RNAi formulations targeting the MARC1 gene and their applications

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. More specifically, it relates to an antisense strand having sequence complementarity to the MARC1 mRNA sequence, an RNAi preparation comprising 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 Art

[0002] Recently, along with the increasing prevalence of metabolic syndrome due to the increase in the obese population, the prevalence of non-alcoholic fatty liver disease has also been on the rise. Fatty liver is a disease in which neutral fat accumulates in hepatocytes and is reported to be induced as a complication of obesity or by various other causes such as alcohol, diabetes, malnutrition, drug abuse, etc. Generally speaking, it can be classified 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. The non-alcoholic fatty liver disease includes simple steatosis and non-alcoholic steatohepatitis (NASH).

[0003] Simple fatty liver disease generally has a relatively good prognosis. However, if fat accumulation becomes severe, it progresses to inflammation, or steatohepatitis. In livers with extensive steatohepatitis, hepatocyte necrosis occurs, and stellate cells are activated, leading to fibrosis. Perisinusoidal fibrosis is particularly severe 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. Liver fibrosis or cirrhosis is observed in 15-50% of patients with non-alcoholic fatty liver disease, and cirrhosis is observed in 30% of patients with fibrosis after 10 years.

[0004] Non-alcoholic fatty liver disease (NAL) is closely associated with a variety of metabolic disorders, including obesity, heart disease, and diabetes. In this context, drugs that suppress metabolic disorders, such as insulin resistance improvers, antioxidants, hyperlipidemia treatments, hepatoprotective agents, and angiotensin II receptor antagonists, have been clinically tested. However, to date, no drugs with clinical evidence for the treatment of NAL have been developed. Currently, only off-label drugs, considered second-best options, exist, but these existing off-label drugs suffer from a lack of clinical evidence and are limited in use due to safety concerns. For example, pioglitazone, an insulin resistance improver, showed promise as a treatment for NAL, but clinically, it failed to demonstrate any significant improvement in liver fibrosis. Furthermore, side effects such as increased fracture risk, weight gain, and worsening and onset of heart failure prevented it from clearly meeting the criteria for therapeutic use. Given the current situation where there are no approved treatments for non-alcoholic fatty liver disease (NALF), the U.S. Food and Drug Administration (FDA) recently presented reduced inflammation, reduced liver fibrosis, and alleviation of NALF symptoms as clinical indicators for NALF.

[0005] Furthermore, the treatment of diseases using RNA interference is attracting attention as a safer therapeutic agent because it utilizes short interfering RNAs (siRNAs) that target mRNA and regulate gene expression at the translational level.

[0006] As a result of diligent research efforts to develop a new and safe drug that shows improvement in clinical indicators of non-alcoholic steatohepatitis, the inventors developed an RNA preparation utilizing RNA interference technology. [Overview of the Initiative] [Problems that the invention aims to solve]

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

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

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

[0010] Another embodiment provides a pharmaceutical composition for the prevention or treatment of liver disease, comprising the RNAi preparation as an active ingredient.

[0011] Another embodiment provides a method for preventing or treating liver disease, comprising the step of administering the RNAi preparation to an individual.

[0012] Another embodiment provides the use of the RNAi preparation for the manufacture of pharmaceuticals for the prevention or treatment of liver disease. [Effects of the Invention]

[0013] One embodiment of the RNAi preparation can suppress the expression of the MARC1 protein by binding to and degrading the mRNA encoding MARC1, while mitigating side effects such as nonspecific immune responses and off-target effects.

[0014] Furthermore, one embodiment of RNAi preparations targets hepatocyte surface receptors and exhibits effects such as mitigating the accumulation of fat in liver tissue, reducing inflammation, reducing liver fibrosis, and alleviating symptoms of non-alcoholic fatty liver disease. Therefore, it can be usefully used as a targeted therapeutic agent for liver diseases, including non-alcoholic fatty liver disease, liver fibrosis, and cirrhosis. [Brief explanation of the drawing]

