RNAi agents targeting APOC3 gene and uses thereof

Abstract

The invention provides an RNAi (Ribonucleic Acid Interference) agent targeting an APOC3 gene and application of the RNAi agent. More specifically, provided are an RNAi agent comprising an antisense strand having sequence complementarity with the APOC3mRNA sequence and a sense strand having sequence complementarity with the antisense strand, and a pharmaceutical composition for preventing or treating a lipid metabolism-related disease comprising the RNAi agent.

Description

Technical Field

[0001] This disclosure relates to an RNAi agent targeting the apolipoprotein C-III (APOC3) gene and its use, and more particularly, to an RNAi agent comprising an antisense strand that is sequence complementary to the APOC3 mRNA sequence and a sense strand that is sequence complementary to the antisense strand, and a pharmaceutical composition comprising the RNAi agent for the prevention or treatment of lipid metabolism-related diseases. Background Technology

[0002] Adipose tissue, or body fat, is a loose connective tissue primarily composed of adipocytes. In addition to adipocytes, adipose tissue can also include stromal vascular component (SVF) cells, such as preadipocytes, fibroblasts, vascular endothelial cells, and various immune cells. Adipose tissue is derived from preadipocytes. The main functions of adipose tissue are not only to protect and shield the body from impacts, but also to store energy in the form of lipids. However, excessive accumulation of fat in adipose tissue is undesirable and can lead to pathological conditions such as obesity, liposis, and hyperlipidemia.

[0003] Typically, when cholesterol levels are elevated, cholesterol metabolism is dysregulated, causing changes in the levels of lipoprotein components such as low-density lipoprotein cholesterol (LDL-cholesterol) and high-density lipoprotein cholesterol (HDL-cholesterol). Specifically, LDL-cholesterol is considered a risk factor for cardiovascular disease, and elevated blood triglyceride levels are known to be a major cause of coronary artery disease because they lower HDL-cholesterol levels and increase the amount of chylomicron remnants (lipoprotein particles primarily composed of triglycerides). Furthermore, because lipids cross vascular endothelial cells more easily than other lipids, they are considered a major cause of atherosclerosis, and a rapid increase in postprandial triglyceride levels is known to be closely associated with stroke.

[0004] Meanwhile, apolipoprotein C-III (APOC3) is primarily synthesized in the liver and plays a crucial role in the production, metabolism, and clearance of triglyceride (TG)-rich lipoproteins in plasma. Specifically, increased APOC3 in the liver promotes the secretion of TG-rich very low-density lipoprotein-C (VLDL-C). Furthermore, excessive APOC3 inhibits the activity of lipoprotein lipase and hepatic lipase, contributing to increased serum TG levels by delaying the catabolism of TG-rich lipoproteins. In addition, increased APOC3 can delay the clearance of TG-rich lipoproteins and their residual particles by interfering with the binding of these particles to hepatic receptors. Therefore, elevated APOC3 levels lead to the development of hypertriglyceridemia or result in higher blood TG levels. Furthermore, elevated TG levels can contribute to the development of various diseases, including cardiovascular disease, atherosclerosis, non-alcoholic fatty liver disease, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes (insulin resistance), and hypertension (see KR 10-2020-0074975).

[0005] Therefore, as a result of in-depth research efforts to develop new drugs for the treatment of lipid metabolism-related diseases, the inventors of this invention have developed an RNAi agent capable of binding to APOC3 mRNA to specifically inhibit its expression, thus completing this disclosure. Summary of the Invention

[0006] An RNAi agent that specifically inhibits the expression of apolipoprotein C-III (APOC3) is provided.

[0007] A pharmaceutical composition for the prevention or treatment of lipid metabolism-related diseases is provided, as well as a method for the prevention or treatment of lipid metabolism-related diseases, the pharmaceutical composition comprising an RNAi agent, the method comprising administering the pharmaceutical composition to a subject.

