specifically, the sequence may include the sense sequence of SEQ ID NO: 5 and the antisense sequence of SEQ ID NO: 6.

[0068] The double-stranded oligonucleotide represented by R (or S and AS) may comprise a sequence that targets SARS-CoV-2. The sequence that targets SARS-CoV-2 may comprise, for example, a sequence selected from the group consisting of SEQ ID NOs: 11-30.

[0069] The present invention is suitable for preventing or treating various respiratory diseases, and is particularly suitable as a method for administering a composition for preventing or treating a respiratory viral infection, pulmonary fibrosis caused by a viral infection, or a respiratory disease.

[0070] In the present invention, the respiratory virus may be, but is not limited to, COVID-19. In the present invention, pulmonary fibrosis caused by a viral infection is an example of a sequela caused by a viral infection, and the present invention can be used to administer a composition for preventing or treating pulmonary fibrosis as well as sequelae caused by a viral infection, particularly a respiratory viral infection.

[0071] In the present invention, the respiratory disease may be selected from the group consisting of interstitial lung disease, chronic obstructive pulmonary disease (COPD), pneumonia, asthma, acute and chronic bronchitis, allergic rhinitis, bronchitis, bronchiolitis, pharyngitis, tonsillitis, and laryngitis, but is not limited to these.

[0072] In the present invention, the double-stranded oligonucleotide structure can form self-assembled nanoparticles having a size of 10 to 100 nm and a neutral charge in an aqueous solution for administration. In the present invention, the nanoparticles may be composed of a mixture of double-stranded oligonucleotide structures, including double-stranded oligonucleotides containing different sequences.

[0073] For administration, the composition of the present invention may further comprise one or more pharma- ceutically acceptable carriers in addition to the above-mentioned active ingredient. The pharma-ceutically acceptable carrier must be compatible with the active ingredient, and may be selected from physiological saline, sterile water, Ringer's solution, buffered physiological saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of two or more of them. If necessary, the composition may contain other conventional additives, such as antioxidants, buffers, or bacteriostatic agents. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to the composition to prepare injection preparations such as aqueous solutions, suspensions, and emulsions. In particular, the composition is preferably provided as a lyophilized preparation. For the preparation of the lyophilized preparation, conventional methods known in the field to which the present invention pertains can be used, and stabilizers for lyophilization may also be added. Furthermore, the composition is preferably formulated according to each disease and component by a suitable method known in the art or by a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

[0074] The pharmaceutical composition of the present invention is preferably administered parenterally, and particularly preferably administered to the lungs by inhalation into the bronchi. The dose of the composition of the present invention may vary depending on the patient's body weight, age, sex, health condition and diet, administration period, administration mode, excretion rate, severity of the disease, etc., and can be easily determined by those skilled in the art.

[0075] [Example] The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of describing the present invention in more detail, and the scope of the present invention is not limited by these examples. Therefore, the essential scope of the present invention is defined by the appended claims and their equivalents. Example 1. Synthesis of double-stranded oligonucleotide constructs The double-stranded oligonucleotide construct (SAMiRNA) produced in the present invention has the following structural formula: [ka] It has a structure represented by:

[0076] To synthesize the sense strand of the monoSAMiRNA double-stranded oligo structure, 3,4,6-triacetyl-1-hexa(ethylene glycol)-CPG was used as a support, and three demethoxytrityl (DMT) hexaethylene glycol phosphoramidates were successively bound to the support as hydrophilic monomers by reaction. DNA synthesis was then carried out, followed by the synthesis of hydrophobic C containing disulfide bonds. 24 (C 18 -S-S-C6) was attached to the 5'-terminal region to form hexaethylene glycol-(-PO4 - Hexaethylene glycol) 3 is bound to the 3' end, and C 24 (C 18 A sense strand having 5'-S-S-C6) bound to the 5' end was synthesized.

[0077] After the synthesis was completed, the synthesized single-stranded DNA and DNA-polymer constructs were cleaved from the CPG by treatment with 28% (v / v) ammonia in a water bath at 60°C, and then the protecting groups were removed by deprotection. The single-stranded DNA, DNA-polymer constructs, and ligand-bound DNA-polymer constructs were separated from the reaction products by high performance liquid chromatography (HPLC), and their molecular weights were measured using a MALDI-TOF mass spectrometer (MALDI TOF-MS, Shimadzu Corporation, Japan) to confirm whether they matched with the nucleotide sequences and DNA-polymer constructs to be synthesized.