[0015] [Figure 1] This is the result of treating Huh7 cells with 50 different monomorphic MARC1 asiRNAs at a concentration of 10 nM each, and then checking the expression level of MARC1 mRNA. [Figure 2] This report shows the results of treating primary hepatocytes derived from C57BL / 6 mice with 23 monomorphic MARC1 GalNAc-asiRNAs at a concentration of 200 nM each, and then examining the expression levels of MARC1 mRNA. [Figure 3]In this study, the expression levels of MARC1 mRNA and MARC2 mRNA were examined after administering OLX-003-1 or OLX-031-1 to animal models fed a high-fat diet (#3-1, #31-1). A shows the results for examining the expression level of MARC1 mRNA, and B shows the results for examining the expression level of MARC2 mRNA. [Figure 4] This is a single-phase analysis of the results obtained by visually examining the liver appearance after administering OLX-003-1 or OLX-031-1 to animal models fed a high-fat diet (#3-1, #31-1). [Figure 5] This is a monochromatic image showing the level of lipid vacuoles in the liver parenchyma after administration of OLX-003-1 or OLX-031-1 to animal models fed a high-fat diet (#3-1, #31-1), confirmed via H&E staining. [Figure 6] This is a single-phase study showing the level of lipid vacuoles in the liver parenchyma after administration of OLX-003-1 or OLX-031-1 to animal models fed a high-fat diet (#3-1, #31-1), confirmed via oil red O staining. [Figure 7] After administering OLX-031-1 in a single phase to an animal model fed a high-fat diet (#031-1), the expression levels of MARC1 mRNA and MARC2 mRNA were examined. A shows the results of examining the expression level of MARC1 mRNA, and B shows the results of examining the expression level of MARC2 mRNA. [Figure 8] This is the result of visually examining the liver appearance after administering OLX-031-1 (#031-1) in a monomorphic form to an animal model fed a high-fat diet. [Figure 9] This is the result of confirming the level of lipid vacuoles in the liver parenchyma via H&E staining after administering monomorphic OLX-031-1 (#031-1) to an animal model fed a high-fat diet. [Figure 10]This is the result of confirming the levels of fat vacuoles and collagen deposition in the liver parenchyma via picrosirius red staining after administering OLX-031-1 in a uniform manner to an animal model provided with a high-fat diet (#31-1). [Figure 11] This is the evaluation of the expression levels of liver fibrosis-related factors after administering OLX-031-1 in a uniform manner to an animal model provided with a high-fat diet (#031-1). A is the result of confirming the expression level of α-SMA mRNA, and B is the result of confirming the expression level of Col1α1 mRNA. [Figure 12] This is the result of confirming the level of neutral fat in the liver tissue after administering OLX-031-1 in a uniform manner to an animal model provided with a high-fat diet (#031-1). [Figure 13] This is the confirmation of the changes in liver injury indicator factors after administering OLX-031-1 in a uniform manner to an animal model provided with a high-fat diet (#031-1). A is the result of confirming the serum AST level, B is the result of confirming the serum ALT level, and C is the result of calculating the ratio of serum AST / ALT. [Figure 14] This is the confirmation of the changes in serum lipid indicator substances after administering OLX-031-2 in a uniform manner to an animal model provided with a high-fat diet (CDHFD 031-2, CDHFD-NCD 031-2). A is the result of confirming the cholesterol level, B is the result of confirming the neutral fat level, C is the result of confirming the low-density lipoprotein level, and D is the result of confirming the high-density lipoprotein level. [Figure 15] This is the result of confirming the levels of fat vacuoles and inflammatory foci in the liver parenchyma via H&E staining after administering OLX-031-2 in a uniform manner to an animal model provided with a high-fat diet (CDHFD 031-2, CDHFD-NCD 031-2). [Figure 16]After administering OLX-031-2 in a uniform manner to an animal model provided with a high-fat diet (CDHFD 031-2, CDHFD-NCD 031-2), the levels of fat vacuoles and collagen deposition in the liver parenchyma were confirmed via Sirius red staining. [Figure 17] After administering OLX-031-2 in a uniform manner to an animal model provided with a high-fat diet (CDHFD 031-2, CDHFD-NCD 031-2), the PSR staining area (%) was compared, and the content of the collagen component in the liver parenchyma was compared. [Figure 18] After administering OLX-031-2 in a uniform manner to an animal model provided with a high-fat diet (CDHFD 031-2, CDHFD-NCD 031-2), the expression levels of liver fibrosis-related factors were evaluated. A is the result of confirming the expression level of Col1α1 mRNA, B is the result of confirming the expression level of α-SMA mRNA, and C is the result of confirming the expression level of TIMP1 mRNA. [Figure 19] After administering OLX-031-2 in a uniform manner to an animal model provided with a high-fat diet (CDHFD 031-2, CDHFD-NCD 031-2), the expression level of MARC1 mRNA was confirmed. [Figure 20A] After treating Huh7 cells with some of the 134 types of MARC1 asiRNA in a uniform manner at a concentration of 1 nM each, the expression level of MARC1 mRNA was confirmed. [Figure 20B] After treating Huh7 cells with some of the 134 types of MARC1 asiRNA in a uniform manner at a concentration of 1 nM each, the expression level of MARC1 mRNA was confirmed. [Figure 20C] After treating Huh7 cells with some of the 134 types of MARC1 asiRNA in a uniform manner at a concentration of 1 nM each, the expression level of MARC1 mRNA was confirmed. [Figure 21]This shows the results of treating Huh7 cells with 41 different MARC1 asiRNAs at a concentration of 0.1 nM each, and then checking the expression levels of MARC1 mRNA. [Figure 22] This is the result of treating Huh7 cells with 30 different MARC1 asiRNAs, each at a concentration of 1 nM, and then checking the expression level of the MARC1 protein. [Figure 23] This is the result of treating Huh7 cells with 41 monomorphic MARC1 GalNAc-asiRNAs at a concentration of 100 nM each, and then checking the expression level of MARC1 mRNA. [Figure 24] This is the result of treating human-derived primary hepatocytes with 41 monomorphic MARC1 GalNAc-asiRNAs at a concentration of 500 nM each, and then confirming the expression level of MARC1 mRNA. [Figure 25] This study shows the results of treating human-derived primary hepatocytes with 41 monomorphic MARC1 GalNAc-asiRNAs at concentrations of 20 nM or 100 nM, and then examining the expression levels of MARC1 mRNA. [Figure 26] This report shows the results of treating human-derived primary hepatocytes with 17 monomorphic MARC1 GalNAc-asiRNAs at concentrations of 10 nM or 100 nM, and then examining the expression levels of MARC1 mRNA. [Figure 27] This is the result of administering OLX-031-2 or OLX-075-2 to monkeys at concentrations of 2.5 mpk, 5 mpk, or 10 mpk, respectively, and then checking the expression level of MARC1 mRNA. [Figure 28] This study describes the results of administering nine monomorphic MARC1 GalNAc-asiRNAs to an animal model transfected with pSELECT-mSEAP-hMARC1, and then confirming the expression level of human MARC1 mRNA via the SEAP reporter fluorescence level. [Figure 29]This study describes the results of administering 10 monomorphic MARC1 GalNAc-asiRNAs to an animal model transfected with psiCHECK-2-hMARC1, and then confirming the expression level of human MARC1 mRNA via the level of luciferase reporter fluorescence. [Modes for carrying out the invention]

[0016] Each description and embodiment disclosed in this application may also apply to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions described below do not limit the scope of this application.

[0017] The present invention provides an RNAi formulation comprising an antisense strand, which is a nucleotide (nt) of 19 to 21 nt in length and sequence-complementary to the MARC1 (mitochondrial amidoxime reducing component 1) mRNA sequence, and a sense strand, which is sequence-complementary to the antisense strand and has a length of 15 to 17 nt, wherein the 5' end of the antisense strand and the 3' end of the sense strand form a blunt end. RNAi formulations

[0018] In this specification, the term "RNA interference (RNAi)" refers to a mechanism by which the expression of a target gene is suppressed by introducing double-stranded RNA (dsRNA), which consists of a strand having a sequence homologous to the mRNA of the target gene and a strand having a complementary sequence, into cells or other organisms, thereby inducing the degradation of the target gene mRNA.

[0019] In this specification, the terms “RNAi agents” or “nucleic acid molecules inducing RNAi” refer to any agent or nucleic acid molecule capable of suppressing or downwardly regulating gene expression or viral replication by mediating RNA interference in a sequence-specific manner. The aforementioned terms may refer to individual nucleic acid molecules, a group of such nucleic acid molecules, or a pool of such nucleic acid molecules. In one specific example, the RNAi agent is also an siRNA.

[0020] In this specification, the term "small interfering RNA (siRNA)" refers to short double-stranded RNA (dsRNA) that mediates sequence-specific and efficient gene silencing.

[0021] In this specification, the term “gene” should be understood in its broadest sense and may include structural proteins or regulatory proteins. In this case, the regulatory protein includes transcription factors, thermal shock proteins, or proteins involved in DNA / RNA replication, transcription, and / or translation. In this invention, the target gene to be repressed is endogenous to the viral genome and may be integrated into animal genes or exist as an extrachromosomal component.

[0022] In this specification, the term “antisense strand” refers to a polynucleotide that is substantially or 100% complementary to a target nucleic acid of interest, and is complementary, either as a whole or in part, to, for example, mRNA (messenger RNA), non-mRNA RNA sequences (e.g., microRNA, piwiRNA, tRNA, rRNA, and hnRNA), or coding DNA sequences or non-coding DNA sequences.

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

[0024] In this specification, the terms “complementarity” or “complementary” refer to the meanings generally accepted in the industry. These terms can generally refer to the formation or presence of hydrogen bonds between one nucleic acid sequence and another, by traditional Watson-Crick or other non-traditional types of bonding described herein. Perfect complementarity may mean that all adjacent residues in one nucleic acid sequence are hydrogen-bonded to the same number of adjacent residues in the second nucleic acid sequence. Partial complementarity may also include a variety of mismatches or non-based paired nucleotides within the nucleic acid molecule (e.g., one, two, three, four, five, six, seven, eight, nine, or more mismatches, e.g., one to three mismatches, non-nucleotide linkers, or non-based paired nucleotides). The aforementioned partial complementarity may result in bulges, loops, overhangs, or blunt ends between the sense strands 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] In this specification, the term “blunt end” has the meaning generally accepted in the industry. In relation to RNAi preparations or nucleic acid molecules as described herein, the term may refer to the end of a double-stranded siRNA molecule that lacks overhanging nucleotides. The siRNA molecules described herein also have blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand. RNAi agents to suppress MARC1 expression

[0026] MARC1 (mitochondrial amidoxime reducing component 1) is a mammalian molybdenum-containing enzyme, also known as MTARC1 or MOSC1. MARC1 enzyme deficiency is associated with low blood cholesterol and liver enzyme levels, reduced liver fat, and a reduced risk of developing cirrhosis, and is known to be a potential therapeutic target for liver disease. The aforementioned MARC1 protein is interpreted to include naturally occurring wild-type MARC1 and its functional variants. The sequences of the aforementioned MARC1 protein, or the genes encoding it, can be obtained from publicly known databases such as GenBank of the U.S. National Institutes of Health.