[0008] Other aspects will be set forth in part in the description which follows, and will be apparent in part from the description, or may be learned by practicing the embodiments presented in this disclosure.

[0009] According to this disclosure, the RNAi agent includes an antisense strand that is sequence complementary to the APOC3 mRNA sequence and has a length of 19 nucleotides (nt) to 21 nt, and a sense strand that is sequence complementary to the antisense strand and has a length of 15 nt to 17 nt, wherein the 5' end of the antisense strand and the 3' end of the sense strand form a blunt end.

[0010] According to another aspect of this disclosure, pharmaceutical compositions for the prevention or treatment of lipid metabolism-related diseases include an RNAi agent as an active ingredient.

[0011] According to another aspect of this disclosure, methods for preventing or treating lipid metabolism-related diseases include administering RNAi agents to subjects.

[0012] According to another aspect of this disclosure, the use of RNAi agents in the preparation of pharmaceutical products for the prevention or treatment of lipid metabolism-related diseases is provided. Attached Figure Description

[0013] The above and other aspects, features and advantages of certain embodiments of this disclosure will become more apparent from the following description taken in conjunction with the accompanying drawings, wherein:

[0014] Figure 1 This is a schematic diagram of the structure of APOC3 asiRNA, which consists of a 16-mer sense strand and a 21-mer antisense strand.

[0015] Figure 2a The results show the results of confirming the expression level of APOC3 mRNA after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-001 to asiAPOC3-032) according to one aspect at a concentration of 1 nM;

[0016] Figure 2b The results show the results of confirming the expression level of APOC3 mRNA after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-033 to asiAPOC3-064) at a concentration of 1 nM, according to one aspect.

[0017] Figure 2c The results show the results of confirming the expression level of APOC3 mRNA after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-065 to asiAPOC3-096) according to one aspect at a concentration of 1 nM;

[0018] Figure 3a The results show the results of confirming the expression level of APOC3 mRNA after treating Huh-7 cells with 34 APOC3 asiRNAs (asiAPOC3-097 to asiAPOC3-130) according to one aspect at a concentration of 1 nM;

[0019] Figure 3b The results show the results of confirming the expression level of APOC3 mRNA after treating Huh-7 cells with 34 APOC3 asiRNAs (asiAPOC3-131 to asiAPOC3-164) according to one aspect at a concentration of 1 nM;

[0020] Figure 3cThe results show the results of confirming the expression level of APOC3 mRNA after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-165 to asiAPOC3-196) according to one aspect at a concentration of 1 nM;

[0021] Figure 4a The results show the results of confirming the expression level of APOC3 mRNA after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-001 to asiAPOC3-032) according to one aspect at a concentration of 0.1 nM;

[0022] Figure 4b The results show the results of confirming the expression level of APOC3 mRNA after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-033 to asiAPOC3-064) according to one aspect at a concentration of 0.1 nM;

[0023] Figure 4c The results show the results of confirming the expression level of APOC3 mRNA after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-065 to asiAPOC3-096) according to one aspect at a concentration of 0.1 nM;

[0024] Figure 5a The results show the results of confirming the expression level of APOC3 mRNA after treating Huh-7 cells with 34 APOC3 asiRNAs (asiAPOC3-097 to asiAPOC3-130) according to one aspect at a concentration of 0.1 nM;

[0025] Figure 5b The results show the results of confirming the expression level of APOC3 mRNA after treating Huh-7 cells with 34 APOC3 asiRNAs (asiAPOC3-131 to asiAPOC3-164) according to one aspect at a concentration of 0.1 nM;

[0026] Figure 5c The results show the results of confirming the expression level of APOC3 mRNA after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-165 to asiAPOC3-196) according to one aspect at a concentration of 0.1 nM;

[0027] Figure 6 The results show the results of confirming the expression level of APOC3 mRNA after treating primary human hepatocytes with 40 APOC3 asiRNAs according to one aspect at a concentration of 1 nM;

[0028] Figure 7 The results show the results of confirming the expression level of APOC3 mRNA after treating primary human hepatocytes with 40 APOC3 asiRNAs according to one aspect at a concentration of 0.1 nM;

[0029] Figure 8a and Figure 8b The results show the results of confirming the expression level of APOC3 mRNA after treating primary human hepatocytes with 40 and 44 chemically modified APOC3 GalNAc-asiRNAs according to one aspect at a concentration of 200 nM.