[0078] For the synthesis of the antisense strand, RNA having a sequence complementary to the sense strand was synthesized using a support to which a linker (UnyLinker (trademark)) was bound, and then the antisense strand was synthesized with a phosphate group (PO4) bound to the 5'-terminal region.

[0079] After the synthesis was completed, the synthesized RNA single strand and RNA-polymer construct were cleaved from CPG by treatment with 28% (v / v) ammonia in a water bath at 60 °C, and then the protecting groups were removed by deprotection reaction. After removing the protecting groups, the RNA single strand and RNA-polymer construct were treated with N-methylpyrrolidone, trimethylamine, and triethylamine trihydrofluoride in a volume ratio of 10:3:4 in an oven at 70 °C to remove 2'-TBDMS (tert-butyldimethylsilyl). The RNA single strand, RNA-polymer construct, and ligand-bound RNA-polymer construct were separated from the reaction product group by high performance liquid chromatography (HPLC), and their molecular weights were measured using a MALDI-TOF mass spectrometer (MALDI TOF-MS, Shimadzu Corporation) to confirm whether they matched with the nucleotide sequence and RNA-polymer construct to be synthesized. Then, to create individual double-stranded oligo structures, the sense and antisense strands were mixed in equal amounts and added to 1x PBS (phosphate buffered saline, 30 mg / 1 ml (1x) PBS) annealing buffer (30 mM HEPES, 100 mM potassium acetate, 2 mM magnesium acetate, pH 7.0-7.5), reacted in a 90°C water bath for 5 minutes, and gradually cooled to room temperature to produce the desired SAMiRNA. Annealing of the produced double-stranded oligo RNA structures was confirmed by high performance liquid chromatography (HPLC, non-denaturing). Information on the sequences of the SAMiRNA-AREG synthesized for efficacy evaluation is shown in Table 2 below.

[0080] [Table 2] [Table 2]

[0081] Example 2. Recovery of SAMiRNA nanoparticles from a nebulizer In the present invention, SAMiRNA was recovered from both an ultrasonic nebulizer and a compressor-type nebulizer. 1ml of SAMiRNA was placed in the drug chamber of an ultrasonic nebulizer (Meshnet2, China), and the mouthpiece was connected to the chamber. The aerosol inhaler was operated with the tip of the mouthpiece in the collection test tube. To collect the nebulized SAMiRNA, the test tube was placed in ice in an icebox, and the nebulizer was operated for 10 minutes, and the collection of the nebulized SAMiRNA was carried out for a total of 20 minutes, taking into account the liquefaction time of the nebulized SAMiRNA.

[0082] 1 ml of SAMiRNA was placed in the drug chamber of a compressor nebulizer (Philips non-heated nebulizer, USA), a rubber hose was connected to the chamber, and a collection tube was connected to the end of the rubber hose. To collect the nebulized SAMiRNA, the test tube was placed in ice in an ice box, and the nebulizer was operated for 10 minutes. The collection of the nebulized SAMiRNA was carried out for a total of 20 minutes, taking into account the liquefaction time of the nebulized SAMiRNA.

[0083] Example 3. Analysis of the physical properties of SAMiRNA nanoparticles recovered from each nebulizer From each recovered SAMiRNA, RNA single strands, RNA-polymer constructs and ligand-bound RNA-polymer constructs were separated by high performance liquid chromatography and analyzed using MALDI-TOF mass spectrometry to confirm the presence of the intended sequence. To confirm whether the osmolality of the captured SAMiRNA was the same as that of saline, which does not cause osmotic changes when directly introduced into the blood vessel, the osmolality of the recovered SAMiRNA was measured using an Osmomat™ 3000 (Gonotec, Federal Republic of Germany). The particle size of the recovered SAMiRNA was analyzed using dynamic light scattering (DLS) devices Zetasizer NANO-ZS (Malvern, UK) and qNano Gold (IZON, New Zealand) with tunable resistive pulse sensing (TRPS) according to the manufacturer's protocol.

