RNAi formulations targeting the MARC1 gene and their uses

Abstract

The present invention relates to an RNAi preparation targeting the MARC1 (mitochondrial amidoxime reducing component 1) gene and its use, and more specifically, to an RNAi preparation comprising an antisense strand having sequence complementarity to the MARC1 mRNA sequence and a sense strand having sequence complementarity to the antisense strand, and a pharmaceutical composition for preventing or treating liver diseases such as non-alcoholic fatty liver disease, comprising the RNAi preparation.

Description

[Technical field]

[0001] The present invention relates to an RNAi preparation targeting the MARC1 (mitochondrial amidoxime reducing component 1) gene and its use, and more particularly to an RNAi preparation comprising an antisense strand having sequence complementarity to the MARC1 mRNA sequence and a sense strand having sequence complementarity to the antisense strand, and a pharmaceutical composition for preventing or treating liver diseases such as non-alcoholic fatty liver disease, comprising the RNAi preparation. [Background technology]

[0002] Recently, the prevalence of metabolic syndromes has been increasing due to the increase in the obese population, and the prevalence of non-alcoholic fatty liver disease has also been increasing. Fatty liver is a disease in which neutral fat accumulates in liver cells, and it has been reported that it is induced as a complication of obesity or by various other causes, such as alcohol, diabetes, malnutrition, drug abuse, etc., but it is broadly divided into alcoholic fatty liver disease caused by excessive alcohol intake and non-alcoholic fatty liver disease (NAFLD) in which neutral fat accumulates in the liver regardless of alcohol intake, and the non-alcoholic fatty liver disease includes simple steatosis and non-alcoholic steatohepatitis (NASH).

[0003] Simple fatty liver disease has a relatively good prognosis, but if fat accumulation becomes excessive, it progresses to inflammation, i.e., steatohepatitis. In livers with extensive steatohepatitis, hepatocyte necrosis is observed, and stellate cells are activated, causing fibrosis. Perisinusoidal fibrosis is particularly observed in areas other than the hepatic vein. Therefore, if steatohepatitis is left untreated, it will develop into liver fibrosis or cirrhosis, resulting in a poor prognosis. 15-50% of patients with nonalcoholic steatohepatitis will show liver fibrosis or cirrhosis, and 30% of fibrotic patients will show cirrhosis after 10 years.

[0004] Non-alcoholic fatty liver disease is closely related to various metabolic syndromes such as obesity, heart disease, and diabetes, and in this respect, drugs that have inhibitory effects on metabolic syndromes, such as insulin sensitizers, antioxidants, hyperlipidemic drugs, liver protectors, and angiotensin II receptor antagonists, have been clinically attempted. However, so far, no drugs with clinical evidence for the treatment of non-alcoholic fatty liver disease have been developed, and all drugs currently in use are only off-label drugs that can be selected as a second-best option, but such existing off-label drugs have limitations in that their use is restricted due to lack of clinical evidence and safety issues. For example, pioglitazone, an insulin sensitizer, was promising as a therapeutic agent for non-alcoholic steatohepatitis, but in clinical trials, not only did it not show a real improvement effect on liver fibrosis, but it also had side effects such as bone fracture risk, weight gain, and worsening and onset of heart failure, so it could not clearly meet the criteria for use as a therapeutic agent. In the absence of any approved treatments for NAFLD, the U.S. Food and Drug Administration (FDA) recently proposed reduced inflammation, reduced liver fibrosis, and alleviation of NAFLD symptoms as clinical indicators for NAFLD.

[0005] In addition, disease treatment using the phenomenon of RNA interference is attracting attention as a safer therapeutic agent because it uses small interfering RNA (siRNA) that targets mRNA and regulates gene expression at the translational level.

[0006] Therefore, the inventors have conducted intensive research and efforts to develop a new safe drug that shows an improving effect on clinical indicators of non-alcoholic steatohepatitis, and as a result, have developed an RNA formulation utilizing RNA interference technology. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide an RNAi preparation that specifically suppresses the expression of MARC1.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating liver disease, or a method for preventing or treating liver disease, comprising the RNAi formulation. [Means for solving the problem]

[0009] To achieve the above-mentioned objective, one embodiment provides an RNAi formulation comprising an antisense strand having a length of 19 to 21 nucleotides (nt) that has sequence complementarity to the MARC1 (mitochondrial amidoxime reducing component 1) mRNA sequence, and a sense strand having a length of 15 to 17 nt that has sequence complementarity to the antisense strand, wherein the 5' end of the antisense strand and the 3' end of the sense strand form blunt ends.

[0010] In another embodiment, a pharmaceutical composition for preventing or treating a liver disease is provided, which comprises the RNAi preparation as an active ingredient.

[0011] Yet another embodiment provides a method for preventing or treating a liver disease, comprising administering the RNAi formulation to an individual.

[0012] Yet another embodiment provides the use of said RNAi formulation for the manufacture of a medicament for preventing or treating liver disease. Effect of the Invention

[0013] In one embodiment, the RNAi formulation can bind to and degrade the mRNA encoding MARC1, thereby inhibiting the expression of the protein, while mitigating side effects such as non-specific immune responses and off-target effects.

[0014] In addition, the RNAi preparation according to one embodiment targets hepatic cell surface receptors and exhibits effects such as alleviating fat accumulation in liver tissue, reducing inflammation, reducing liver fibrosis, and alleviating non-alcoholic fatty liver inflammation symptoms, and can therefore be usefully used as a targeted therapeutic agent for liver diseases including non-alcoholic fatty liver disease, hepatic fibrosis, and cirrhosis. [Brief description of the drawings]