[0030] Figure 9a and Figure 9b The results show the results of confirming the expression level of APOC3 mRNA after treating primary human hepatocytes with 21 chemically modified APOC3 GalNAc-asiRNAs according to one aspect at concentrations of 20 nM or 100 nM.

[0031] Figure 10 The results show the results of confirming the level of APOC3 in mouse serum after a single subcutaneous injection of 1 mg / kg (mpk) into transgenic mice expressing APOC3 according to one of 10 chemically modified APOC3 GalNAc-asiRNAs.

[0032] Figure 11 The results show the results of confirming the level of APOC3 in mouse serum after a single subcutaneous injection of 1 mg / kg (mpk) into transgenic mice expressing APOC3 according to eight chemically modified APOC3 GalNAc-asiRNAs according to one aspect;

[0033] Figure 12 The results show the results of confirming serum triglyceride levels in mice after a single subcutaneous injection of 1 mg / kg (mpk) into transgenic mice expressing APOC3 according to eight chemically modified APOC3 GalNAc-asiRNAs.

[0034] Figure 13 The results show the total cholesterol levels in mouse serum confirmed after a single subcutaneous injection of 1 mg / kg (mpk) into transgenic mice expressing APOC3 according to eight chemically modified APOC3 GalNAc-asiRNAs.

[0035] Figure 14a The results show the results of confirming the expression level of APOC3 mRNA after treating primary human hepatocytes with 12 chemically modified APOC3GalNAc-asiRNAs according to one aspect at a concentration of 200 nM;

[0036] Figure 14b The results show the results of confirming the expression level of APOC3 mRNA after treating primary human hepatocytes with 12 chemically modified APOC3GalNAc-asiRNAs according to one aspect at a concentration of 200 nM;

[0037] Figure 14c The results show the results of confirming the expression level of APOC3 mRNA after treating primary human hepatocytes with 12 chemically modified APOC3GalNAc-asiRNAs according to one aspect at a concentration of 200 nM;

[0038] Figure 15a The results show the results of confirming the expression level of APOC3 mRNA after treating primary human hepatocytes with 12 chemically modified APOC3GalNAc-asiRNAs according to one aspect at a concentration of 20 nM;

[0039] Figure 15b The results show the results of confirming the expression level of APOC3 mRNA after treating primary human hepatocytes with 12 chemically modified APOC3GalNAc-asiRNAs according to one aspect at a concentration of 20 nM;

[0040] Figure 15c The results show the results of confirming the expression level of APOC3 mRNA after treating primary human hepatocytes with 12 chemically modified APOC3GalNAc-asiRNAs according to one aspect at a concentration of 20 nM;

[0041] Figure 16a The results show the results of confirming the expression level of APOC3 mRNA after treating primary human hepatocytes with 12 chemically modified APOC3GalNAc-asiRNAs according to one aspect at a concentration of 2 nM;

[0042] Figure 16b The results show the results of confirming the expression level of APOC3 mRNA after treating primary human hepatocytes with 12 chemically modified APOC3GalNAc-asiRNAs according to one aspect at a concentration of 2 nM;

[0043] Figure 16c The results show the results of confirming the expression level of APOC3 mRNA after treating primary human hepatocytes with 12 chemically modified APOC3GalNAc-asiRNAs according to one aspect at a concentration of 2 nM;

[0044] Figure 17The results show the results of confirming the level of APOC3 in mouse serum after a single subcutaneous injection of 0.25 mg / kg (mpk) into transgenic mice expressing APOC3 according to seven chemically modified APOC3 GalNAc-asiRNAs according to one aspect;