[0084] As a result, it was confirmed that the concentration, molecular weight, purity, nanoparticle size, and osmolality of the SAMiRNA nanoparticles recovered from the ultrasonic nebulizer were the same as those of the SAMiRNA before passing through the ultrasonic nebulizer, whereas in the case of the SAMiRNA recovered from the compressor nebulizer, the nanoparticle size, molecular weight, and purity of the SAMiRNA were the same as those of the SAMiRNA before passing through the compressor nebulizer, but the concentration was 3.6-fold lower (8.3 mg / ml) and the osmolality was significantly lower than that of physiological saline (Figure 1).

[0085] Example 4. Evaluation of cytotoxicity of SAMiRNA nanoparticles recovered from each nebulizer Human lung cancer cell line A549 (CCL-185, ATCC, USA) and human nasal epithelial cell line RPMI2650 (Korea Cell Line Bank, Korea) were used to evaluate the cytotoxicity of SAMiRNA nanoparticles recovered from each nebulizer. A549 cells were cultured at 37°C under 5% CO2 in F12K medium (Gibco, USA) containing 10% fetal bovine serum (Hyclone, USA) and 1% penicillin-streptomycin (Hyclone, USA). RPMI2650 cells were cultured at 37°C under 5% CO2 in RPMI1640 medium (Hyclone, USA) containing 10% fetal bovine serum (Hyclone, USA) and 1% penicillin-streptomycin (Hyclone, USA). A549 cells were cultured at 3 × 10 cells / well in 96-well plates (Falcon, USA). 3 RPMI2650 cells were seeded at a density of 1 × 10 cells / well in a 96-well plate (Falcon, USA). 4 The cells were seeded at a density of 1000 cells / well. The next day, the cells were treated with various concentrations (0, 1, 5, 10, 20, and 50 μM) of SAM iRNA collected from each nebulizer. After 96 h of incubation, experimental analysis was performed using a WST assay kit (DOGEN, Korea) according to the manufacturer's protocol to measure the cytotoxicity of SAM iRNA.

[0086] As a result, no cytotoxicity was found in the two cell lines before or after passing through the ultrasonic nebulizer at concentrations up to 20 μM, and in the case of RPMI2650 cells, slight cytotoxicity was found only at a concentration of 50 μM (Figure 2).

[0087] <Example 5. Evaluation of the target gene expression inhibitory activity of SAMiRNA nanoparticles recovered from each ultrasonic nebulizer> 5-1. Cell treatment using SAMiRNA nanoparticles recovered from an ultrasonic nebulizer A549 cells (CCL-185, ATCC, USA) and RPMI2650 cells (Korea Cell Line Bank, Korea) were used to analyze whether the SAMiRNA nanoparticles recovered from the ultrasonic nebulizer effectively inhibited the expression of target genes. A549 cells were cultured at 37°C under 5% CO2 in F12K medium (Gibco, USA) containing 10% fetal bovine serum (Hyclone, USA) and 1% penicillin-streptomycin (Hyclone, USA). RPMI2650 cells were cultured at 37°C under 5% CO2 in RPMI1640 medium (Hyclone, USA) containing 10% fetal bovine serum (Hyclone, USA) and 1% penicillin-streptomycin (Hyclone, USA). A549 cells were cultured at 5 × 10 cells in 12-well plates (Falcon, USA). 4 RPMI2650 cells were seeded at a density of 1.2 × 10 cells / well in 12-well plates (Falcon, USA). 5 The cells were seeded at a density of 1000 cells / well. The next day, the cells were treated with various concentrations (0, 0.1, 0.5, 1, 5, and 10 μM) of SAMiRNA recovered from the ultrasonic nebulizer.

[0088] 5-2. Analysis of target gene expression using SAMiRNA nanoparticles recovered from an ultrasonic nebulizer According to the method described in Example 5-1, each cell line was treated with SAMiRNA nanoparticles collected from an ultrasonic nebulizer. After 24 hours of incubation, total RNA was extracted from the cell lysate using a universal RNA extraction kit (Bioneer, Korea). Using this RNA as a template, the mRNA expression levels of human AREG and RPL13A (human reference qPCR primer set, Bioneer, Korea) were analyzed by qRT-PCR using AccuPower® GreenStar™ RT-qPCR Master Mix (Bioneer, Korea) according to the manufacturer's protocol. Based on the Ct values ​​of the two genes obtained after qPCR array, the relative amount (rate of change) of AREG mRNA in the test group compared with the control group was analyzed by the 2(-Delta Delta C(T)) method [Livak KJ, Schmittgen TD., Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001.Dec, Vol. 25, No. 4, p.402-8]. The primer sequences for each gene are as follows (Table 3):