[0015] [Figure 1] The results show that 50 types of MARC1 asiRNAs produced by the same method were administered to Huh7 cells at a concentration of 10 nM each, and then the expression level of MARC1 mRNA was confirmed. [Diagram 2] The results show that 23 types of MARC1 GalNAc-asiRNAs from a single molecule were administered to primary hepatocytes derived from C57BL / 6 mice at a concentration of 200 nM each, and then the expression level of MARC1 mRNA was confirmed. [Diagram 3]In one aspect, OLX-003-1 or OLX-031-1 was administered to an animal model provided with a high-fat diet (#3-1, #31-1), and the expression levels of MARC1 mRNA and MARC2 mRNA were confirmed. (A) shows the results of confirming the expression level of MARC1 mRNA, and (B) shows the results of confirming the expression level of MARC2 mRNA. [Figure 4] FIG. 1 shows the results of macroscopic observation of the liver appearance after administration of OLX-003-1 or OLX-031-1 to animal models provided with a high-fat diet (#3-1, #31-1). [Diagram 5] In one aspect, after administration of OLX-003-1 or OLX-031-1 to an animal model provided with a high-fat diet (#3-1, #31-1), the level of lipid vacuoles in the liver parenchyma was confirmed via H&E staining. [Figure 6] In one aspect, after administration of OLX-003-1 or OLX-031-1 to an animal model provided with a high-fat diet (#3-1, #31-1), the level of lipid vacuoles in the liver parenchyma was confirmed via Oil Red O staining. [Figure 7] After administering OLX-031-1 (#031-1) to an animal model fed a high-fat diet, the expression levels of MARC1 mRNA and MARC2 mRNA were confirmed. A shows the results of confirming the expression level of MARC1 mRNA, and B shows the results of confirming the expression level of MARC2 mRNA. [Figure 8] This is the result of macroscopic observation of the appearance of the liver after administration of OLX-031-1 (#031-1) in a single-phase animal model provided with a high-fat diet. [Figure 9] The results are as follows: After administration of OLX-031-1 (#031-1) to an animal model fed a high-fat diet, the level of lipid vacuoles in the liver parenchyma was confirmed via H&E staining. [Figure 10]After administration of OLX-031-1 (#31-1) to an animal model fed a high-fat diet, the levels of lipid vacuoles and collagen deposition in the liver parenchyma were confirmed using picrosirius red staining. [Figure 11] The expression levels of liver fibrosis-related factors were evaluated after administration of OLX-031-1 (#031-1) to an animal model fed a high-fat diet. (A) shows the expression levels of α-SMA mRNA, and (B) shows the expression levels of Col1α1 mRNA. [Figure 12] The results show that OLX-031-1, a monoclonal antibody, was administered to an animal model fed a high-fat diet, after which the levels of neutral fats in liver tissue were examined. [Figure 13] After administration of OLX-031-1 (#031-1) in a single phase to an animal model fed a high-fat diet, changes in liver damage indicator factors were observed. A shows the results of checking serum AST levels, B shows the results of checking serum ALT levels, and C shows the results of calculating the serum AST / ALT ratio. [Figure 14] The results show that after OLX-031-1 was administered to an animal model (#31-1) fed a high-fat diet, changes in serum lipid indicators were observed. A shows the results of confirming cholesterol levels, B shows the results of confirming triglyceride levels, C shows the results of confirming low-density lipid protein levels, and D shows the results of confirming high-density lipid protein levels. [Figure 15] The levels of fatty vacuoles and inflammatory foci in the liver parenchyma were confirmed by H&E staining after administration of OLX-031-2, a monoclonal antibody, to animal models provided with a high-fat diet (CDHFD 031-2, CDHFD-NCD 031-2). [Figure 16]The levels of lipid vacuoles and collagen deposition in the liver parenchyma were confirmed by picrosirius red staining after administration of OLX-031-2 in a single dose to animal models provided with a high-fat diet (CDHFD 031-2, CDHFD-NCD 031-2). [Figure 17] After administration of OLX-031-2 in a single dose to animal models provided with a high-fat diet (CDHFD 031-2, CDHFD-NCD 031-2), the PSR stained area (%) was compared to compare the content of collagen components in the liver parenchyma. [Figure 18] After administering OLX-031-2 in a single dose to animal models provided with a high-fat diet (CDHFD 031-2, CDHFD-NCD 031-2), the expression levels of liver fibrosis-related factors were evaluated. A shows the expression level of Col1α1 mRNA, B shows the expression level of α-SMA mRNA, and C shows the expression level of TIMP1 mRNA. [Figure 19] The results are as follows: MARC1 mRNA expression levels were examined after administration of OLX-031-2, a monoclonal antibody, to animal models fed a high-fat diet (CDHFD 031-2, CDHFD-NCD 031-2). [Figure 20A] The results are shown in Table 1. Huh7 cells were treated with some of the 134 types of MARC1 asiRNAs developed by the same company at a concentration of 1 nM, and then the expression level of MARC1 mRNA was confirmed. [Figure 20B] The results are shown in Table 1. Huh7 cells were treated with some of the 134 types of MARC1 asiRNAs developed by the same company at a concentration of 1 nM, and then the expression level of MARC1 mRNA was confirmed. [Figure 20C] The results are shown in Table 1. Huh7 cells were treated with some of the 134 types of MARC1 asiRNAs developed by the same company at a concentration of 1 nM, and then the expression level of MARC1 mRNA was confirmed. [Figure 21]The results show that 41 types of MARC1 asiRNAs from a single entity were administered to Huh7 cells at a concentration of 0.1 nM, and then the expression level of MARC1 mRNA was examined. [Figure 22] The results show that 30 types of MARC1 asiRNAs from a single phase were administered to Huh7 cells at a concentration of 1 nM each, and then the expression level of MARC1 protein was confirmed. [Figure 23] The results show that 41 types of MARC1 GalNAc-asiRNAs from a single molecule were administered to Huh7 cells at a concentration of 100 nM, and then the expression level of MARC1 mRNA was confirmed. [Figure 24] The results show that 41 types of MARC1 GalNAc-asiRNAs from a single molecule were administered to primary human hepatocytes at a concentration of 500 nM each, and then the expression level of MARC1 mRNA was examined. [Diagram 25] The results show that 41 types of MARC1 GalNAc-asiRNAs from a single molecule were administered to primary human hepatocytes at concentrations of 20 nM or 100 nM, after which the expression levels of MARC1 mRNA were examined. [Figure 26] The results show that 17 types of MARC1 GalNAc-asiRNAs from a single molecule were administered to primary human hepatocytes at a concentration of 10 nM or 100 nM, and then the expression level of MARC1 mRNA was examined. [Figure 27] In one embodiment, OLX-031-2 or OLX-075-2 was administered to monkeys at concentrations of 2.5 mpk, 5 mpk, or 10 mpk, respectively, and the expression level of MARC1 mRNA was then examined. [Figure 28] Nine types of uniform MARC1 GalNAc-asiRNA were administered to an animal model transfected with pSELECT-mSEAP-hMARC1, and the expression level of human MARC1 mRNA was confirmed via the level of SEAP reporter fluorescence. [Figure 29]Ten types of uniform MARC1 GalNAc-asiRNA were administered to an animal model transfected with psiCHECK-2-hMARC1, and the expression level of human MARC1 mRNA was confirmed via the level of luciferase reporter fluorescence. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Each description and embodiment disclosed in this application may be applied to each other description and embodiment. In other words, all combinations of various elements disclosed in this application belong to the scope of this application. In addition, the specific descriptions described below do not limit the scope of this application.

[0017] One aspect provides an RNAi formulation comprising an antisense strand having sequence complementarity to the MARC1 (mitochondrial amidoxime reducing component 1) mRNA sequence and a length of 19 to 21 nt nucleotides (nt), and a sense strand having sequence complementarity to the antisense strand and a length of 15 to 17 nt, wherein the 5' end of the antisense strand and the 3' end of the sense strand form blunt ends. RNAi preparations

[0018] As used herein, the term "RNA interference (RNAi)" refers to a mechanism in which double-stranded RNA (dsRNA) consisting of a strand having a sequence homologous to the mRNA of a target gene and a strand having a complementary sequence is introduced into a cell or the like to suppress the expression of the target gene by inducing degradation of the mRNA of the target gene.

[0019] As used herein, the term "RNAi agents" or "nucleic acid molecules inducing RNAi" refers to any agent or nucleic acid molecule capable of suppressing or down-regulating gene expression or viral replication by mediating said RNA interference in a sequence-specific manner. The above terms may refer to either an individual nucleic acid molecule, a plurality of said nucleic acid molecules, or a pool of said nucleic acid molecules. In one embodiment, said RNAi agent is also an siRNA.

[0020] As used herein, the term "small interfering RNA (siRNA)" refers to short double-stranded RNA (dsRNA) that mediates efficient gene silencing in a sequence-specific manner.

[0021] The term "gene" as used herein should be considered in its broadest sense and may code for structural or regulatory proteins, including transcription factors, heat shock proteins, or proteins involved in DNA / RNA replication, transcription and / or translation. In the present invention, the gene of interest whose expression is to be silenced may be endogenous to the viral genome, integrated into the animal genome, or may exist as an extrachromosomal entity.

[0022] As used herein, the term "antisense strand" refers to a polynucleotide that is substantially or 100% complementary to a target nucleic acid of interest, such as, in whole or in part, a messenger RNA (mRNA), a non-mRNA RNA sequence (e.g., microRNA, piwiRNA, tRNA, rRNA, and hnRNA), or a coding or non-coding DNA sequence.

[0023] As used herein, the term "sense strand" refers to a polynucleotide having a nucleic acid sequence identical to a nucleic acid of interest, which is identical in whole or in part to an mRNA (messenger RNA), a non-mRNA RNA sequence (e.g., microRNA, piwiRNA, tRNA, rRNA, and hnRNA), or a coding or non-coding DNA sequence.

[0024] The term "complementarity" or "complementary" as used herein refers to the meaning generally accepted in the art. The term may generally refer to the formation or presence of hydrogen bonds between one nucleic acid sequence and another nucleic acid sequence, either by traditional Watson-Crick or other non-traditional types of binding as described herein. Perfect complementarity may mean that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Partial complementarity may also include multiple mismatches or non-based paired nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mismatches, e.g., 1 to 3 mismatches, non-nucleotide linkers, or non-base paired nucleotides) within a nucleic acid molecule. The partial complementarity can result in bulges, loops, overhangs or blunt ends between the sense and antisense strands of a nucleic acid molecule, or between the antisense strand of a nucleic acid molecule and its corresponding target nucleic acid molecule.

[0025] The term "blunt end" as used herein refers to its commonly accepted meaning in the art. In the context of the RNAi formulations or nucleic acid molecules herein, the term may refer to the ends of a double-stranded siRNA molecule that are free of overhanging nucleotides. The siRNA molecules described herein are also those in which the 5' end of the antisense strand and the 3' end of the sense strand form a blunt end. RNAi formulations to suppress MARC1 expression

[0026] "MARC1 (mitochondrial amidoxime reducing component 1) is a molybdenum-containing enzyme in mammals, also called MTARC1 or MOSC1. Deficiency of MARC1 enzyme is associated with lower blood cholesterol levels, blood liver enzyme levels, reduced liver fat, and reduced risk of developing liver cirrhosis, and is known to be a potential therapeutic target for liver diseases. The MARC1 protein may be interpreted as including naturally occurring wild-type MARC1 and its functional variants, and the sequence of the MARC1 protein or the gene encoding it may be obtained from a publicly known database such as GenBank of the National Institutes of Health.