[0045] Figure 18 The results show the serum triglyceride levels in mice after a single subcutaneous injection of 0.25 mg / kg (mpk) into transgenic mice expressing APOC3, based on seven chemically modified APOC3GalNAc-asiRNAs according to one aspect; and

[0046] Figure 19 The results show the results of confirming the total cholesterol level in mouse serum after a single subcutaneous injection of 0.25 mg / kg (mpk) into transgenic mice expressing APOC3 according to seven chemically modified APOC3GalNAc-asiRNAs. Detailed Implementation

[0047] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein the same reference numerals refer to the same elements throughout. In this respect, embodiments of the invention may take different forms and should not be construed as limited to the descriptions listed herein. Therefore, the embodiments are described below by reference only to the figures to explain aspects of this description. As used herein, the term “and / or” includes any and all combinations of one or more of the related items listed. When an expression (such as “at least one,”) precedes a list of elements, it modifies the entire list of elements, not a single element in the list.

[0048] Each description and embodiment disclosed in this application can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this application are within the scope of this application. Furthermore, it should not be construed that the scope of this application is limited to the detailed description described below.

[0049] On the one hand, an RNAi agent is provided, comprising: an antisense strand with sequence complementarity to the apolipoprotein C-III (APOC3) mRNA sequence and a length of 19 nucleotides (nt) to 21 nt; and a sense strand with sequence complementarity to the antisense strand and a length of 15 nt to 17 nt, wherein the 5' end of the antisense strand and the 3' end of the sense strand form a blunt end.

[0050] RNAi agents

[0051] The term "RNA interference (RNAi)" refers to a mechanism that inhibits the expression of a target gene by introducing a double-stranded RNA (dsRNA) consisting of a strand that is sequence homologous to the mRNA of the target gene and a strand that has a sequence complementary to the aforementioned strand, thereby inducing the degradation of the target gene's mRNA.

[0052] As used in this specification, the term "RNAi agent" or "RNAi-inducing nucleic acid molecule" refers to any agent or nucleic acid molecule capable of inhibiting or downregulating gene expression or viral replication by mediating RNA interference in a sequence-specific manner. This term can refer to a single nucleic acid molecule, multiple nucleic acid molecules, or a pool of nucleic acid molecules. In the examples, the RNAi agent may be siRNA.

[0053] The term “small interfering RNA (siRNA; short interfering RNA)” used in this article refers to short double-stranded RNA (dsRNA) that mediates effective gene silencing with sequence specificity.

[0054] As used in this specification, the term "gene" should be considered in its broadest sense and may encode structural or regulatory proteins. Here, regulatory proteins include transcription factors, heat shock proteins, or proteins involved in DNA / RNA replication, transcription, and / or translation. In this disclosure, target genes subject to expression repression are inherent to the viral genome and may be integrated into the animal genome or exist as extrachromosomal components.

[0055] As used in this article, the term "antisense strand" refers to a polynucleotide that is substantially or 100% complementary to the target nucleic acid and can be fully or partially complementary to, for example, messenger RNA (mRNA), non-mRNA RNA sequences (e.g., microRNA, piwiRNA, tRNA, rRNA, and hnRNA), or coding or non-coding DNA sequences.

[0056] As used in this article, the term "sense chain" refers to a polynucleotide that has the same nucleic acid sequence as the target nucleic acid, or a polynucleotide that is completely or partially identical to, for example, messenger RNA (mRNA), non-mRNA sequences (e.g., microRNA, piwiRNA, tRNA, rRNA, and hnRNA), or coding or non-coding DNA sequences.