[0089] [Table 3] [Table 3]

[0090] As a result, it was observed that both the SAMiRNA nanoparticles before and after passing through the ultrasonic nebulizer exhibited a concentration-dependent inhibitory effect on AREG mRNA in the two cell lines (Figure 3). In short, it was confirmed that the SAMiRNA nanoparticles recovered from the ultrasonic nebulizer maintained their physical and chemical properties, did not exhibit cytotoxicity, and maintained the same target gene inhibitory activity as before passing through the ultrasonic nebulizer.

[0091] Example 6. Evaluation of the delivery efficiency of SAMiRNA nanoparticles administered using an ultrasonic nebulizer in an animal model. 6-1. Characterization of fluorescently labeled SAMiRNA (SAMiRNA-Cy5) nanoparticles recovered after passing through an ultrasonic nebulizer The SAMiRNA-Cy5 nanoparticles recovered from the ultrasonic nebulizer according to the method of Example 2 were analyzed according to the method of Example 3. The results confirmed that the SAMiRNA-Cy5 nanoparticles recovered from the ultrasonic nebulizer had the same fluorescence value, concentration, molecular weight, purity, nanoparticle size, and osmolality as the material before passing through the aerosol nebulizer (Figure 4).

[0092] 6-2. Evaluation of the delivery efficiency of SAMiRNA-Cy5 nanoparticles administered using an ultrasonic nebulizer in a hamster animal model Hamsters were used as experimental animals. Hamsters (5 weeks old, male) were purchased from Central Laboratory Animal Inc. and acclimatized for 1 week before use in the experiment. After attaching the mouthpiece of the ultrasonic nebulizer to the face of the hamster, SAMiRNA-Cy5 (5 mg / ml) or 1 ml of PBS was placed in the drug chamber, and the ultrasonic nebulizer was operated for 2 minutes and 30 seconds while the hamster was administered with the nebulizer. 24 hours after ultrasonic nebulizer administration, the lungs, liver, spleen, and kidneys were collected and weighed. Analysis of the fluorescence images and fluorescence values ​​of each organ was performed using a Davinch-Invivo™ imaging system (Davinch-K, Korea).

[0093] As a result, it was observed that the weights and body weight ratios of the lungs, spleen, liver, and kidneys collected from hamsters administered SAMiRNA-Cy5 by ultrasonic nebulizer were unchanged compared to those of hamsters administered PBS by ultrasonic nebulizer (Figure 5a). Furthermore, the fluorescence images of the lungs, spleen, liver, and kidney tissues collected from hamsters administered SAMiRNA-Cy5 by ultrasonic nebulizer were analyzed using the Davinch-Invivo™ system, and it was confirmed that there was almost no fluorescence in the liver, spleen, and kidneys, but strong fluorescence in the lungs (Figure 5b), indicating that SAMiRNA nanoparticles were efficiently delivered to the lungs through the airways by ultrasonic nebulizer.

[0094] 6-3. Analysis of lung tissue distribution of SAMiRNA nanoparticles administered by ultrasonic nebulizer in a hamster animal model Immunofluorescence staining was performed on lung tissues collected from hamsters administered PBS and SAMiRNA-Cy5 in Example 6-2 by ultrasonic nebulizer. The tissues were placed in 10% neutral buffered formalin (Sigma, USA) for fixation for one day, and then dehydrated sequentially in 10%, 20%, and 30% sucrose (Sigma, USA) solutions. Each lung tissue sample was placed in a base mold (Thermo Scientific, USA) containing OCT compound (Sakura Finetek, USA), and a stainless steel plate was placed in a container containing liquid nitrogen on which the base mold was placed to completely freeze the OCT compound. The frozen tissues were stored at -70°C and placed at -20°C for 30 minutes before sectioning with a microtome to facilitate tissue sectioning. Tissue sections with a thickness of 14 μm were placed on slides and dried for 1 hour. The tissue was then incubated with 0.1% Triton-X100 (Sigma, USA) solution for 10 min to permeabilize the cells, blocked with a solution containing 5% normal goat serum (Abcam, UK) and 1% BSA (Sigma, USA) for 1 h, and incubated with alpha-actin-2 antibody (Sigma, USA) for 1 day at 4°C. After washing with PBS, the tissue was incubated with a secondary antibody, Alexa Fluor® 488-labeled anti-mouse antibody (Invitrogen, USA), for 1 h and then washed. The tissue was incubated with 1 μM DAPI (Sigma, USA) solution for 10 min, washed, and covered with a cover glass (VWR, USA) on which a drop of mounting medium solution (Thermo Scientific, USA) was applied. For fluorescence analysis of stained tissues, the stained tissues were analyzed using spinning disk confocal microscopy (Dragonfly high speed confocal imaging platform, Andor, UK).