[0027] As used herein, the term "expression" refers to its meaning generally accepted in the art. The term generally refers to the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, splicing, post-transcriptional modification, or translation. As used herein, expression levels may be determined or monitored by detection of mRNA levels or protein levels.

[0028] The terms "suppression" or "reduction" as used in reference to MARC1 gene expression in an individual refers to a statistically significant reduction compared to an untreated or normal control group, such as a reduction of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 60%, 65%, 70%, 70%, 80%, 85%, 90% or 95% or more, but also below the level of detection depending on the method of detection or measurement.

[0029] siRNA is a small interfering RNA that is involved in the RNAi (RNA interference) effect. RNAi is an intracellular gene regulation mechanism that was first discovered in Caenorhabditis elegans in 1998. Its mechanism of action is known to induce target gene degradation by complementary binding of the antisense strand to the target gene's mRNA in the RNA double strand introduced into the cell, and it is currently the most popular new drug development candidate technology.

[0030] However, contrary to such possibilities, side effects and disadvantages of siRNA have been continuously reported. In order to develop an RNAi-based therapeutic agent, it is necessary to overcome obstacles such as 1) absence of an effective delivery system, 2) off-target effects, 3) induction of immune response, and 4) saturation of the intracellular RNAi mechanism. Although siRNA is an effective method for directly regulating the expression of target genes, such problems have caused difficulties in the development of therapeutic agents. In this regard, asymmetric siRNA (asiRNA: asymmetric shorter duplex siRNA) is an asymmetric RNAi-inducing structure with a shorter double helix length compared to the (19+2) structure of conventional siRNA. This technology overcomes problems such as off-target effects, saturation of the RNAi mechanism, and immune response due to TLR3, which are confirmed in existing siRNA structure technology, and therefore makes it possible to develop new RNAi drugs with low side effects.

[0031] Based on this, in this embodiment, an asymmetric siRNA is presented, which comprises a sense strand and an antisense strand complementary to the sense strand, and the siRNA according to one embodiment can effectively suppress expression of the MARC1 gene to the desired extent without causing problems such as off-target effects and saturation of the RNAi mechanism, while maintaining stable and high delivery efficiency.

[0032] In one embodiment, asymmetric siRNA (asiRNA) targeting MARC1 was designed and prepared, and the asiRNA was transfected into cells expressing MARC1, and then a nucleic acid molecule for inducing RNAi with excellent knockdown efficiency, i.e., MARC1 asiRNA, was selected.

[0033] In one embodiment, the RNAi formulation is characterized in that the sense strand has a length of 15 to 17 nt and the antisense strand has a length of 19 to 21 nt.More preferably, the sense strand length is 16 nt and the complementary antisense strand length is 19 nt, 20 nt or 21 nt, but is not limited thereto.

[0034] The 5' end of the antisense strand and the 3' end of the sense strand may form a blunt end. The 3' end of the antisense strand may include an overhang of, for example, 2 to 6 nt.

[0035] In one embodiment, the sense strand is also a sequence selected from the antisense strand sequences listed in Table 1, Table 10, Table 11, Table 12, Table 13, and Table 14, and the antisense strand also comprises a sequence selected from the sense strand sequences listed in Table 1, Table 10, Table 11, Table 12, Table 13, and Table 14.

[0036] In one embodiment, the sense strand may be any one selected from the group consisting of, for example, SEQ ID NO:149, SEQ ID NO:159, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:183, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:227, SEQ ID NO:247, SEQ ID NO:251, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:273, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:331, SEQ ID NO:353, SEQ ID NO:387, SEQ ID NO:391, SEQ ID NO:399, SEQ ID NO:423, SEQ ID NO:429, SEQ ID NO:431, SEQ ID NO:435, SEQ ID NO:439, SEQ ID NO:443, SEQ ID NO:445, SEQ ID NO:447, and SEQ ID NO:527, for example, any one selected from the group consisting of SEQ ID NO:61, SEQ ID NO:149, SEQ ID NO:183, SEQ ID NO:227, SEQ ID NO:423, SEQ ID NO:431, SEQ ID NO:435, SEQ ID NO:439, SEQ ID NO:447, SEQ ID NO:527, and SEQ ID NO:551.

[0037] In one embodiment, the antisense strand may be any one selected from the group consisting of, for example, SEQ ID NO:150, SEQ ID NO:160, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:184, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:228, SEQ ID NO:248, SEQ ID NO:252, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:274, SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:332, SEQ ID NO:354, SEQ ID NO:388, SEQ ID NO:392, SEQ ID NO:400, SEQ ID NO:424, SEQ ID NO:430, SEQ ID NO:432, SEQ ID NO:436, SEQ ID NO:440, SEQ ID NO:444, SEQ ID NO:446, SEQ ID NO:448, and SEQ ID NO:528, for example, any one selected from the group consisting of SEQ ID NO:62, SEQ ID NO:150, SEQ ID NO:184, SEQ ID NO:228, SEQ ID NO:424, SEQ ID NO:432, SEQ ID NO:436, SEQ ID NO:440, SEQ ID NO:448, SEQ ID NO:528, and SEQ ID NO:552.

[0038] Chemically modified RNAi formulations In the RNAi formulation, the sense strand or the antisense strand may also include one or more chemical modifications.

[0039] General siRNA cannot pass through cell membranes due to the high negative charge caused by the phosphate backbone structure and the high molecular weight, and is quickly degraded and removed in the blood, making it difficult to deliver a sufficient amount for RNAi induction to the actual target site. Currently, many highly efficient delivery methods using cationic lipids and cationic polymers have been developed for in vitro delivery, but in vivo, it is difficult to deliver siRNA as efficiently as in vitro, and there is a problem that the siRNA delivery efficiency is reduced due to interactions with various proteins present in the body.

[0040] Therefore, in this embodiment, chemical modifications are introduced into an asymmetric siRNA structure to provide an RNAi formulation with improved hepatocyte targeted delivery ability, and more specifically, an asymmetric siRNA structure (GalNAc-asiRNA: GalNAc asymmetric siRNA) that can be effectively delivered into hepatocytes without a separate delivery vehicle is provided.

[0041] In the present invention, in the sense strand or the antisense strand, the chemical modification may include one or more selected from the group consisting of: binding with an N-acetylgalactosamine (GalNAc) derivative; modification of a nucleotide bond with a phosphorothioate, boranophosphate or methylphosphonate; replacement of an -OH group at the 2' carbon position of a sugar structure in a nucleotide with -CH3 (methyl), -OCH3 (methoxy), -NH2, -F, -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl or -O-3-dimethylaminopropyl; binding of a phosphate group, E-vinylphosphonate or a cell-penetrating peptide.

[0042] In one embodiment, the N-acetylgalactosamine (GalNAc) derivative has a structure represented by the following formula 1. The N-acetylgalactosamine (GalNAc) derivative recognizes the ASGPR (asialoglycoprotien receptor) on the surface of liver cells and helps the RNAi preparation to enter the liver cells, i.e., acts as an ASGPR targeting moiety. Therefore, the RNAi preparation having the N-acetylgalactosamine (GalNAc) derivative bound to its terminus has improved delivery to liver cells, providing an effective targeted therapy for liver diseases. [ka]

[0043] In one embodiment, the sense strand also includes one or more chemical modifications selected from the following: modification of two to four nucleotide bonds adjacent to the 5' end with phosphorothioate, boranophosphate, or methylphosphonate; replacement of an -OH group at the 2' carbon position of the sugar structure in one or more nucleotides with -CH3 (methyl), -OCH3 (methoxy), -NH2, -F, -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; and a bond to an N-acetylgalactosamine (GalNAc) derivative or a cell-penetrating peptide at the 3' end.

[0044] In one embodiment, the antisense strand also includes any one or more chemical modifications selected from the following: modification of 2 to 7 nucleotide bonds adjacent to the 3'-terminus or 5'-terminus with phosphorothioate, boranophosphate, or methylphosphonate; replacement of an -OH group at the 2'-carbon position of the sugar structure in one or more nucleotides with -CH3 (methyl), -OCH3 (methoxy), -NH2, -F, -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; and a bond to a phosphate group, E-vinylphosphonate, or a cell-penetrating peptide at the 5'-terminus.