[0057] As used herein, the term "complementarity" or "complementarity" has the meaning generally accepted in the art. This term typically refers to the formation or presence of one or more hydrogen bonds between one nucleic acid sequence and another via conventional Watson-Crick bonding or other non-traditional types of bonding as described herein. Complete complementarity can refer to all consecutive residues of a nucleic acid sequence forming hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. Partial complementarity within a nucleic acid molecule may include multiple mismatched or non-base-paired nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mismatches, such as 1 to 3 mismatches, non-nucleotide linkers, or non-base-paired nucleotides). Partial complementarity can result in protrusions, loops, overhangs, or flat ends between the sense and antisense strands of a nucleic acid molecule or between the antisense strand of a nucleic acid molecule and the corresponding target nucleic acid molecule.

[0058] As used herein, the term "flat-ended" has the meaning generally accepted in the art. Regarding RNAi agents or nucleic acid molecules as used herein, the term may refer to the ends of a double-stranded siRNA molecule lacking protruding nucleotides. The siRNA molecules described herein allow for a flat-ended 5' end of the antisense strand and a flat-ended 3' end of the sense strand.

[0059] RNAi agents for inhibiting APOC3 expression

[0060] The term "apolipoprotein C-III (APOC3)" describes a component of very low-density lipoprotein (VLDL), which plays a crucial role in the production, metabolism, and clearance of triglyceride-rich lipoproteins in plasma. Specifically, APOC3 inhibits lipoprotein lipase, which hydrolyzes triglycerides into free fatty acids. Additionally, APOC3 inhibits ApoE-mediated hepatic uptake of triglyceride-rich lipoproteins via the low-density lipoprotein receptor (LDLR) and LDL receptor-associated protein (LRP), as well as via receptor-independent endocytosis, and promotes hepatic secretion of VLDL. Furthermore, according to recent studies, at least one mutation in the APOC3 gene has been reported to be highly associated with improved lipid profiles. Therefore, the APOC3 gene may be a potential therapeutic target for lipid metabolism-related diseases. APOC3 can be interpreted as including naturally occurring wild-type APOC3 and its functional variants, and sequences of APOC3 or the genes encoding it can be obtained from known databases such as GenBank of the US National Institutes of Health.

[0061] As used herein, the term "expression" has any meaning generally accepted in the art. The term typically 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 in this specification, expression levels can be determined or monitored by detecting mRNA or protein levels.

[0062] The terms “inhibition” or “reduction” (as used when referring to APOC3 gene expression in subjects) refer to a statistically significant reduction compared to the untreated or normal control group. The reduction can be, for example, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or higher, but may be below the detection limit depending on the detection or measurement method.

[0063] siRNA is a small interfering RNA that participates in RNA interference (RNAi). RNAi is an intracellular gene regulation mechanism first discovered in Caenorhabditis elegans in 1998. Its mechanism of action is known to be the induction of target gene degradation through an antisense strand, which is one of the RNA double strands introduced into the cell and binds complementary to the mRNA of the target gene. Recently, RNAi has become one of the most popular candidate technologies for developing new drugs.

[0064] However, contrary to this potential, side effects and drawbacks of siRNA are constantly being reported. To develop RNAi-based therapeutics, several obstacles must be overcome, such as 1) the lack of efficient delivery systems, 2) off-target effects, 3) induction of immune responses, and 4) saturation of RNAi mechanisms in cells. Although siRNA is an effective method for directly regulating target gene expression, these problems hinder the development of therapeutics. In this regard, asymmetric shorter double-stranded siRNA (asiRNA) is an asymmetric RNAi-inducing structure with a shorter double helix length compared to the 19+2 structure of existing siRNAs. asiRNA technology overcomes the problems of off-target effects, RNAi mechanism saturation, and TLR3 immune responses present in existing siRNA structural technologies, thus potentially enabling the development of new RNAi drugs with lower side effects.

[0065] Based on this, this embodiment presents an asymmetric siRNA comprising a sense strand and an antisense strand complementary to the sense strand. Since the siRNA according to this embodiment does not cause off-target effects or RNAi mechanism saturation, the siRNA can effectively suppress the expression of the APOC3 gene to the desired level while stably maintaining high delivery efficiency.