[0095] As a result, fluorescence (SAMiRNA-Cy5) was found throughout the lungs of hamsters administered SAMiRNA-Cy5 by ultrasonic nebulizer. Observation of each part at high magnification confirmed that SAMiRNA-Cy5 was sufficiently delivered to most cells that make up the lungs, and particularly strong fluorescence was found in the alveoli and bronchi (Figure 6). It was confirmed that when SAMiRNA nanoparticles were administered using an ultrasonic nebulizer, the nanoparticles were efficiently delivered to the lungs, suggesting that double-stranded oligonucleotide structures and nanoparticles can serve as the basis for optimized siRNA for ultrasonic nebulizers.

[0096] <6-4. Time-dependent fluorescence imaging analysis of tissues after administration of SAMiRNA-Cy5 nanoparticles using an ultrasonic nebulizer in a mouse animal model>

[0097] C57BL / 6 mice were used as experimental animals. Mice (6 weeks old, male) were purchased from Daehan Biolink and were allowed to acclimate for one week before the experiment. The materials used in Example 6-1 were used as fluorescently labeled SAMiRNA-Cy5. After attaching the mouthpiece of an ultrasonic nebulizer to the face of each mouse, SAMiRNA-Cy5 (2 mg) or PBS was placed in the drug chamber and administered by operating the ultrasonic nebulizer for 30 seconds (15 seconds of inhalation-15 seconds of rest-15 seconds of inhalation) (Figure 7a). After administration, the nasal cavity, lungs, liver, spleen, kidneys, and heart were collected and weighed at various time points (0, 1, 24, 48, 96, and 168 hours), and then fluorescence image analysis was performed using a Davinch-Invivo™ imaging system (Davinci-K, Korea). Fluorescence image analysis was performed under the same conditions (fluorescence intensity, exposure time, etc.) for all tissues (taken at various time points). As a result, it was confirmed that the strongest fluorescence was found in the mouse nasal and lung tissues collected 1 hour after administration by ultrasonic nebulizer, indicating that SAMiRNA was efficiently delivered to the nasal and lung tissues. On the other hand, it was confirmed that no fluorescent signal was found in the liver, kidney, heart, spleen, brain, blood, etc. After 24 hours, the fluorescence intensity of the nasal and lung tissues was significantly lower than that after 1 hour, and after 48 hours, the fluorescence intensity of the nasal and lung tissues began to gradually decrease, and after 168 hours, almost no fluorescence was observed even in the lung tissue (Figure 7b).