[0027] In this specification, the term “expression” has the meaning generally accepted in the industry. The term can generally mean 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 at the mRNA level or protein level.

[0028] The terms “suppression” or “reduction” used in relation to MARC1 gene expression in individuals refer to a statistically significant reduction compared to the untreated group or the normal control group. Such reductions may be, for example, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 60%, 65%, 70%, 70%, 80%, 85%, 90%, or 95% or greater, but may also be below the detection level depending on the detection or measurement method.

[0029] siRNA is a short interfering RNA (SRNA) involved in RNA interference (RNAi). RNAi is an intracellular gene regulatory mechanism first discovered in Caenorhabditis elegans in 1998. Its mechanism of action is known to involve antisense strands binding complementaryly to the mRNA of a target gene in a double-stranded RNA introduced into the cell, thereby inducing the degradation of the target gene. It is currently one of the most promising new drug development technologies.

[0030] However, contrary to such potential, side effects and disadvantages of siRNA have been continuously reported. For RNAi-based therapeutics to be developed, it is necessary to overcome obstacles such as 1) the absence of an effective signaling system, 2) off-target effects, 3) induction of immune responses, and 4) saturation of intracellular RNAi mechanisms. Despite siRNA being an effective method that can directly regulate the expression of target genes, therapeutic drug development has been difficult due to these problems. In this regard, asymmetric shorter duplex siRNA (asiRNA) 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 RNAi mechanisms, and immune responses mediated by TLR3 that have been observed in existing siRNA structural technologies, thereby enabling the development of new RNAi drugs with fewer side effects.

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

[0032] In one example, an asymmetric siRNA (asiRNA) targeting MARC1 was designed and prepared. Cells expressing MARC1 were then transfected with the asiRNA, and a nucleic acid molecule for RNAi induction with excellent knockdown efficiency, i.e., MARC1 asiRNA, was selected.

[0033] In one specific example, the RNAi preparation 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 the preparation is not limited thereto.

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

[0035] In one specific example, the sense strand is a sequence selected from among the antisense strand sequences listed in Tables 1, 10, 11, 12, 13, and 14, and the antisense strand also includes a sequence selected from among the sense strand sequences listed in Tables 1, 10, 11, 12, 13, and 14.

[0036] In one specific example, the sense strand may be any one selected from the group consisting of, for example, SEQ ID NOs: 149, 159, 177, 179, 183, 197, 199, 227, 247, 251, 257, 259, 261, 273, 285, 287, 331, 353, 387, 391, 399, 423, 429, 431, 435, 439, 443, 445, 447, and 527, or it may be any one selected from the group consisting of, for example, SEQ ID NOs: 61, 149, 183, 227, 423, 431, 435, 439, 447, 527, and 551.

[0037] In one specific example, the antisense strand may be any one selected from the group consisting of, for example, SEQ ID NOs: 150, 160, 178, 180, 184, 198, 200, 228, 248, 252, 258, 260, 262, 274, 286, 288, 332, 354, 388, 392, 400, 424, 430, 432, 436, 440, 444, 446, 448, and 528, or any one selected from the group consisting of, for example, SEQ ID NOs: 62, 150, 184, 228, 424, 432, 436, 440, 448, 528, and 552.

[0038] chemical modification RNAi preparations that have been introduced In the RNAi preparation, the sense strand or the antisense strand is one or more chemical modification This also includes (chemical modification).

[0039] Conventional siRNAs, due to their high negative charge from their phosphate backbone structure and high molecular weight, are unable to pass through cell membranes. However, in the blood, they are rapidly degraded and removed, making it difficult to deliver a sufficient amount to the actual target site for RNAi induction. Currently, many highly efficient delivery methods using cationic lipids and cationic polymers have been developed for in vitro delivery. However, in vivo delivery of siRNA is difficult to achieve with the same high efficiency as in vitro, and there are problems with reduced siRNA delivery efficiency due to interactions with various proteins present in the body.

[0040] Therefore, in this embodiment, the asymmetric siRNA structure chemical modification We introduce an RNAi preparation with improved hepatocyte-targeted delivery ability, and more specifically, we present an asymmetric siRNA structure (GalNAc-asiRNA: GalNAc asymmetric siRNA) that can effectively deliver signals into hepatocytes without the need for a separate signaling molecule.

[0041] In the present invention, in the sense strand or the antisense strand, chemical modification This also includes one or more selected from the following groups: binding with N-acetylgalactosamine (GalNAc) derivatives; nucleotide binding with phosphorothioate, boranophosphate, or methylphosphonate. qualification; Substitution of the -OH group at the 2' carbon position of the sugar structure within the 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 cell-penetrating peptide.

[0042] In one specific example, the N-acetylgalactosamine (GalNAc) derivative also has the structure shown in Chemical Formula 1 below. The N-acetylgalactosamine (GalNAc) derivative recognizes the ASGPR (asialoglycoprotien receptor) on the surface of hepatocytes and plays a role in helping RNAi preparations to flow into hepatocytes; in other words, it acts as an ASGPR targeting moisture. Therefore, RNAi preparations to which the N-acetylgalactosamine (GalNAc) derivative is bound at the terminal have improved transmissibility to hepatocytes and provide effective targeted therapy for liver disease. [ka]

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

[0044] In one specific example, the antisense strand is one or more selected from the following: chemical modification It also includes: 2 to 7 nucleotide bonds adjacent to the 3' or 5' end, with phosphorothioate, boranophosphate, or methylphosphonate. qualification ; at the 2' carbon position of the sugar structure within one or more nucleotides, the -OH group is substituted 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 5' end, binding to a phosphate group, E-vinylphosphonate, or cell-penetrating peptide.

[0045] In other specific examples, the RNAi preparation has two to seven adjacent nucleotide bonds from the 3' or 5' end of the sense strand or antisense strand, with a phosphorothioate. qualification ; In one or more nucleotides of a sense strand or antisense strand, the -OH group at the 2' carbon position of the sugar structure is replaced with -OCH3(methoxy) or -F. qualificationOne or more selected from the group consisting of: ; and binding to an N-acetylgalactosamine (GalNAc) derivative at the 3' end of the sense strand; and binding to a phosphate group or E-vinylphosphonate at the 5' end of the antisense strand; qualification It also includes that.

[0046] In one specific example, the sense strand is a sequence selected from among the antisense strand sequences listed in Tables 2, 15, 16, and 17, and the antisense strand also includes a sequence selected from among the sense strand sequences listed in Tables 2, 15, 16, and 17.