[0066] In the embodiments, asymmetric siRNA (asiRNA) targeting APOC3 was designed and prepared, and nucleic acid molecules that induce RNAi with excellent knockdown efficiency, namely APOC3asiRNA, were screened by using cell or animal models expressing APOC3.

[0067] In embodiments, the RNAi agent may include a sense strand of 15 to 17 nt in length and an antisense strand of 19 to 21 nt in length. In embodiments, the sense strand may be 16 nt in length, and the complementary antisense strand may be 19 nt, 20 nt, or 21 nt in length, but the embodiments are not particularly limited thereto.

[0068] The 5' end of the antisense chain and the 3' end of the sense chain can form a flat end. The 3' end of the antisense chain may include, for example, a protruding end of 2 to 6 nt.

[0069] In an embodiment, the sense chain may have a sequence selected from the sense chain sequences listed in Tables 1 to 4, and the antisense chain may have a sequence selected from the antisense chain sequences listed in Tables 1 to 4.

[0070] In embodiments, the sense chain may have any sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:45, SEQ ID NO:165, SEQ ID NO:171, SEQ ID NO:187, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:201, SEQ ID NO:207, SEQ ID NO:255, SEQ ID NO:287, SEQ ID NO:335, SEQ ID NO:337, SEQ ID NO:339, SEQ ID NO:341, SEQ ID NO:343, SEQ ID NO:355, SEQ ID NO:357, SEQ ID NO:361, SEQ ID NO:363, and SEQ ID NO:387; preferably, the sense chain has any sequence selected from the group consisting of: SEQ ID NO:337, SEQ ID NO:339, and SEQ ID NO:343.

[0071] In an embodiment, the antisense chain may have any sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:46, SEQ ID NO:166, SEQ ID NO:172, SEQ ID NO:188, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:202, SEQ ID NO:208, SEQ ID NO:256, SEQ ID NO:288, SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:362, SEQ ID NO:364, and SEQ ID NO:388. Preferably, the antisense chain has any sequence selected from the group consisting of: SEQ ID NO:338, SEQ ID NO:340, and SEQ ID NO:344.

[0072] In an embodiment, the sense chain has any one of the sequences selected from the group consisting of: SEQ ID NO:337, SEQ ID NO:339 and SEQ ID NO:343, and the antisense chain has any one of the sequences selected from the group consisting of: SEQ ID NO:338, SEQ ID NO:340 and SEQ ID NO:344.

[0073] In one embodiment, the sense strand has the sequence of SEQ ID NO:337, and the antisense strand has the sequence of SEQ ID NO:338. In another embodiment, the sense strand has the sequence of SEQ ID NO:339, and the antisense strand has the sequence of SEQ ID NO:340. In yet another embodiment, the sense strand has the sequence of SEQ ID NO:343, and the antisense strand has the sequence of SEQ ID NO:344.

[0074] Introducing chemically modified RNAi agents

[0075] In RNAi agents, the sense strand or antisense strand may include one or more chemical modifications.

[0076] Conventional siRNAs, due to their high negative charge and high molecular weight phosphate backbone, cannot cross cell membranes and are rapidly degraded and eliminated from the bloodstream, making it difficult to deliver sufficient quantities to the actual target site for RNAi induction. Currently, many efficient delivery methods using cationic lipids and cationic polymers have been developed for in vitro delivery; however, in vivo siRNA delivery is difficult to achieve the same efficiency as in vitro, and there is a problem of reduced siRNA delivery efficiency due to interactions with various proteins present in the organism.

[0077] Therefore, this example provides an RNAi agent with improved hepatocyte-targeted delivery capability by introducing chemical modifications into the asymmetric siRNA structure, and more particularly, provides an asymmetric siRNA construct (GalNAc asymmetric siRNA, GalNAc-asiRNA) capable of efficient intracellular delivery without a separate carrier.