[0098] 6-5. Quantitative analysis of SAMiRNA nanoparticles administered by ultrasonic nebulizer in nasal and lung tissues of a mouse animal model Fluorescence quantitative analysis and PK analysis were performed using nasal and lung tissues collected from mice administered PBS and SAMiRNA-Cy5 by ultrasonic nebulizer, respectively, in Example 6-4. Each collected tissue was measured, and the whole tissue was placed in a round-bottom tube (SPL, Korea). 1 ml of tissue lysis buffer (Bioneer, Korea) was added to the tissue, which was then homogenized using a homogenizer (IKA, Federal Republic of Germany). After incubation on ice for 15 min, the tissue was centrifuged at 14,000 rpm at 4°C for 15 min, and the supernatant was then transferred to a 1.5 ml amber microcentrifuge tube (Axigen, USA) and stored. Standard samples for fluorescence quantitative analysis were prepared by adding SAMiRNA-Cy5 at various concentrations (0.1, 0.5, 0.25, and 0.125 μg / ml) to the lysates of PBS-treated tissues. 100 μl of each standard and sample was dispensed into a black 96-well microplate (Corning Costar, USA), and then the fluorescence intensity (excitation wavelength 645 nm, emission wavelength 675 nm) was measured using a microplate reader (TECAN, Switzerland) and the amount of SAMiRNA-Cy5 remaining in each tissue was measured by substituting it into the determined standard curve. For PK analysis of SAMiRNA, the lysed tissue samples were homogenized once more using QIAshredder (QIAGEN). Ago2 immunoprecipitation was performed from the tissue lysates using MagListo™ Protein G Kit (Bioneer, Korea) conjugated with anti-mouse AGO2 antibody (Sigma, USA). The resulting RISC-loaded siRNA was subjected to absolute quantification analysis by stem-loop RT-qPCR.Conversion to cDNA was performed using Taqman™ microRNA Reverse Transcription Kit (Applied Biosystem, USA), Mygenie™ 96 (Bionia, Korea), and SAMiRNA-mRelA antisense RT primer (Bionia, Korea), and then qPCR was performed using AccuPower® Plus DualStar™ qPCR Master Mix (Bionia, Korea), 600 nM qPCR primers (Bionia, Korea), 300 nM probe, and Exicycler™ 96 (Bionia, Korea). Standards used for quantification were added to untreated samples and then used for analysis in the same manner. The sequences of the RT and qPCR primers used for analysis are as follows (Table 4).

[0099] [Table 4] [Table 4]

[0100] As a result, the amount of SAMiRNA-Cy5 detected in the lung samples taken after administration was confirmed to be 144.18 μg / g (1.3%) at 1 h, 1.27 μg / g (0.01%) at 24 h, and 0.77 μg / g (0.006%) at 48 h. The amount of SAMiRNA-Cy5 detected in the nasal cavity was 661.8 μg / g (8.2%) at 1 h, and 0.7 μg / g (0.01%) at 24 and 48 h. It was confirmed that the maximum amount of SAMiRNA delivered to the nasal and lung tissues by the ultrasonic nebulizer was detected 1 h after administration, and the amount of detected SAMiRNA gradually decreased over time (Figure 8a). The results of PK analysis of lung tissue, similar to the results of fluorescent quantitative analysis of lung tissue, confirmed that the copy number of the antisense strand of SAMiRNA was highest (3.E+11) 1 hour after administration, 2.E+08 at 24 hours, and 6.E+07 at 48 hours, indicating that the copy number of the antisense strand of SAMiRNA gradually decreased over time (Figure 8b).

[0101] For SARS-CoV-2-targeting SAMiRNA, it is possible to test whether SAMiRNA can be delivered to nasal and lung tissues using the same method as described above. The sequences targeting SARS-CoV-2 are shown in Table 5 below.

[0102] [Table 5] [Table 5]

[0103] 6-6. Analysis of lung tissue distribution of SAMiRNA nanoparticles administered by ultrasonic nebulizer in a mouse animal model Immunofluorescence staining was performed on lung tissues collected from mice administered PBS and SAMiRNA-Cy5 in Example 6-4 by ultrasonic nebulizer. The lung tissues were placed in 10% neutral buffered formalin (Sigma, USA) for fixation for one day, and then dehydrated sequentially in 10%, 20%, and 30% sucrose (Sigma, USA) solutions. Each lung tissue sample was divided into left and right lobes, then placed in a base mold (Thermo Scientific, USA) containing OCT compound (Sakura Finetech, USA), and a stainless steel plate was placed in a container containing liquid nitrogen on which the base mold was placed, allowing the OCT compound to freeze completely. The frozen tissues were stored at -70°C and placed at -20°C for 30 minutes before sectioning with a microtome to facilitate tissue sectioning. Tissue sections with a thickness of 8 μm were placed on slides and dried for 1 hour. The tissue was then incubated with 0.1% Triton-X100 (Sigma, USA) solution for 10 min for cell permeabilization, and then blocked with a solution containing 5% normal goat serum (Abcam, UK) and 1% BSA (Sigma, USA) for 1 h. The tissue was then incubated with 1 μM DAPI (Sigma, USA) solution for 10 min, washed, and then covered with a cover glass (VWR, USA) with a drop of mounting medium solution (Thermo Scientific, USA) on top. For fluorescence analysis of the stained tissue, the stained tissue was analyzed using spinning disk confocal microscopy (Dragonfly high-speed confocal imaging platform, Andor, UK). To accurately analyze the distribution of SAMiRNA over time in lung tissue, confocal microscopy was performed under the same conditions (fluorescence intensity, exposure time, etc.) for all lung tissue samples.