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

[0048] TIFF0007853399000002.tif220154

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

[0050] TIFF0007853399000003.tif111155

[0051] Specifically, the sense strand and antisense strand of the RNAi preparation are also the sense strand sequence and antisense strand sequence selected from the table above.

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

[0053] Another embodiment provides the use of the RNAi preparation for the manufacture of pharmaceuticals for the prevention or treatment of liver disease.

[0054] The aforementioned pharmaceutical composition either contains the aforementioned RNAi preparation as is, or utilizes it. In order to avoid excessive complexity in this specification, any common elements between the two are omitted from the description.

[0055] Liver disease The aforementioned pharmaceutical composition can also be used as an active ingredient in pharmaceutical compositions for the prevention or treatment of liver disease by suppressing MARC1 gene expression.

[0056] The aforementioned liver diseases include fatty liver, hepatic fibrosis, and cirrhosis. Here, "fatty liver" refers to a condition in which triglycerides, which are not present in normal cells, appear to be abnormally deposited within liver cells. A normal liver is composed of approximately 5% adipose tissue, with triglycerides, fatty acids, phospholipids, cholesterol, and cholesterol esters being the main components of fat. However, once fatty liver develops, most of these components are replaced by triglycerides, and if the amount of triglycerides exceeds 5% of the liver weight, it is diagnosed as fatty liver. The aforementioned fatty liver is also known as "non-alcoholic fatty liver disease (NAFLD)," which is a disease in which triglycerides accumulate in the liver regardless of alcohol consumption. It can be defined as a condition in which fatty acids accumulate in the form of triglycerides in the parenchymal cells of the liver at a rate of 5% or more. The aforementioned non-alcoholic fatty liver disease is also known as simple steatosis or non-alcoholic steatohepatitis (NASH). Pathologically, this non-alcoholic fatty liver disease is classified into simple steatosis and non-alcoholic steatohepatitis (NASH) with inflammation, although NASH can be considered a severe form of NAFLD. Fatty liver disease with significant fat accumulation progresses to inflammation, i.e., steatohepatitis, and if left untreated for a long period, can develop into serious liver diseases such as hepatitis, liver fibrosis, and cirrhosis. The incidence of simple fatty liver disease is 10-15% in people with normal body weight, but it rises sharply to 80% in overweight individuals. Therefore, for non-alcoholic steatohepatitis to be effectively treated, it is important to effectively reduce triglyceride accumulation in the liver, suppress the progression of non-alcoholic steatohepatitis to fatty liver disease, and improve the inflammation of fatty liver disease, thereby preventing progression to the next stage.

[0057] Pharmaceutical composition In this specification, the term "effective ingredient" means an appropriate effective amount of an ingredient that is beneficial or has an effect on a desirable clinical or biochemical outcome. Specifically, it may mean an effective amount of a formulation, activator, or RNAi formulation.

[0058] The effective dose is also an appropriate amount administered once or more to prevent the disease or to relieve symptoms, reduce the extent of the disease, stabilize the disease (i.e., prevent exacerbation), slow or reduce the rate of disease progression, improve the disease, or provide temporary relief and reduction (partial or overall) of the disease state, without limitation.

[0059] In this specification, the term "prevention" means all actions that block the onset of a disease, suppress a disease, or delay its progression. For example, it means preventing or interfering with the onset of the liver disease or its characteristic features, or defending against or protecting against the onset of the liver disease or its characteristic features.

[0060] In this specification, the term "treatment" means both therapeutic treatment and preventive or protective measures. It also means all actions that improve or favorably alter the symptoms of a disease. For example, preventing, reducing or improving the liver disease or its characteristic features, or slowing (weakening) the progression of the liver disease or its characteristic features in an individual.

[0061] In this specification, the term “effective amount” refers to the meaning generally accepted in the industry. The term may generally mean the amount of a molecule, compound, or component that elicits an intended biological response (e.g., a beneficial response) in a cell, tissue, system, animal, or human being pursued by researchers, veterinarians, physicians, or other clinicians. Specifically, “therapeutic effective amount” may mean the amount of a molecule, compound, or component that elicits a desirable medical response such that a therapeutically relevant change occurs in a measurable parameter related to a disease or disorder, and a particular clinical treatment may be considered effective. The therapeutically effective amount of a drug for the treatment of the disease or disorder is also the amount required to produce a therapeutically relevant change in the parameter.

[0062] The method of administering the aforementioned pharmaceutical composition can be determined by a professional in the art based on the typical symptoms of a patient and the severity of the disease. Furthermore, it can be formulated in various forms such as powders, tablets, capsules, liquids, injections, ointments, and syrups, and can also be provided in single-dose or multi-dose containers, such as sealed ampoules and bottles.

[0063] The pharmaceutical compositions of the present invention can be administered orally or parenterally. The administration routes of the compositions according to the present invention are not limited to the following, but can include, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, sublingual, or topical administration. The dosage of the compositions according to the present invention varies widely depending on the patient's weight, age, sex, health status, diet, administration time, method, excretion rate, or disease severity, and can be easily determined by a typical expert in the art. Furthermore, for clinical administration, the compositions of the present invention can be formulated into suitable dosage forms using known techniques.

[0064] Another embodiment provides a method for preventing or treating liver disease, which includes the step of administering the RNAi preparation to an individual.

[0065] The methods for treating the aforementioned liver diseases either include the aforementioned RNAi preparations or pharmaceutical compositions as they are, or utilize them. To avoid excessive complexity in this specification, any commonalities between them are omitted.

[0066] In this specification, the term “individual” means an object requiring treatment for a disease, specifically liver disease, and more specifically, includes any primate, mouse, dog, cat, horse, cattle, sheep, pig, goat, camel, or geese, whether human or non-human.

[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 RNAi induction and evaluation of therapeutic efficacy 1-1. Evaluation of 50 asiRNA designs and their inhibitory effects on MARC1 mRNA expression. In this example, in order to secure a double-stranded nucleic acid molecule that induces highly efficient RNAi interference targeting MARC1, asiRNA (MARC1 asymmetric siRNA) was designed after selecting a target sequence for the MARC1 gene (sense strand (16-mer), antisense strand (19-mer)). Specifically, after obtaining MARC1 gene information via an NCBI database search, and considering animal experiments, a total of 50 asiRNAs were designed using a nucleotide sequence criterion that has a common target with mice, showing a certain level of homology. These were then synthesized on a 10 nmole scale at IDT Korea, Inc. Subsequently, the synthesized asiRNAs were annealed via incubation at 95°C for 5 minutes and 37°C for 25 minutes, followed by 12% polyacrylamide gel electrophoresis (PAGE), and then quality control (QC) was performed via a ChemiDoc UV transilluminator (Biorad).

[0069] The sequence information for the MARC1 asiRNA designed using the method described above is shown in Table 1 below.