[0045] In another specific example, the RNAi formulation includes one or more modifications selected from the group consisting of: modification of 2 to 7 nucleotide bonds adjacent to the 3' or 5' end in the sense strand or antisense strand with phosphorothioate; a modification in which an -OH group at the 2' carbon position of a sugar structure in one or more nucleotides in the sense strand or antisense strand is replaced with -OCH3 (methoxy) or -F; and a bond with an N-acetylgalactosamine (GalNAc) derivative at the 3' end of the sense strand; and a phosphate group or an E-vinylphosphonate bond at the 5' end of the antisense strand.

[0046] In one embodiment, the sense strand is also a sequence selected from the antisense strand sequences listed in Table 2, Table 15, Table 16, and Table 17, and the antisense strand also comprises a sequence selected from the sense strand sequences listed in Table 2, Table 15, Table 16, and Table 17.

[0047] In one specific example, the sense strand is any one selected from the group consisting of (A) to (O) in the table below, and the antisense strand is any one selected from the group consisting of (a) to (q) in the table below.

[0048] TIFF2024525078000003.tif220154

[0049] In the above sequence, * means a phosphorothioated bond, m means 2'-O-methyl, f means 2'-fluoro, GalNAc means a trivalent GalNAc derivative of chemical formula 1, P means a 5'-phosphate group, and EVP means a 5'-E-vinylphosphonate bond.

[0050] TIFF2024525078000004.tif111155

[0051] Specifically, the sense strand and the antisense strand of the RNAi formulation are also the following sense strand sequences and antisense strand sequences selected in the table.

[0052] Another embodiment provides a pharmaceutical composition for preventing or treating a liver disease, comprising the RNAi preparation as an active ingredient.

[0053] Yet another embodiment provides the use of said RNAi formulation for the manufacture of a medicament for preventing or treating liver disease.

[0054] The pharmaceutical composition may directly contain or utilize the above-mentioned RNAi formulation, and therefore, the details common to the two are omitted here in order to avoid overcomplicating this specification.

[0055] Liver disease The pharmaceutical composition can also be used as an active ingredient in a pharmaceutical composition for preventing or treating liver diseases by suppressing the expression of the MARC1 gene.

[0056] The above-mentioned liver diseases include fatty liver, hepatic fibrosis, and hepatic cirrhosis, and here, "fatty liver" refers to a phenomenon in which neutral fat, which does not exist in normal cells, appears to be abnormally deposited in liver cells. A normal liver is composed of about 5% adipose tissue, and neutral fat, fatty acid, phospholipid, cholesterol, and cholesterol ester are the main components of fat. Once fatty liver occurs, most of the components are replaced by neutral fat, and if the amount of neutral fat is 5% or more of the liver weight, it is diagnosed as fatty liver. The fatty liver is also called "non-alcoholic fatty liver disease (NAFLD)", which is a disease in which neutral fat accumulates in the liver regardless of alcohol consumption, and can be defined as the case in which fatty acids accumulate in the form of neutral fat in the liver parenchymal cells at 5% or more. The non-alcoholic fatty liver disease is also called simple steatosis or non-alcoholic steatohepatitis (NASH). The non-alcoholic fatty liver disease is pathologically classified into simple steatosis and non-alcoholic steatohepatitis (NASH) accompanied by inflammation, and NASH can be said to be a severe form of NAFLD. Fatty liver disease with severe fat accumulation progresses to inflammation, i.e., steatohepatitis, and if left untreated for a long period of time, it can progress to serious liver diseases such as hepatitis, liver fibrosis, and liver cirrhosis. The incidence of simple fatty liver disease is 10-15% in people with normal weight, but increases to as much as 80% in people with overweight. Therefore, in order to effectively treat non-alcoholic fatty liver disease, it is important to effectively reduce neutral fat accumulation in the liver and inhibit the progression of non-alcoholic fatty liver disease to steatohepatitis, while improving inflammation of steatohepatitis, thereby inhibiting progression to the next stage.

[0057] Pharmaceutical Compositions As used herein, the term "effective ingredient" refers to an appropriate effective amount of an ingredient that is beneficial or that affects a desired clinical or biochemical result. Specifically, it can refer to an effective amount of a pharmaceutical agent, an active agent, or an RNAi agent.

[0058] The effective amount is also an amount administered one or more times that is adequate to prevent a disease or to provide, without limitation, relief of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., that it does not worsen), slowing or reducing the rate of disease progression, or amelioration, or palliation and relief (partial or total) of the disease state.

[0059] As used herein, the term "prevention" refers to any action of blocking the onset of a disease, suppressing a disease, or delaying its progression, such as preventing or hindering the onset of the liver disease or its characteristic features, or guarding or protecting against the onset of the liver disease or its characteristic features.

[0060] As used herein, the term "treatment" refers to both therapeutic treatment and preventive or prophylactic measures. It also refers to any action that improves or favorably alters the symptoms of a disease, such as preventing, reducing or ameliorating the liver disease or its characteristic features, or slowing down (attenuating) the progression of the liver disease or its characteristic features in an individual.

[0061] The term "effective amount" as used herein refers to the meaning generally accepted in the art. The term may generally refer to an amount of a molecule, compound, or construct that elicits an intended biological response (e.g., a beneficial response) in a cell, tissue, system, animal, or human that is sought by a researcher, veterinarian, physician, or other clinician. Specifically, a "therapeutically effective amount" may refer to an amount of a molecule, compound, or construct that is capable of eliciting a desired medical response, such as a therapeutically relevant change in a measurable parameter associated with a disease or disorder, such that a particular clinical treatment may be considered effective. A therapeutically effective amount of a drug for treating the disease or disorder is also the amount necessary to produce a therapeutically relevant change in the parameter.

[0062] The method of administration of the pharmaceutical composition can be determined by a person skilled in the art based on the symptoms of a patient and the severity of the disease. The pharmaceutical composition can be formulated into various forms such as powder, tablet, capsule, liquid, injection, ointment, syrup, etc., and can be provided in a unit dose container or a multi-dose container, such as a sealed ampule or bottle.

[0063] The pharmaceutical composition of the present invention can be administered orally or parenterally. The administration route of the composition of the present invention is not limited to the following, but may be, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intestinal, sublingual or topical. The dosage of the composition of the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate, or disease severity, and can be easily determined by a person skilled in the art. In addition, the composition of the present invention can be formulated into a suitable dosage form using known techniques for clinical administration.

[0064] Yet another embodiment provides a method of preventing or treating liver disease comprising administering to an individual the RNAi formulation.

[0065] The above-mentioned methods for treating liver diseases include or utilize the above-mentioned RNAi formulations or pharmaceutical compositions, and therefore, the common content therebetween will be omitted in order to avoid overcomplicating this specification.

[0066] As used herein, the term "individual" refers to a subject in need of treatment for a disease, specifically a liver disease, and more specifically includes any mammal, such as a human or non-human primate, mouse, dog, cat, horse, cow, sheep, pig, goat, camel, or goat.

[0067] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0068] Example 1: Screening of nucleic acid molecules for inducing RNAi and evaluation of therapeutic efficacy 1-1. Design of 50 asiRNAs and evaluation of their suppressive effect on MARC1 mRNA expression In this embodiment, in order to secure a double-stranded nucleic acid molecule that induces highly efficient RNAi interference targeting MARC1, a target sequence for the MARC1 gene was selected, and a corresponding asiRNA (MARC1 asymmetric siRNA) was designed (sense strand (16-mer), antisense strand (19-mer)). Specifically, after obtaining MARC1 gene information through a NCBI database search, a total of 50 asiRNAs were designed and synthesized at a 10 nmole scale at IDT Korea, Inc., after selecting sequences showing a certain level of homology based on a sequence standard with a common target with mice, taking into consideration animal experiments. The synthesized asiRNAs were then annealed through an incubation process at 95°C for 5 minutes and 37°C for 25 minutes, and subjected to 12% polyacrylamide gel electrophoresis (PAGE), followed by QC (quality control) through a ChemiDoc UV transilluminator (Biorad).

[0069] The sequence information of the MARC1 asiRNA designed by the above-mentioned method is shown in Table 1 below.