[0104] As a result, it was observed that the fluorescence of SAMiRNA-Cy5 was highest at 1 hour after administration and gradually decreased with time, which was consistent with the results of the fluorescence quantitative analysis and PK analysis of lung tissue. SAMiRNA was observed to be distributed uniformly throughout the lung tissue at 1 hour, 24 hours, and 48 hours, when fluorescence was observed in the lung tissue (Figure 9a). Observation of the left and right lung tissues 1 hour after administration confirmed that SAMiRNA was distributed uniformly throughout the left and right lungs, indicating that it was delivered efficiently (Figure 9b). In addition, observation of the lung tissues 1 hour after administration at a high magnification confirmed that SAMiRNA-Cy5 was found in most cells that make up the lung tissue, and was uniformly distributed in the bronchiole, bronchiolar epithelial cells, alveolar epithelial cells, alveolar sacs, etc., indicating that it was delivered efficiently (Figure 9b). [Industrial Applicability]

[0105] The double-stranded oligonucleotide structure according to the present invention forms self-assembled nanoparticles with a size of 90 nm and neutral charge in aqueous solution, and therefore can serve as an optimized siRNA platform for nebulizers that nebulize the structure in the form of fine particles of 5 μm or less.In particular, when ultrasonic nebulizers are used, it is possible to efficiently deliver pharmaceutical compositions to the nasal cavity and lungs (alveoli) without changes during administration by maintaining the same concentration, molecular weight, purity, nanoparticle size, and osmolality as well as target gene inhibitory activity as the stock material to be administered.

[0106] Therefore, the present invention is suitable for the treatment of respiratory viral infections, pulmonary fibrosis or pneumonia caused by viral infection, or other respiratory diseases, and in particular, when applied to a therapeutic agent for COVID-19 caused by the new respiratory virus SARS-CoV-2, which is causing explosive infections and deaths around the world, the present invention has the advantage that it enables patients to easily and conveniently self-administer the double-stranded oligonucleotide structure, enabling efficient in vivo delivery of the double-stranded oligonucleotide structure.

[0107] Although the present invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that the description is merely of the preferred embodiments of the present invention and is not intended to limit the scope of the present invention. Thus, the true scope of the present invention is to be defined by the appended claims and their equivalents.

Claims

1. The following structural formula 1: [Structural formula 1] A-X-R-Y-B (wherein A represents a hydrophilic compound, B represents a hydrophobic compound, X and Y each independently represent a simple covalent bond or a covalent bond via a linker, and R represents a double-stranded oligonucleotide). A pharmaceutical composition for preventing or treating a respiratory viral infection, pulmonary fibrosis caused by a viral infection, or a respiratory disease, comprising a double-stranded oligonucleotide structure comprising a structure represented by:

2. The pharmaceutical composition of claim 1 , wherein the nebulizer is an ultrasonic nebulizer.

3. The pharmaceutical composition of claim 1 , wherein the double-stranded oligonucleotide comprises a sense strand and an antisense strand comprising a sequence complementary to the sense strand.

4. The pharmaceutical composition according to claim 3, wherein the sense strand or the antisense strand consists of 19 to 31 nucleotides.

5. The pharmaceutical composition of claim 3 , wherein the sense strand or the antisense strand is independently DNA or RNA.

6. The pharmaceutical composition of claim 3 , wherein the sense strand or the antisense strand comprises a chemical modification.

7. Chemical modification is The OH group at the 2' position of the sugar structure of one or more nucleotides is replaced with a methyl group (-CH ３ ), methoxy group (-OCH ３ ), amine group (-NH ２ ), a modification substituted with any one selected from the group consisting of fluorine (-F), an -O-2-methoxyethyl group, an -O-propyl group, an -O-2-methylthioethyl group, an -O-3-aminopropyl group, an -O-3-dimethylaminopropyl group, an -O-N-methylacetamide group, and an -O-dimethylamidooxyethyl group; A modification in which oxygen in the sugar structure of a nucleotide is replaced by sulfur; The internucleotide bond is modified to be any one bond selected from the group consisting of phosphorothioate bond, boranophosphate bond, and methylphosphonate bond; and Modifications made to PNA (peptide nucleic acid), LNA (locked nucleic acid) or UNA (unlocked nucleic acid) The pharmaceutical composition according to claim 6, which is any one or more selected from the group consisting of:

8. The pharmaceutical composition of claim 3 , wherein at least one phosphate group is attached to the 5′ end of the antisense strand.