[0070] [Table 1] TIFF0007853399000005.tif253161TIFF0007853399000006.tif187163

[0071] Subsequently, to confirm the expression repression efficiency at the mRNA level, Huh7 cells were plasma-infected with the MARC1 asiRNA, and then qRT-PCR was performed to measure the expression level of MARC1 mRNA. Specifically, the Huh7 cells were placed in a 96-well plate in 8 × 10⁶ wells. 3Cells were seeded into wells, and MARC1 asiRNA (10nM (OliX Inc.)) and RNAiMax (2μl / ml (Invitrogen Inc. 13778150)) were added. Then, phenotypic infection was performed according to the protocol provided by Invitrogen. Subsequently, all RNA was extracted using the RNeasy Plus Mini Kit (Qiagen 74136), and cDNA was synthesized via reverse transcription using the mRNA contained therein as a template. The PCR mixture was then 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 (NT) was a group that received only the phenotypic infection reagent. As a result, as shown in Figure 1, we confirmed the effect of treatment with 50 types of MARC1 asiRNA on suppressing MARC1 mRNA expression. 1-2. Evaluation of 23 GalNAc-asiRNA designs and their inhibitory effects on MARC1 mRNA expression.

[0072] In this embodiment, 23 types of MARC1 asiRNA produced in Example 1 were targeted, and a GalNAc ligand and chemical modification We designed asymmetric siRNAs incorporating diverse chemical modification MARC1 GalNAc-asiRNA was produced by introducing (2'OMe, PS, Fluoro, etc.) and attaching a derivative labeled "trivalent GalNAc" to the 3' end of the sense strand.

[0073] [Table 2] TIFF0007853399000008.tif84163

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

[0075] [Table 3]

[0076] Specifically, in Table 3 above, "*" means that the existing phosphodiester bond is replaced by a phosphorothioate bond, and "m" means that the existing 2'-OH is replaced by a 2'-O-methyl group. Also, "f" means, for example, in the case of fG, that the existing G (guanine) 2'-OH is replaced by a fluoro group, and "P" means that a phosphate group is attached to the 5' end (a phosphate group is attached to the oxygen bonded to the 5th carbon in the 5' terminal base). Furthermore, "GalNAc" means that a trivalent GalNAc derivative of chemical formula 1 below is attached 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 transfection-infected primary hepatocytes derived from C57BL / 6 mice, followed by qRT-PCR to measure the expression level of MARC1 mRNA. Specifically, the primary hepatocytes were placed in a 24-well plate in a 7.5 × 10⁶ format. 4Cells were seeded into wells and treated with MARC1 GalNAc-asiRNA (200nM (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 in the lysates as a template. Subsequently, quantitative PCR was performed using the synthesized cDNA as a template, employing THUNDERBIRD® Probe qPCR Mix (TOYOBO QPS-101) and Probe (Hs00224227_m1, Hs03928985_g1 (Applied Biosystems)). The expression level of MARC1 mRNA was then confirmed using the CFX Connect Real-Time PCR System (Bio-Rad). In this example, the control group consisted of a group not subjected to phenotypic infection (NT), a negative control group consisting of a group subjected to phenotypic infection using the protocol provided by Invitrogen after adding OLX700A-001-8 (10nM) and RNAiMAX (2μl / ml (Invitrogen Inc. 13778150)), and a positive control group (PC) consisting of asiMARC1-30 (10nM) and RNAiMAX (2μl / ml (Invitrogen Inc. 13778150)). After adding 13778150), a group (PC) was subjected to phenotypic infection according to the protocol provided by Invitrogen. The sequence information for OLX700A-001-8 is shown in Table 4, where "*", "m", "f", "P", and "GalNAc" are denoted. chemical modification This is as shown in Table 3 above.

[0079] [Table 4]

[0080] As a result, as shown in Figure 2, we confirmed the inhibitory effect of treatment with 23 types of MARC1 GalNAc-asiRNA on MARC1 mRNA expression. Among these, the inhibitory effects of OLX-002-1, OLX003-1, and OLX-031-1 were particularly outstanding.

[0081] 1-3. Evaluation of therapeutic efficacy using animal models In this example, C57BL / 6 mice (male, n=15) were administered MARC1 GalNAc-asiRNA, whose expression suppression effect was confirmed in Examples 1-2, and the therapeutic efficacy 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)). These groups were further classified according to the substance administered: a group administered 1X PBS (VC), a group administered OLX-001-8 (NC), a group administered OLX-003-1 subcutaneously (#3-1), and a group administered 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] Furthermore, MARC1 GalNAc-asiRNA was administered 16 weeks after the introduction of a high-fat diet, and MARC1 asiRNA was administered again 2 weeks later. Two weeks after the second administration, the livers were removed from the target mice, and liver tissue samples and sections were obtained therefrom.

[0084] (1) Confirmation of the expression patterns of MARC1 and MARC2 The expression levels of MARC1 mRNA and MARC2 mRNA were evaluated in liver tissue samples obtained from the aforementioned mice using the same method as in Examples 1-2.

[0085] As a result, as shown in Figure 3, the groups administered OLX-003-1 or OLX-031-1 according to one example (#3-1, #31-1) showed suppression of MARC1 mRNA expression compared to the other groups (VC, NC) that were provided with a high-fat diet, while MARC2 mRNA expression was not affected.

[0086] (2) Visual evaluation of the liver The livers obtained from the aforementioned mice were observed with the naked eye, and changes in the lesion sites of the liver were evaluated.

[0087] As a result, as shown in Figure 4, the groups provided with a high-fat diet (VC, NC) did not have sharp liver margins, i.e., lesion sites, while the groups administered OLX-003-1 or OLX-031-1 according to one example (#3-1, #31-1) showed morphological characteristics similar to the normal group.

[0088] (3) Histopathological evaluation Liver tissue samples obtained from the aforementioned mice were immobilized in a 10% NBF (neutral buffered formalin) solution to prepare paraffin-embedded blocks, from which 4 μm thick tissue sections were prepared. Subsequently, these tissue sections were stained with H&E using Mayer's hematoxylin reagent. Furthermore, 4 μm thick frozen sections obtained using OCT embedding medium were stained with Oil Red O.

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

[0090] Example 2. Evaluation of therapeutic efficacy using an animal model. In this example, C57BL / 6 mice were used as a template, with asiMARC1-031, whose expression suppression effect was confirmed in Example 1, being used as the subject. The MARC1 GalNAc-asiRNAs listed in Table 6 below were administered subcutaneously, and the therapeutic efficacy was evaluated.

[0091] [Table 6]

[0092] Specifically, in Table 6 above, the terms "*", "m", "f", "P", "GalNAc", and "EVP" are used. chemical modification This is as shown in Table 7 below.

[0093] [Table 7]

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

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

[0096] [Table 8]

[0097] Furthermore, OLX-031-1 was administered 28 weeks after the introduction of a high-fat diet, and thereafter, OLX-031-1 was administered a total of three times at one-week intervals. One week after the completion of the four total doses, the livers were removed from the target mice, and liver tissue samples, sections thereof, and serum samples were obtained.

[0098] (1) Confirmation of the expression patterns of MARC1 and MARC2 The expression levels of MARC1 mRNA and MARC2 mRNA were evaluated in liver tissue samples obtained from the aforementioned mice using the same method as in Examples 1-2.

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

[0100] (2) Visual evaluation of the liver The livers obtained from the aforementioned mice were observed with the naked eye, and changes in the lesion sites of the liver were evaluated.