[0070] [Table 1] TIFF2024525078000006.tif253161TIFF2024525078000007.tif187163

[0071] Then, to confirm the efficiency of expression suppression at the mRNA level, the MARC1 asiRNA was transfected into Huh7 cells, and the expression level of MARC1 mRNA was measured by qRT-PCR. 3Cells / well were seeded, MARC1 asiRNA (10 nM (OliX Inc.)) and RNAiMax (2 μl / ml (Invitrogen Inc. 13778150)) were added thereto, and transfection was performed according to the protocol provided by Invitrogen. Then, total RNA was extracted using RNeasy Plus Mini Kit (Qiagen 74136), and cDNA was synthesized through reverse transcription using the mRNA contained therein as a template. Then, PCR mixture was prepared using TB Green (Takara RR820A), and quantitative PCR was performed using the StepOne Real-Time PCR system according to the manufacturer's guidelines. In this example, the control group was a group (NT) to which only the transfection reagent was added. As a result, as shown in FIG. 1, the effect of suppressing MARC1 mRNA expression by treatment with 50 types of MARC1 asiRNA was confirmed. 1-2. Design of 23 GalNAc-asiRNAs and evaluation of their inhibitory effect on MARC1 mRNA expression

[0072] In this example, asymmetric siRNAs into which GalNAc ligands and chemical modifications were introduced were designed for 23 types of MARC1 asiRNAs among the MARC1 asiRNAs produced in Example 1. Specifically, various chemical modifications (2'OMe, PS, Fluoro, etc.) were introduced, and a derivative referred to as "trivalent GalNAc" was bound to the 3' end of the sense strand to produce MARC1 GalNAc-asiRNAs.

[0073] [Table 2] TIFF2024525078000009.tif84163

[0074] Specifically, the chemical modifications indicated as "*", "m", "f", "P" and "GalNAc" in Table 2 above are as shown in Table 3 below.

[0075] [Table 3]

[0076] Specifically, in Table 3, "*" means that the existing phosphodiester bond is replaced with a phosphorothioate bond, "m" means that the existing 2'-OH is replaced with 2'-O-methyl, "f" means that, for example, in the case of fG, the 2'-OH of the existing G (guanine) is replaced with fluoro, and "P" means that a phosphate group is bound to the 5' end (a phosphate group is bound to the oxygen bound to the 5th carbon in the 5'-end base), and "GalNAc" means that a trivalent GalNAc derivative of the following chemical formula 1 is bound to the 3' end of the sense strand. The trivalent GalNAc derivative structure is as shown in Chemical Formula 1 below.

[0077] [ka]

[0078] To confirm the efficiency of expression suppression at the mRNA level, the MARC1 GalNAc-asiRNA was transfected into primary hepatocytes derived from C57BL / 6 mice, and then qRT-PCR was performed to measure the expression level of MARC1 mRNA. 4Cells / well were seeded and treated with MARC1 GalNAc-asiRNA (200 nM (OliX US, Inc.)). After 24 hours, cell lysates were prepared using SuperPrep™ Cell lysis & RT kit for qPCR Kit II (TOYOBO SCQ-401), and cDNA was synthesized via reverse transcription using the mRNA contained therein as a template. Then, quantitative PCR was performed using the synthesized cDNA as a template and THUNDERBIRD (registered trademark) Probe qPCR Mix (TOYOBO QPS-101) and Probes (Hs00224227_m1, Hs03928985_g1 (Applied Biosystems). Then, the expression level of MARC1 mRNA was confirmed using a CFX Connect Real-Time PCR System (Bio-Rad). In this example, the control group was a group that was not transfected (NT), the negative control group was a group that was transfected according to the protocol provided by Invitrogen after adding OLX700A-001-8 (10 nM) and RNAiMAX (2 μl / ml (Invitrogen Inc. 13778150)), and the positive control group was a group that was transfected according to the protocol provided by Invitrogen (NC). The OLX700A-001-8 was transfected according to the protocol provided by Invitrogen after adding 13778150) to the OLX700A-001-8. The sequence information of the OLX700A-001-8 is shown in Table 4, and the chemical modifications indicated as "*", "m", "f", "P" and "GalNAc" in Table 4 are as shown in Table 3.

[0079] [Table 4]

[0080] As a result, as shown in Figure 2, the effect of suppressing MARC1 mRNA expression was confirmed by treatment with 23 types of MARC1 GalNAc-asiRNA. Among them, the expression suppression effect of OLX-002-1, OLX003-1, and OLX-031-1 was even more excellent.

[0081] 1-3. Evaluation of therapeutic efficacy using animal models In this example, MARC1 GalNAc-asiRNA, the expression suppression effect of which was confirmed in Example 1-2, was administered to C57BL / 6 mice (male, n=15), and the therapeutic efficacy thereof was evaluated. To this end, the animal model groups were first classified into a group provided with a normal diet (normal chow) and a group provided with a high fat diet (HFD (high fat diet) (Research Diet D12492)), and the groups were classified according to the substance administered into a group administered with 1X PBS (VC), a group administered with OLX-001-8 (NC), a group administered with OLX-003-1 subcutaneously (#3-1), and a group administered with OLX-031-1 subcutaneously (#31-1). In this example, the specific classification of the animal model groups is shown in Table 5 below.

[0082] [Table 5]

[0083] MARC1 GalNAc-asiRNA was administered 16 weeks after the high-fat diet was provided, and MARC1 asiRNA was administered again 2 weeks after the second administration. Two weeks after the second administration, the liver was excised from the subject mice, and liver tissue samples and sections thereof were obtained.

[0084] (1) Expression patterns of MARC1 and MARC2 The expression levels of MARC1 mRNA and MARC2 mRNA were evaluated for liver tissue samples obtained from the mice in the same manner as in Example 1-2.

[0085] As a result, as shown in Figure 3, it was confirmed that the groups (#3-1, #31-1) administered OLX-003-1 or OLX-031-1 according to one embodiment showed suppression of MARC1 mRNA expression compared to the other groups (VC, NC) provided with a high-fat diet, while there was no effect on the expression of MARC2 mRNA.

[0086] (2) Evaluation of the appearance of the liver The livers obtained from the mice were observed with the naked eye to evaluate changes in lesion sites in the liver.

[0087] As a result, as shown in Figure 4, the groups provided with a high-fat diet (VC, NC) had less sharp lesions at the edges of the liver, while the groups administered OLX-003-1 or OLX-031-1 according to one embodiment (#3-1, #31-1) showed morphological characteristics similar to those of the normal group.

[0088] (3) Histopathological evaluation The liver tissue samples obtained from the mice were fixed in 10% NBF (neutral buffered formalin) solution to prepare paraffin-embedded blocks, from which 4 μm-thick tissue sections were prepared. The tissue sections were then stained with Mayer's hematoxylin reagent for H&E. Oil red O staining was also performed on 4 μm-thick frozen sections obtained using OCT embedding medium.

[0089] As a result, as shown in Figures 5 and 6, numerous lipid vacuoles were observed to form in the liver parenchyma in the groups provided with a high-fat diet (VC, NC), while it was confirmed that the formation of lipid vacuoles was significantly reduced in the groups administered OLX-003-1 or OLX-031-1 according to one embodiment (#3-1, #31-1).

[0090] Example 2. Evaluation of therapeutic efficacy using animal models In this example, C57BL / 6 mice were used as subjects, and MARC1 GalNAc-asiRNA shown in Table 6 below was subcutaneously administered using asiMARC1-031, the expression suppression effect of which was confirmed in Example 1, as a template, and the therapeutic efficacy was evaluated.

[0091] [Table 6]

[0092] Specifically, the chemical modifications designated "*", "m", "f", "P", "GalNAc", and "EVP" in Table 6 above are as shown in Table 7 below.

[0093] [Table 7]

[0094] Specifically, in Table 7, "*", "m", "f", "P" and "GalNAc" are the same as those described above. Also, "EVP" means a form in which (E)vinylphosphonate is bound to the 5'-terminus (forming a double bond including the 5th carbon in the 5'-terminus base), for example, in the case of EVP-mU, it means a form in which 2'-OH of the existing U (guanine) is replaced with 2'-O-methyl and (E)vinylphosphonate is bound to the 5'-terminus.

[0095] 2-1.Evaluation of the therapeutic efficacy of OLX-031-1 in HFD-induced NASH mouse model The animal model groups were divided into a group provided with a normal diet (normal chow) and a group provided with a high fat diet (HFD), and the groups were divided into a group provided with 1X PBS (VC), a group provided with OLX700A-001-8 (NC), and a group provided with OLX-031-1 (#31-1) according to the substance administered. In this example, the specific classification of the animal model groups is shown in Table 8 below.

[0096] [Table 8]

[0097] OLX-031-1 was administered 28 weeks after the high-fat diet was provided, and then OLX-031-1 was administered three times at weekly intervals. One week after the four administrations were completed, the livers were excised from the mice, and liver tissue samples, sections thereof, and serum samples were obtained.

[0098] (1) Expression patterns of MARC1 and MARC2 The expression levels of MARC1 mRNA and MARC2 mRNA were evaluated for liver tissue samples obtained from the mice in the same manner as in Example 1-2.