9. The pharmaceutical composition of claim 1 , wherein the double-stranded oligonucleotide is an siRNA, shRNA or miRNA.

10. The following structural formula 2: [Structural formula 2] 【Chemistry 1】 (wherein S and AS represent the sense and antisense strands of the double-stranded oligonucleotide, respectively, and A, B, X, and Y are as defined in claim 1). The pharmaceutical composition of claim 1 , comprising a structure represented by:

11. The following structural formula 3: [Structural formula 3] 【Chemistry 2】 (wherein A, B, X, Y, S and AS are as defined in claim 10, and 5' and 3' represent the 5' and 3' ends of the sense strand, respectively). The pharmaceutical composition of claim 10, comprising a structure represented by:

12. The hydrophilic compound is represented by the following structural formula 4 or 5: [Structural formula 4] A' ｍ -J) ｎ [Structural formula 5] (J-A' ｍ ) ｎ (In the formula, A' represents a hydrophilic monomer, J represents a linker that connects m hydrophilic monomers together or connects m hydrophilic monomers to an siRNA, m is an integer ranging from 1 to 15, and n is an integer ranging from 1 to 10. The hydrophilic monomer A' is any of the following compounds (1) to (3): [Compound (1)] 【Chemistry 3】 (Wherein, G is CH ２ , O, S and NH; [Compound (2)] 【Chemistry 4】 [Compound (3)] 【Chemistry 5】 and the linker (J) is PO ３ － , S.O. ３ and CO ２ (selected from 2. The pharmaceutical composition of claim 1 having a structure represented by:

13. The pharmaceutical composition according to claim 1, wherein the hydrophilic compound has a molecular weight of 200 to 10,000.

14. 2. The pharmaceutical composition according to claim 1, wherein the hydrophilic compound is any one selected from the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone, and polyoxazoline.

15. 2. The pharmaceutical composition according to claim 1, wherein the hydrophobic compound has a molecular weight of 250 to 1,000.

16. The hydrophobic compounds include steroid derivatives, glyceride derivatives, glycerol ethers, polypropylene glycols, C １２ -C ５０ 2. The pharmaceutical composition according to claim 1, wherein the lipid component is any one selected from the group consisting of unsaturated or saturated hydrocarbons, diacylphosphatidylcholine, fatty acids, phospholipids, lipopolyamines, lipids, tocopherols, and tocotrienols.

17. 17. The pharmaceutical composition according to claim 16, wherein the steroid derivative is any one selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholestanylamine.

18. The pharmaceutical composition according to claim 16, wherein the glyceride derivative is any one selected from the group consisting of monoglycerides, diglycerides, and triglycerides.

19. 2. The pharmaceutical composition of claim 1, wherein the covalent bonds represented by X and Y, respectively, are either non-degradable or degradable bonds.

20. 20. The pharmaceutical composition of claim 19, wherein the non-degradable bond is an amide bond or a phosphate bond.

21. 20. The pharmaceutical composition of claim 19, wherein the degradable bond is any one selected from the group consisting of a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, and an enzymatically degradable bond.

22. 2. The pharmaceutical composition of claim 1, wherein the respiratory disease is selected from the group consisting of interstitial lung disease, chronic obstructive pulmonary disease (COPD), pneumonia, asthma, acute and chronic bronchitis, allergic rhinitis, bronchitis, bronchiolitis, pharyngitis, tonsillitis, and laryngitis.

23. 2. The pharmaceutical composition of claim 1, wherein the double-stranded oligonucleotide specifically inhibits the expression of a gene selected from the group consisting of amphiregulin, RelA / p65, and SARS-CoV-2.

24. 2. The pharmaceutical composition of claim 1, wherein the double-stranded oligonucleotide construct forms self-assembled nanoparticles having a size of 10-100 nm and a neutral charge in an aqueous solution for administration.

25. 25. The pharmaceutical composition of claim 24, wherein the nanoparticles are composed of a mixture of double-stranded oligonucleotide structures, the double-stranded oligonucleotides comprising different sequences.

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