[0101] As a result, as shown in Figure 8, the groups provided with a high-fat diet (HFDVC, HFDNC) did not have sharp liver margins, i.e., lesion sites, while the group administered OLX-31-1 according to one example (#31-1) showed morphological characteristics similar to the normal group.

[0102] (3) Histopathological evaluation Liver tissue samples obtained from the aforementioned mice were immobilized in a 10% NBF (neutral buffered formalin) solution to prepare paraffin-embedded blocks, from which 4 μm thick tissue sections were prepared. Subsequently, these tissue sections were subjected to H&E staining using Mayer's hematoxylin reagent and picrosilius red staining.

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

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

[0105] As a result, as shown in Figure 11, the expression level of α-SMA mRNA, a liver fibrosis-related factor, increased in the group provided with a high-fat diet (VC, NC), while in the group administered OLX-031-1 according to the first example (#31-1), such increases in α-SMA and mCol1α1 mRNA expression were reduced. Furthermore, as shown in Figure 12, the level of triglycerides in liver tissue was also reduced by the administration of OLX-031-1 according to the first example.

[0106] (5) Serological evaluation Serum samples obtained from the aforementioned mice were used to examine 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 lipid proteins (LDL), and high-density lipid proteins (HDL), which are lipid indicators.

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

[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 and duration of their diet into four groups: a group fed a normal diet for 12 weeks (NCD 12w), a group fed a normal diet for 16 weeks (NCD 16w), a group fed a high-fat diet for 12 weeks (CDHFD 12w), a group fed a high-fat diet for 16 weeks (CDHFD 16w), and a group fed a high-fat diet for 12 weeks and a normal diet for 4 weeks (CDHFD-NCD). These groups were further classified according to the substance administered: a group fed 1X PBS (VC) and a group fed OLX-031-2 (#31-2). In this example, the specific classification of the animal model groups is shown in Table 9 below.

[0109] [Table 9]

[0110] Furthermore, OLX-031-2 was administered 12 weeks after the introduction of the high-fat diet, and then again 2 weeks later. Two weeks after the second administration, the livers were removed from the target mice, and liver tissue samples and sections were obtained. In addition, in animal model groups 1 and 3 mentioned above, mice were sacrificed 12 weeks after the introduction of the high-fat diet to obtain the aforementioned samples.

[0111] (1) Histopathological evaluation Using the same method as in (3) of Example 2-1, H&E staining and picrosilius red staining were performed on tissue sections, and changes such as numerous lipid vacuoles and collagen deposition formed in the liver parenchyma were confirmed.

[0112] As a result, as shown in Figure 15, the groups provided with a high-fat diet (CDHFD 12w, CDHFD 16w) showed numerous lipid vacuoles and inflammatory foci in the liver parenchyma, while the groups administered OLX-031-2 according to one example (CDHFD 031-2, CDHFD-NCD 031-2) showed a reduction in such lipid vacuoles and inflammatory foci. Furthermore, as shown in Figures 16 and 17, lipid component and collagen deposition were significantly reduced in the groups administered OLX-031-2 according to one example (CDHFD 031-2, CDHFD-NCD 031-2). In particular, Group 6 (CDHFD 16w 031-2), which was administered OLX-031-2, showed reduced levels of lipid vacuoles and collagen deposition compared to Groups 3 and 4 (CDHFD 12w, CDHFD 16w), which were provided with a high-fat diet, indicating effective therapeutic efficacy against non-alcoholic steatohepatitis.

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

[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 group provided with a high-fat diet (CDHFD, CDHFD-NCD), while in the group administered OLX-031-2 according to one example (#CDHFD 031-2, CDHFD-NCD 031-2), such increases in the expression of α-SMA, mCol1α1, and TIMP1 mRNA were reduced. Also, similar to the experimental results mentioned above, group 6 (031-2 CDHFD), which was administered OLX-031-2, showed a significant difference compared to groups 3 and 4 (CDHFD 12w, CDHFD 16w), which were provided with a high-fat diet.

[0115] (3) Evaluation of the inhibitory effect on MARC1 mRNA expression The expression level of MARC1 mRNA was evaluated using the same method as in Examples 1-2, with respect to liver tissue samples obtained from the aforementioned mice.

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

[0117] Example 3: Secondary screening of double-stranded nucleic acid molecules for RNAi induction 3-1. Design and fabrication of 250 types of asiRNA In this embodiment, using the same method as described in 1-2 above, MARC1 gene information was obtained via an NCBI database search. Then, for both human and monkey-derived MARC1, sequences showing a certain level of homology were selected based on a nucleotide sequence criterion having a common target, and a total of 250 types of asiRNA were designed (sense strand (16-mer), antisense strand (21-mer)). These were then synthesized on a 10 nmole scale in a Bioneer. The sequence information of the MARC1 asiRNAs designed by the above method is shown in Tables 10 to 14 below.

[0118] [Table 10] TIFF0007853399000018.tif253147TIFF0007853399000019.tif187147

[0119] [Table 11] TIFF0007853399000021.tif252147TIFF0007853399000022.tif188148

[0120] [Table 12] TIFF0007853399000024.tif252148TIFF0007853399000025.tif186147

[0121] [Table 13] TIFF0007853399000027.tif252147TIFF0007853399000028.tif187147

[0122] [Table 14] TIFF0007853399000030.tif253148TIFF0007853399000031.tif187146

[0123] 3-2. Evaluation of the inhibitory effect on MARC1 mRNA expression To confirm the expression suppression efficiency at the mRNA level, Huh7 cells were plasma-infected with the MARC1 asiRNA, and then qRT-PCR was performed to measure the expression level of MARC1 mRNA. Specifically, the Huh7 cells were placed in a 96-well plate in 8 × 10⁶ wells. 3 Cells were seeded into wells, and then MARC1 asiRNA (10nM (OliX Inc.)) and RNAiMax (2μl / ml (Invitrogen Inc. 13778150)) were added. Plasma infection was then performed according to the protocol provided by Invitrogen. After 24 hours, cell lysates were prepared using the SuperPrep® Cell lysis & RT kit for qPCR Kit II (TOYOBO SCQ-401), and cDNA was synthesized via reverse transcription using the mRNA contained in the lysates as a template. Subsequently, quantitative PCR was performed using the synthesized cDNA as a template, employing THUNDERBIRD® Probe qPCR Mix (TOYOBO QPS-101) and Probe (Hs00224227_m1, Hs03928985_g1 (Applied Biosystems)). Based on the mRNA expression suppression level, a total of 134 MARC1 asiRNAs were initially selected (not shown). In this example, the control group consisted of a group to which only the phenotypic infection reagent was added (Mock), and a positive control group (PC1) was used, in which asiMARC1-30 was used and phenotypic infection was performed in the same manner as in Example 1-2. Subsequently, the MARC1 mRNA expression levels were evaluated for the 134 MARC1 asiRNAs selected as described above, while sequentially changing only the MARC1 asiRNA treatment concentration to 1 nM or 0.1 nM using the same method as described above.