[0099] As a result, as shown in Figure 7, it was confirmed that the group (#31-1) administered OLX-031-1 according to one embodiment showed suppression of MARC1 mRNA expression compared to the other groups (VC, NC) provided with a high-fat diet, while it had no effect on the expression of MARC2 mRNA.

[0100] (2) Evaluation of the appearance of the liver The livers obtained from the mice were observed with the naked eye to evaluate changes in lesion sites in the liver.

[0101] As a result, as shown in FIG. 8, it was confirmed that the groups provided with a high-fat diet (HFDVC, HFDNC) had less sharp lesions at the edges of the liver, whereas the group administered OLX-31-1 according to one embodiment (#31-1) showed morphological characteristics similar to those of the normal group.

[0102] (3) Histopathological evaluation The liver tissue samples obtained from the mice were fixed in 10% NBF (neutral buffered formalin) solution to prepare paraffin-embedded blocks, from which 4 μm-thick tissue sections were prepared, and then the tissue sections were stained with Mayer's hematoxylin reagent (H&E) and picrosirius red.

[0103] As a result, as shown in Figures 9 and 10, in the groups provided with a high-fat diet (VC, NC), numerous fatty vacuoles formed in the liver parenchyma and collagen deposition were observed, while in the group administered with OLX-031-1 according to one embodiment (#31-1), it was confirmed that such fatty vacuole formation and collagen deposition were significantly reduced.

[0104] (4) Evaluation of the expression of fibrosis-related factors and triglyceride levels in liver tissue The expression levels of hepatic fibrosis-related factors and the level of triglycerides were evaluated for liver tissue samples obtained from the mice. Specifically, total RNA was extracted from cells derived from liver tissue using a Tri-RNA reagent (FAVORGEN FATRR 001), and cDNA was synthesized through reverse transcription using the total RNA as a template (High-capacity cDNA Reverse Transcription Kit (Applied Biosystems 4368814)). Then, quantitative PCR was performed using the synthesized cDNA as a template using TB Green Premix Ex Taq (Takara RR420A). The expression levels of α-SMA and mCol1α1 mRNA were then assessed using the StepOne™ Real-Time PCR System (Applied Biosystems™) and the expression levels of the constitutive gene RPL32. Liver tissue samples were also homogenized in 5% NP-40 / UPW using a homogenizer (100 mg / mL, 1:10 dilution). The homogenized samples were then subjected to measurement of triglyceride levels using the Triglyceride Assay Kit (Abcam ab65336).

[0105] As a result, as shown in Figure 11, it was confirmed that the expression level of α-SMA mRNA, a factor associated with liver fibrosis, increased in the groups (VC, NC) provided with a high-fat diet, while such an increase in the expression of α-SMA and mCol1α1 mRNA was reduced in the group (#31-1) administered with OLX-031-1 according to one embodiment. In addition, as shown in Figure 12, it was confirmed that the level of neutral fat in liver tissue was also reduced by administration of OLX-031-1 according to one embodiment.

[0106] (5) Serological evaluation Serum samples obtained from the mice were examined for the levels of AST (aspartate aminotransferase) and ALT (alanine aminotransferase), which are indicators of liver damage, as well as the levels of cholesterol (CHOL), triglycerides (TG), low-density lipoproteins (LDL), and high-density lipoproteins (HDL), which are lipid indicators.

[0107] As a result, as shown in Figure 13, AST and ALT levels were significantly increased in the groups (VC, NC) that were provided with a high-fat diet, while such increases in AST and ALT levels were reduced in the group (#31-1) that was administered OLX-031-1 according to one embodiment. Also, as shown in Figure 14, it was confirmed that the levels of lipid indicator substances were also reduced in the group (#31-1) that was administered OLX-031-1 according to one embodiment.

[0108] 2-2.Evaluation of the therapeutic efficacy of OLX-031-2 in a CDHFD-induced NASH mouse model The animal model groups were classified according to the type of diet and the duration of the diet into a group provided with a normal diet for 12 weeks (NCD 12w), a group provided with a normal diet for 16 weeks (NCD 16w), a group provided with a high-fat diet for 12 weeks (CDHFD 12w), a group provided with a high-fat diet for 16 weeks (CDHFD 16w), and a group provided with a high-fat diet for 12 weeks and a normal diet for 4 weeks (CDHFD-NCD), and the groups were further classified according to the substances administered into a group administered with 1X PBS (VC) and a group administered with OLX-031-2 (#31-2). In this embodiment, the specific classification of the animal model groups is shown in Table 9 below.

[0109] [Table 9]

[0110] OLX-031-2 was administered 12 weeks after the high-fat diet was provided, and OLX-031-2 was administered again 2 weeks after that. Two weeks after the second administration, the liver was excised from the subject mice, and liver tissue samples and sections thereof were obtained. In the animal model groups 1 and 3, the mice were sacrificed 12 weeks after the high-fat diet was provided, and the above-mentioned samples were obtained.

[0111] (1) Histopathological evaluation The tissue sections were stained with H&E and picrosirius red in the same manner as in Example 2-1(3) above, and changes such as numerous fatty vacuoles formed in the liver parenchyma and collagen deposition were confirmed.

[0112] As a result, as shown in Figure 15, in the groups provided with a high fat diet (CDHFD 12w, CDHFD 16w), numerous fatty vacuoles and inflammatory foci were observed in the liver parenchyma, while in the groups administered with OLX-031-2 according to an embodiment (CDHFD 031-2, CDHFD-NCD 031-2), such fatty vacuoles and inflammatory foci were confirmed to be reduced. In addition, as shown in Figures 16 and 17, in the groups administered with OLX-031-2 according to an embodiment (CDHFD 031-2, CDHFD-NCD 031-2), lipid components and collagen deposition were confirmed to be significantly reduced. Notably, Group 6 treated with OLX-031-2 (CDHFD 16w 031-2) had reduced levels of fatty vacuoles and collagen deposition compared to Groups 3 and 4 (CDHFD 12w, CDHFD 16w) provided with a high fat diet, indicating its effective therapeutic efficacy against NASH.

[0113] (2) Evaluation of the expression of fibrosis-related factors in liver tissue The expression levels of liver fibrosis-related factors were evaluated in the same manner as in Example 2-1(4).

[0114] As a result, as shown in Figure 18, the expression levels of α-SMA, mCol1α1 and TIMP1 mRNA, which are liver fibrosis-related factors, increased in the groups provided with a high-fat diet (CDHFD, CDHFD-NCD), while the increase in the expression of such α-SMA, mCol1α1 and TIMP1 mRNA was reduced in the groups administered with OLX-031-2 according to one embodiment (#CDHFD 031-2, CDHFD-NCD 031-2). As in the above experimental results, Group 6 administered with OLX-031-2 (031-2 CDHFD) showed a significant difference from Groups 3 and 4 provided with a high-fat diet (CDHFD 12w, CDHFD16w).

[0115] (3) Evaluation of the effect of suppressing MARC1 mRNA expression The expression level of MARC1 mRNA was evaluated using liver tissue samples obtained from the mice in the same manner as in Example 1-2.

[0116] As a result, as shown in Figure 19, it was confirmed that the expression level of MARC1 mRNA was significantly lower in the groups administered OLX-031-2 according to one embodiment (#CDHFD 031-2, CDHFD-NCD 031-2) compared to the other groups.

[0117] Example 3: Secondary screening of double-stranded nucleic acid molecules for inducing RNAi 3-1. Design and construction of 250 asiRNAs In this example, the MARC1 gene information was obtained through NCBI database search in the same manner as in 1-2 above, and then base sequences showing a certain level of homology were selected for each of human-derived and monkey-derived MARC1 based on base sequence standards with a common target, and a total of 250 asiRNAs were designed (sense strand (16-mer), antisense strand (21-mer)), which were synthesized at a 10 nmole scale at Bioneer. The sequence information of the MARC1 asiRNAs designed by the above-mentioned method is as shown in Tables 10 to 14 below.

[0118] [Table 10] TIFF2024525078000019.tif253147TIFF2024525078000020.tif187147

[0119] [Table 11] TIFF2024525078000022.tif252147TIFF2024525078000023.tif188148

[0120] [Table 12] TIFF2024525078000025.tif252148TIFF2024525078000026.tif186147

[0121] [Table 13] TIFF2024525078000028.tif252147TIFF2024525078000029.tif187147

[0122] [Table 14] TIFF2024525078000031.tif253148TIFF2024525078000032.tif187146

[0123] 3-2.Evaluation of MARC1 mRNA expression suppression effect To confirm the efficiency of expression suppression at the mRNA level, the MARC1 asiRNA was transfected into Huh7 cells, and then qRT-PCR was performed to measure the expression level of MARC1 mRNA. 3 Cells / well were seeded, MARC1 asiRNA (10 nM (OliX Inc.)) and RNAiMax (2 μl / ml (Invitrogen Inc. 13778150)) were added thereto, and transfection was performed according to the protocol provided by Invitrogen. 24 hours later, cell lysates were prepared using SuperPrep™ Cell lysis & RT kit for qPCR Kit II (TOYOBO SCQ-401), and cDNA was synthesized via reverse transcription using the mRNA contained therein as a template. Then, quantitative PCR was performed using the synthesized cDNA as a template with THUNDERBIRD (registered trademark) Probe qPCR Mix (TOYOBO QPS-101) and Probe (Hs00224227_m1, Hs03928985_g1 (Applied Biosystems)), and a total of 134 kinds of MARC1 asiRNA were initially selected based on the mRNA expression suppression level (not shown). In addition, in this example, the control group was a group (Mock) to which only the transfection reagent was added, and the positive control group was a group (PC1) to which asiMARC1-30 was used and transfection was performed in the same manner as in Example 1-2. Then, the MARC1 mRNA expression level was evaluated for the 134 kinds of MARC1 asiRNA selected as described above, while sequentially changing only the MARC1 asiRNA treatment concentration to 1 nM or 0.1 nM, in the same manner as described above.

[0124] As a result, as shown in FIG. 20A to FIG. 20C, the expression of MARC1 mRNA was suppressed by treatment with 134 kinds of MARC1 asiRNA at 1 nM. Among them, the top 41 kinds of MARC1 asiRNA with excellent MARC1 expression suppression efficiency were selected. asiRNAs were selected (#54, #75, #80, #89, #90, #92, #97, #99, #100, #114, #124, #126, #129, #130, #131, #137, #143, #144, #161, #166, #177, #194, #195, #196, #200, #201, #202, #210, #211, #212, #215, #216, #217, #218, #220, #222, #223, #224, #233, #264, #268). Furthermore, as shown in Figure 21, the MARC1 mRNA expression suppression effect by treatment with 0.1 nM of 41 types of MARC1 asiRNA whose expression suppression effect was confirmed was confirmed, and from these, the top 30 MARC1 asiRNAs with excellent MARC1 expression suppression efficiency were selected (#75, #80, #89, #90, #92, #99, #100, #114, #124, #126, #129, #130, #131, #137, #143, #144, #166, #177, #194, #196, #200, #212, #215, #216, #218, #220, #222, #223, #224, #264).

[0125] 3-3.Evaluation of the effect of suppressing MARC1 protein expression To confirm the efficiency of expression suppression at the protein level, the 30 types of MARC1 asiRNA selected in Example 3-2 were transfected into Huh7 cells, and then Western blot was performed to measure the expression level of MARC1 protein. 4Cells / well were seeded, MARC1 asiRNA (1 nM (OliX Inc.) and RNAiMax (2 μl / ml (Invitrogen Inc. 13778150)) was added, and transfection was performed according to the protocol provided by Invitrogen. After 48 hours, the transfected cells were disrupted to obtain 15 μg of cell lysate for each sample. Western blots were performed on the obtained cell lysates using 10% SDS-polyacrylamide gel, MARC1 rabbit polyclonal antibody (1:1,000 dilution in 3% BSA (Abcepta AP9754c)) and vinculin mouse monoclonal antibody (1:1,000 dilution in 3% BSA (Santa Cruz Biotechnology sc-73614)) and ChemiDoc XRS+. The expression level of MARC1 protein was confirmed using a ELISA System (Bio-Rad). In this example, the control group was a group (M) to which only the transfection reagent was added, and the positive control groups were groups (PC1 and PC2) to which asiMARC1-30 or asiMARC1-31 was used and transfection was carried out in the same manner as in Example 1-2.

[0126] As a result, as shown in FIG. 22, it was confirmed that all of the 30 MARC1 asiRNAs selected in Example 3-2 suppressed the expression of MARC1 protein. Example 4: Preparation of chemically modified nucleic acid molecules for inducing RNAi

[0127] 4-1.Design and construction of 41 MARC1 GalNAc-asiRNAs In this example, an asymmetric siRNA (GalNAc-asiRNA) was designed by introducing a GalNAc ligand and chemical modifications into MARC1 asiRNA, the expression-suppressing effect of which was confirmed in Examples 1 and 3. The designed GalNAc-asiRNA is an asymmetric siRNA in which various chemical modifications (2'OMe, PS, Fluoro, GalNAc ligand, etc.) have been introduced compared to the asiRNA, and it has improved delivery into liver cells. The sequence information of the 41 types of MARC1 GalNAc-asiRNAs produced in this Example is as shown in Table 15 below.

[0128] [Table 15] TIFF2024525078000034.tif253163TIFF2024525078000035.tif70162

[0129] Specifically, the chemical variations designated "*", "m", "f", "P" and "GalNAc" in Table 15 above are as shown in Table 7 above.

[0130] 4-2.Evaluation of MARC1 mRNA expression suppression effect To confirm the expression suppression efficiency at the mRNA level, each of the 41 types of MARC1 GalNAc-asiRNA (100 nM) prepared in Example 4-1 was treated with primary cultured mouse hepatocytes (PMH) derived from C57BL / 6 mice (Koatech) (n=3), and qRT-PCR was performed in the same manner as in Example 1-2 to measure the expression level of MARC1 mRNA. In this example, the control group was a group not treated with any substance (NT), the negative control group was a group treated with OLX700A-001-8 (100 nM) (NC), and the positive control group was a group treated with OLX-031-1 (10 nM) (PC).

[0131] As a result, as shown in Figure 23, the effect of suppressing MARC1 mRNA expression was confirmed by treatment with 41 types of MARC1 GalNAc-asiRNA. Among them, the expression suppression effects of OLX-031-1, OLX-031-9, OLX-274-1, OLX-231-3, OLX-281-1, OLX-031-8, OLX-031-6, OLX-237-1, and OLX-031-4 were even more excellent.

[0132] 4-3. Evaluation of the effect of suppressing MARC1 mRNA expression in human hepatocytes To confirm the efficiency of expression suppression at the mRNA level, each of the 41 types of MARC1 GalNAc-asiRNA prepared in Example 4-1 was treated with primary human hepatocytes (F00995-P) (n=3), and then qRT-PCR was performed to measure the expression level of MARC1 mRNA. 4 Cells were seeded per well and treated with MARC1 GalNAc-asiRNA (500 nM (OliX Inc.)). qRT-PCR was then performed in the same manner as in Example 1-2 to measure the expression level of MARC1 mRNA. In this example, the control group was a group that was not treated with any substance (NT), the negative control group was a group that was treated with OLX700A-001-8 (500 nM) (NC), and the positive control group was a group that was treated with OLX-031-1 (10 nM) (PC).

[0133] In order to confirm the efficiency of expression suppression at the mRNA level depending on the treatment concentration of MARC1 GalNAc-asiRNA, the primary hepatocytes derived from the human were cultured in a 96-well plate at 3 × 10 4Cells were seeded per well and treated with 20 nM or 100 nM MARC1 GalNAc-asiRNA (OliX Inc.) (n=2). Then, qRT-PCR was performed in the same manner as in Example 1-2 to measure the expression level of MARC1 mRNA. In this example, the control group was a group not treated with any substance (NT), the negative control group was a group treated with OLX700A-001-8 (100 nM) (NC), and the positive control groups were groups treated with OLX-075-1 or OLX-218-1 (10 nM) (PC1 and PC2).

[0134] As a result, as shown in Figure 24, the effect of suppressing MARC1 mRNA expression by treatment with 41 types of MARC1 GalNAc-asiRNA was confirmed. In addition, as shown in Figure 25, the 41 types of MARC1 GalNAc-asiRNA suppressed MARC1 expression in a concentration-dependent manner, and in particular, a total of 10 types of MARC1 GalNAc-asiRNA (OLX-075-1, OLX-114-1, OLX-212-1, OLX-216-1, OLX-224-1, OLX-218-1, OLX-264-1, OLX-220-1, OLX-31-7, OLX-92-1) were selected that had superior efficacy to OLX-031-1 obtained in Example 1.

[0135] Example 5. Preparation of GalNAc-asiRNA targeting human MARC1 and evaluation of its effect of suppressing MARC1 mRNA expression In this example, based on the experimental results of Example 4, OLX-075-1, which showed excellent effects, was selected to prepare MARC1 GalNAc-asiRNAs with various chemical modifications. The sequence information of the 17 types of MARC1 GalNAc-asiRNAs prepared in this example is shown in Table 16 below.