[0124] As a result, as shown in Figures 20A to 20C, the inhibitory effect of 1nM treatment with 134 types of MARC1 asiRNA on MARC1 mRNA expression was confirmed, and among them, the top 41 types of MARC1 mRNA exhibited superior MARC1 expression inhibitory efficiency were identified. asiRNA was 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 expression suppression effect of 41 types of MARC1 asiRNAs treated with 0.1 nM was confirmed, and from these, the top 30 types of MARC1 asiRNAs with superior 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 inhibitory effect on MARC1 protein expression To confirm the efficiency of expression suppression at the protein level, Huh7 cells were transfected with the 30 MARC1 asiRNAs selected in Example 3-2, and then Western blotting was performed to measure the expression level of the MARC1 protein. Specifically, the Huh7 cells were placed in a 12-well plate with 5 × 10⁶ cells. 4Cells were seeded into wells, and MARC1 asiRNA (1nM (OliX Inc.) and RNAiMax (2 μl / ml (Invitrogen Inc. 13778150))) were added. Transfection was then performed according to the protocol provided by Invitrogen. After 48 hours, the transfected cells were lysed to obtain 15 μg of cell lysate from each sample. Western blotting was performed on the obtained cell lysates using a 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 the MARC1 protein was confirmed using System (Bio-Rad). In this example, the control group consisted of a group to which only the phenotypic infection reagent was added (M), and the positive control group consisted of groups (PC1, PC2) that underwent phenotypic infection using asiMARC1-30 or asiMARC1-31 in the same manner as in Example 1-2.

[0126] As a result, as shown in Figure 22, it was confirmed that all 30 types of MARC1 asiRNA selected in Example 3-2 suppressed the expression of the MARC1 protein. 。

[0127] Example 4: Production of chemically modified nucleic acid molecules for RNAi induction 4-1. Design and fabrication of 41 types of MARC1 GalNAc-asiRNA In this embodiment, the MARC1 asiRNA whose expression suppression effect was confirmed in Examples 1 and 3 was targeted, and GalNAc ligand and chemical modification We designed an asymmetric siRNA (GalNAc-asiRNA) incorporating [a specific compound]. Compared to the aforementioned asiRNA, the designed GalNAc-asiRNA exhibits a diverse range of [various characteristics]. chemical modification These are asymmetric siRNAs into which ligands (such as 2'OMe, PS, Fluoro, and GalNAc) have been introduced, resulting in improved transmission into hepatocytes. The sequence information for all 41 MARC1 GalNAc-asiRNAs prepared in this example is shown in Table 15 below.

[0128] [Table 15] TIFF0007853399000033.tif253163TIFF0007853399000034.tif70162

[0129] Specifically, in Table 15 above, the terms "*", "m", "f", "P", and "GalNAc" are used. chemical modification This is as shown in Table 7 above.

[0130] 4-2. Evaluation of the inhibitory effect on MARC1 mRNA expression To confirm the expression suppression efficiency at the mRNA level, each of the 41 types of MARC1 GalNAc-asiRNA (100nM) 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 using the same method as in Example 1-2 to measure the expression level of MARC1 mRNA. In this example, the control group consisted of a group that was not treated with any substance (NT), a negative control group treated with OLX700A-001-8 (100nM) (NC), and a positive control group treated with OLX-031-1 (10nM) (PC).

[0131] As a result, as shown in Figure 23, we confirmed the inhibitory effect of treatment with 41 types of MARC1 GalNAc-asiRNA on MARC1 mRNA expression. Among these, the inhibitory effects on 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 particularly outstanding.

[0132] 4-3. Evaluation of the inhibitory effect of MARC1 mRNA expression on human-derived 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. Specifically, the primary hepatocytes were placed in a 96-well plate in a 4 × 10⁶ well. 4 Cells were seeded into wells and treated with MARC1 GalNAc-asiRNA (500nM) (OliX Inc.). QRT-PCR was then performed using the same method as in Examples 1-2, and the expression level of MARC1 mRNA was measured. In this example, the control group consisted of an untreated group (NT), a negative control group treated with OLX700A-001-8 (500nM) (NC), and a positive control group treated with OLX-031-1 (10nM) (PC).

[0133] Furthermore, in order to confirm the efficiency of expression suppression at the mRNA level depending on the treatment concentration of MARC1 GalNAc-asiRNA, the aforementioned human-derived primary hepatocytes were placed in a 96-well plate in a 3 × 10⁶ format. 4Cells were seeded into wells and treated with 20 nM or 100 nM MARC1 GalNAc-asiRNA (OliX Inc.), respectively (n=2). Subsequently, qRT-PCR was performed using the same method as in Examples 1-2, and the expression level of MARC1 mRNA was measured. In this example, the control groups were: an untreated group (NT), a negative control group treated with OLX700A-001-8 (100 nM) (NC), and a positive control group treated with OLX-075-1 or OLX-218-1 (10 nM) (PC1 and PC2).

[0134] As a result, as shown in Figure 24, the inhibitory effect of treatment with 41 types of MARC1 GalNAc-asiRNA on MARC1 mRNA expression was confirmed. Furthermore, 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 were selected that had superior efficacy to OLX-031-1 secured in Example 1 (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).

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

[0136] [Table 16]

[0137] Specifically, in Table 16 above, the terms "*", "m", "f", "P", "GalNAc", and "EVP" are used. chemical modification This is as shown in Table 7 above.

[0138] Furthermore, to confirm the expression suppression efficiency at the mRNA level, each of the 17 MARC1 GalNAc-asiRNAs prepared as described 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 placed in a 96-well plate in a 4 × 10⁶ format. 4 Cells were seeded into wells and treated with MARC1 GalNAc-asiRNA (10nM or 100nM (OliX Inc.)). qRT-PCR was then performed using the same method as in Examples 1-2, and the expression level of MARC1 mRNA was measured.

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

[0140] Example 6. Evaluation of the inhibitory effect of MARC1 mRNA expression using a monkey model. In a monkey animal model, OLX-031-2 or OLX-075-2 were administered subcutaneously at various concentrations (2.5 mp, 5 mp, or 10 mp) twice, once a week apart. One week after the second administration, the livers were removed from the monkeys, and liver tissue samples and sections were obtained. Subsequently, qRT-PCR was performed using the same method as in Example 1-2, and the expression level of MARC1 mRNA was measured. In this example, the sequence information for OLX-031-2 and OLX-075-2 is shown in Table 17 below, and the specific classification related to the animal model group is shown in Table 18 below.

[0141] [Table 17]

[0142] [Table 18]

[0143] As a result, as shown in Figure 27, an even better suppression of MARC1 mRNA expression was confirmed in the OLX-075-2 administration group, and such an effect showed a concentration-dependent trend.

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

[0145] [Table 19]

[0146] Furthermore, four weeks after phenotypic infection with the aforementioned plasmid, MARC1 GalNAc-asiRNA was administered subcutaneously. One week later, blood samples were collected, and the level of SEAP reporter fluorescence was confirmed via the Phospha-Light® SEAP Reporter Gene Assay System (Invitrogen® T1015) to compare the levels 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, and among them, the inhibitory effect was even more pronounced in the groups treated with 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 a dual luciferase reporter Six-week-old Balb / c mice were transfected with a plasmid containing the human MARC1 gene (psiCHECK-2-hMARC1), and then subcutaneously administered 3 mpk of the MARC1 GalNAc-asiRNA from 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). Three days later, blood samples were collected, and the level of luciferase reporter fluorescence was confirmed via the Dual-Luciferase® Reporter Assay System (Promega E1980) to compare the levels of suppression of human MARC1 mRNA expression.