[0136] [Table 16]

[0137] Specifically, the chemical variations designated "*", "m", "f", "P", "GalNAc" and "EVP" in Table 16 above are as shown in Table 7 above.

[0138] In addition, to confirm the efficiency of expression suppression at the mRNA level, each of the 17 types of MARC1 GalNAc-asiRNAs prepared above was treated with primary human hepatocytes (F00995-P) (n=3), and then qRT-PCR was performed to measure the expression level of MARC1 mRNA. Specifically, the primary hepatocytes were cultured in a 96-well plate at 4×10 4 The cells / well were seeded and treated with MARC1 GalNAc-asiRNA (10 nM or 100 nM (OliX Inc.)), and then qRT-PCR was performed in the same manner as in Example 1-2 to measure the expression level of MARC1 mRNA.

[0139] As a result, as shown in Figure 26, the effect of suppressing MARC1 mRNA expression by treatment with 17 types of MARC1 GalNAc-asiRNA was confirmed.

[0140] Example 6. Evaluation of MARC1 mRNA expression suppression effect using a monkey model Various concentrations (2.5mp, 5mp, or 10mp) of OLX-031-2 or OLX-075-2 were subcutaneously administered twice weekly to monkey animal models, and one week after the second administration, the liver was excised from the monkey and liver tissue samples and slices were obtained. Then, qRT-PCR was performed in the same manner as in Example 1-2 to measure the expression level of MARC1 mRNA. In this example, the sequence information of the OLX-031-2 and the OLX-075-2 are shown in Table 17 below, and the specific classification of the animal model groups is shown in Table 18 below.

[0141] [Table 17]

[0142] [Table 18]

[0143] As a result, as shown in FIG. 27, a more excellent MARC1 mRNA expression suppression effect was confirmed in the OLX-075-2 administration group, and such an effect showed a tendency to be concentration-dependent.

[0144] Example 7. Evaluation of MARC1 mRNA expression suppression effect using animal models 7-1.Evaluation of expression suppression effect using SEAP reporter Six-week-old Balb / c mice (female) were transfected with a plasmid (pSELECT-mSEAP-hMARC1) containing the human MARC1 gene and a SEAP reporter linked thereto, and the animal model groups were classified according to the substance administered into a 1X PBS-administered group (VC), a MARC1 GalNAc-asiRNA-administered group (OLX-075-2, OLX-075-4, OLX-075-5, OLX-075-6, OLX-075-8, OLX-075-12, OLX-075-16, OLX-075-18) of Example 5, and an OLX-031-2-administered group of Example 6. In this example, the specific classification of the animal model groups is shown in Table 19 below.

[0145] [Table 19]

[0146] Four weeks after transfection with the plasmid, MARC1 GalNAc-asiRNA was administered subcutaneously, and one week later, blood samples were taken and the level of SEAP reporter fluorescence was confirmed using the Phospha-Light™ SEAP Reporter Gene Assay System (Invitrogen™ T1015) to compare the level of suppression of human MARC1 mRNA expression.

[0147] As a result, as shown in Figure 28, treatment with nine types of MARC1 GalNAc-asiRNA demonstrated an inhibitory effect on MARC1 mRNA expression, with the expression inhibitory effect being even superior in the groups administered OLX-075-12, OLX-075-16, OLX-075-17, OLX-075-5, OLX-075-8 or OLX-075-2.

[0148] 7-2. Evaluation of expression suppression effect using dual luciferase reporter Six-week-old Balb / c mice were transfected with a plasmid (psiCHECK-2-hMARC1) containing the human MARC1 gene, and then subcutaneously administered 3 mpk of the MARC1 GalNAc-asiRNA of Example 5 (OLX-075-2, OLX-075-4, OLX-075-5, OLX-075-6, OLX-075-8, OLX-075-12, OLX-075-16, OLX-075-17, OLX-075-18). After 3 days, blood samples were taken and the level of luciferase reporter fluorescence was confirmed via the Dual-Luciferase® Reporter Assay System (Promega E1980) to compare the expression suppression level of human MARC1 mRNA.

[0149] As a result, as shown in Figure 29, all of the MARC1 GalNAc-a siRNA groups of Example 5, which had various chemical modifications, showed excellent MARC1 mRNA expression suppression effects, and among them, the expression suppression effects were even better in the OLX-075-16, OLX-075-17, OLX-075-12, OLX-075-8, and OLX-075-8 groups.

[0150] Although the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that such specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the claims and their equivalents.

Claims

1. An RNAi formulation comprising: an antisense strand having sequence complementarity to the MARC1 (mitochondrial amidoxime reducing component 1) mRNA sequence and consisting of the base sequence shown in SEQ ID NO: 150; and a sense strand having sequence complementarity to the antisense strand and consisting of the base sequence shown in SEQ ID NO: 149, An RNAi formulation, wherein the 5' end of the antisense strand and the 3' end of the sense strand form a blunt end.

2. The RNAi formulation of claim 1, wherein the RNAi formulation suppresses the expression of MARC1.

3. The sense strand has an N-acetylgalactosamine (GalNAc) derivative represented by the following formula (I) at its 3' end: 【Chemistry 1】 The RNAi formulation of claim 1, wherein the RNAi formulation is linked to

4. The RNAi formulation of claim 1 , wherein the sense strand or the antisense strand comprises one or more chemical modifications.

5. 5. The RNAi formulation of claim 4, wherein the sense strand comprises one or more chemical modifications selected from the following: The adjacent two to four nucleotide bonds from the 5' end are modified with phosphorothioate, boranophosphate, or methylphosphonate; a replacement of the —OH group at the 2′ carbon position of the sugar structure in one or more nucleotides with —CH3 (methyl), —OCH3 (methoxy), —NH2, —F, —O-2-methoxyethyl-O-propyl, —O-2-methylthioethyl, —O-3-aminopropyl, or —O-3-dimethylaminopropyl; and At the 3'-end, an N-acetylgalactosamine (GalNAc) derivative represented by the following formula (I): 【Chemistry 2】 or conjugation with cell-penetrating peptides.

6. 5. The RNAi formulation of claim 4, wherein the antisense strand comprises one or more chemical modifications selected from the following: two to seven adjacent nucleotide bonds from the 3'-end or 5'-end are modified with phosphorothioate, boranophosphate, or methylphosphonate; a replacement of the —OH group at the 2′ carbon position of the sugar structure in one or more nucleotides with —CH3 (methyl), —OCH3 (methoxy), —NH2, —F, —O-2-methoxyethyl-O-propyl, —O-2-methylthioethyl, —O-3-aminopropyl, or —O-3-dimethylaminopropyl; and Conjugation at the 5' end with a phosphate group, E-vinylphosphonate, or a cell-penetrating peptide.

7. The RNAi formulation comprises: In the sense strand or antisense strand, adjacent 2 to 7 nucleotide bonds from the 3' end or 5' end, be modified with phosphorothioate; a modification in which the -OH group at the 2' carbon position of the sugar structure in one or more nucleotides of the sense or antisense strand is replaced with -OCH3 (methoxy) or -F; An N-acetylgalactosamine (GalNAc) derivative represented by the following formula (I) at the 3'-end of the sense strand: 【Transformation 3】 Combination with; and The RNAi formulation of claim 4, comprising one or more modifications selected from the group consisting of a phosphate group or an E-vinylphosphonate bond at the 5' end of the antisense strand.

8. The sense strand and the antisense strand are selected from the sequences shown in the table below, and the combination of the sense strand and the antisense strand is: A combination of a sense strand (A) and an antisense strand (a), (b), (c), (d), or (e); A combination of a sense strand (B) and an antisense strand (b), (c), (d), or (e); A combination of a sense strand (C) and an antisense strand (b), (c), (d), or (e); and a combination of a sense strand (C) and an antisense strand (b), (c), (d), or (e); The RNAi formulation of claim 4, wherein the RNAi formulation is selected from the group consisting of: Table 1

9. A pharmaceutical composition for preventing or treating liver disease, comprising an RNAi formulation according to any one of claims 1 to 8 as an active ingredient, wherein the liver disease is fatty liver, liver fibrosis, or cirrhosis.

10. The pharmaceutical composition for preventing or treating liver disease according to claim 9, wherein the fatty liver is non-alcoholic fatty liver disease (NAFLD).

11. 10. The pharmaceutical composition for preventing or treating liver disease according to claim 9, wherein the non-alcoholic fatty liver disease is simple steatosis or non-alcoholic steatohepatitis (NASH).

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