[0149] As a result, as shown in Figure 29, diverse chemical modificationIn Example 5, all MARC1 GalNAc-asiRNA administration groups showed excellent suppression of MARC1 mRNA expression, and among them, the OLX-075-16, OLX-075-17, OLX-075-12, OLX-075-8, and OLX-075-8 administration groups showed even better suppression of expression.

[0150] Although specific aspects of the present invention have been described in detail above, it will be obvious to those with ordinary skill in the art that such specific technologies are merely desirable 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. The present invention encompasses the following embodiments. [1] An RNAi preparation comprising an antisense strand which is a nucleotide (nt) of 19 to 21 nt in length and which is sequence-complementary to the MARC1 (mitochondrial amidoxime reducing component 1) mRNA sequence, and a sense strand which is sequence-complementary to the antisense strand and which is sequence-complementary to the antisense strand and which is 15 to 17 nt in length, wherein the 5' end of the antisense strand and the 3' end of the sense strand form a blunt end. [2] The RNAi preparation according to [1], wherein the sense strand has a sequence selected from among the antisense strand sequences listed in Table 1, Table 10, Table 11, Table 12, Table 13 and Table 14. [3] The RNAi preparation described in [1], wherein the sense strand is selected from the group consisting of SEQ ID NOs: 149, 159, 177, 179, 183, 197, 199, 227, 247, 251, 257, 259, 261, 273, 285, 287, 331, 353, 387, 391, 399, 423, 429, 431, 435, 439, 443, 445, 447, and 527. [4] The RNAi preparation according to [1], wherein the sense strand is 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. [5] The RNAi preparation described in [1], wherein the antisense strand has a sequence selected from the sense strand sequences listed in Tables 1, 10, 11, 12, 13 and 14. [6] The RNAi preparation described in [1], wherein the antisense strand is selected from the group consisting of SEQ ID NOs: 150, 160, 178, 180, 184, 198, 200, 228, 248, 252, 258, 260, 262, 274, 286, 288, 332, 354, 388, 392, 400, 424, 430, 432, 436, 440, 444, 446, 448, and 528. [7] The RNAi preparation according to [1], wherein the antisense strand is 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. [8] The RNAi preparation is the RNAi preparation described in [1] that suppresses the expression of MARC1. [9] The RNAi preparation according to [1], wherein the sense strand is conjugated to an N-acetylgalactosamine (GalNAc) derivative at its 3' end.

[10] The RNAi preparation according to [1], comprising one or more chemical modifications, the sense strand or the antisense strand.

[11] The sense strand comprises one or more chemical modifications selected from the following: the RNAi preparation described in

[10] : The 5' end is modified by altering two to four adjacent nucleotide bonds with a phosphorothioate, boranophosphate, or methylphosphonate; In one or more nucleotides, the -OH group at the 2' carbon position of the sugar structure is substituted with -CH3(methyl), -OCH3(methoxy), -NH2, -F, -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl or -O-3-dimethylaminopropyl; and Binding to an N-acetylgalactosamine (GalNAc) derivative or cell-penetrating peptide at the 3' end.

[12] The RNAi preparation according to

[10] , comprising one or more chemical modifications selected from the following: Deform two to seven adjacent nucleotide bonds from the 3' or 5' end with a phosphorothioate, boranophosphate, or methylphosphonate; The -OH group at the 2' carbon position of the sugar structure in one or more nucleotides is substituted with -CH3(methyl), -OCH3(methoxy), -NH2, -F, -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; and Binding to a phosphate group, E-vinylphosphonate, or cell-penetrating peptide at the 5' end.

[13] The RNAi preparation is In a sense strand or antisense strand, two to seven adjacent nucleotides are linked from the 3' or 5' end, To be deformed by a phosphorothioate; A modification in which the -OH group at the 2' carbon position of a sugar structure in one or more nucleotides of a sense strand or antisense strand is replaced with -OCH3(methoxy) or -F; Binding of N-acetylgalactosamine (GalNAc) derivatives at the 3' terminus of the sense strand; and The RNAi preparation according to

[10] , comprising one or more modifications selected from the group consisting of binding of a phosphate group or E-vinylphosphonate at the 5' end of an antisense strand.

[14] The RNAi preparation according to

[10] , wherein the sense strand has a sequence selected from among the antisense strand sequences listed in Tables 2, 15, 16 and 17.

[15] The RNAi preparation according to

[10] , wherein the antisense strand has a sequence selected from among the antisense strand sequences listed in Tables 2, 15, 16 and 17. A pharmaceutical composition for the prevention or treatment of liver disease, comprising an RNAi preparation described in any of

[16] [1] to

[15] as an active ingredient.

[17] The liver disease is fatty liver, hepatic fibrosis, or cirrhosis, a pharmaceutical composition for the prevention or treatment of the liver disease according to

[16] .

[18] The fatty liver is non-alcoholic fatty liver disease (NAFLD), and the pharmaceutical composition for the prevention or treatment of the liver disease described in

[17] .

[19] The non-alcoholic fatty liver disease is simple steatosis or non-alcoholic steatohepatitis (NASH), a pharmaceutical composition for the prevention or treatment of the liver disease described in

[18] . A method for preventing or treating liver disease, comprising the step of administering an RNAi preparation described in any of

[20] [1] to

[15] to an individual.

[21] Uses of RNAi preparations described in any of [1] to

[15] for the manufacture of pharmaceuticals for the prevention or treatment of liver disease.

Claims

1. An RNAi preparation comprising an antisense strand having sequence complementarity to the MARC1 (mitochondrial amidoxime reducing component 1) mRNA sequence and consisting of the nucleotide sequence shown in SEQ ID NO: 150, and a sense strand having sequence complementarity to the antisense strand and consisting of the nucleotide sequence shown in SEQ ID NO:

149. The 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 preparation according to claim 1, wherein the RNAi preparation suppresses the expression of MARC1.

3. The aforementioned sense strand has an N-acetylgalactosamine (GalNAc: N-acetylgalactosamine) derivative represented by the following formula (I) at its 3' end: 【Chemistry 1】 The RNAi preparation according to claim 1, which is bound to the RNAi preparation according to claim 1.

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

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

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

7. The aforementioned RNAi preparation is In a sense strand or antisense strand, two to seven adjacent nucleotides are linked from the 3' or 5' end, Modification with phosphorothioate; Modifications in which an -OH group is replaced with -OCH3(methoxy) or -F at the 2' carbon position of a sugar structure in one or more nucleotides of a sense strand or antisense strand; The N-acetylgalactosamine (GalNAc: N-acetylgalactosamine) derivative represented by the following formula (I) at the 3' end of the sense strand: 【Transformation 3】 The union with; and The RNAi formulation according to claim 4, comprising one or more modifications selected from the group consisting of binding of a phosphate group or E-vinylphosphonate at the 5' end of an antisense strand.

8. The sense strands and antisense strands are selected from the sequences listed in the table below, and the combinations of sense strands and antisense strands are: 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), An RNAi preparation according to claim 4, selected from the above. Table 1

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

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

11. The pharmaceutical composition for the prevention or treatment of a 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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