[0078] In this disclosure, chemical modifications to the sense or antisense strand may include one or more of the following: binding to an N-acetylgalactosamine (GalNAc) derivative or a cell-penetrating peptide; modifying the nucleotide bond with a thiophosphate, boron phosphate, or methyl phosphonate; replacing the -OH group at the 2' carbon position of the nucleotide's sugar structure with -H, -CH3 (methyl), -OCH3 (-O-methyl), -NH2, -F, -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; replacing any nucleotide with a reverse baseless residue; and replacing the -OH group at the 5' end with an E-vinylphosphonate.

[0079] In this embodiment, an asymmetric siRNA (GalNAC-asiRNA) targeting APOC3 and having the aforementioned chemical modifications was designed and prepared. Subsequently, using cell or animal models expressing APOC3, nucleic acid molecules that induce RNAi with excellent knockdown efficiency and effective reduction of serum triglyceride and total cholesterol levels, namely APOC3 GalNAC-asiRNA, were screened without the application of a separate delivery medium.

[0080] In the embodiments, RNAi agents containing N-acetylgalactosamine (GalNAc) derivatives may have a structure of Formula 1 or Formula 2: the GalNAc derivative recognizes the asialic acid glycoprotein (ASGPR) receptor on the surface of hepatocytes to facilitate the inflow of the RNAi agent into the hepatocytes. That is, the GalNAc derivative acts as the ASGPR targeting moiety; therefore, the RNAi agent (where the GalNAc derivative binds to the terminal portion) can have improved delivery capability to hepatocytes, thereby providing effective targeted therapy for the target disease.

[0081] [Formula 1]

[0082]

[0083] The structure of Formula 2 can be a racemic mixture or an isomer with RR, SS, RS or SR configurations at the stereo center.

[0084] In the embodiments, the sense chain may have one or more chemical modifications selected from the following: modifying one to four nucleotide bonds adjacent to the 3' or 5' end with thiophosphate, boron phosphate, or methyl phosphonate; replacing the -OH group at the 2' carbon position of the sugar structure of at least one nucleotide with -CH3 (methyl), -OCH3 (-O-methyl), -NH2, -F, -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; replacing any nucleotide at the 3' or 5' end with a reverse-base-free residue; and binding the 3' end to a GalNAc derivative or a cell-penetrating peptide.

[0085] In the embodiments, the antisense chain may have one or more chemical modifications selected from the following: modifying 2 to 7 nucleotide bonds adjacent to the 3' or 5' end with thiophosphate, boron phosphate, or methyl phosphonate; replacing the -OH group at the 2' carbon position of the sugar structure of at least one nucleotide with -H, -CH3, -OCH3, -NH2, -F, -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; and replacing the OH group at the 5' end with phosphate or E-vinylphosphonate.

[0086] In one or more embodiments, the RNAi agent may have one or more modifications selected from the group consisting of: 1 to 7 nucleotide bonds adjacent to the 3' or 5' end of the sense or antisense strand with a thiophosphate ester; a -OH group at the 2' carbon position of the sugar structure of at least one nucleotide replaced with -H, -OCH3, or -F; a nucleotide at the 3' and / or 5' end of the sense strand replaced with an inverse abasic residue; the 3' end of the sense strand being bound to an N-acetylgalactosamine derivative; and a 5' OH group at the 5' end of the antisense strand replaced with a phosphate ester or E-vinylphosphonate.

[0087] In the embodiments, the RNAi agent may have the following modifications: in the sense strand, positions 2, 4, 5, 6, 14, and 16 are modified with 2'-fluorine from 5' to 3', and the remaining positions are modified with 2'-OMe; the 5' end has two thiophosphate bonds, and optionally, the 3' end has a nucleotide linked to a GalNAc derivative via a thiophosphate bond or a phosphodiester bond; in the antisense strand, positions 2, 14, and 16 are modified with 2'-fluorine from 5' to 3', the existing 2'-OH groups at positions 5 and 7 are replaced with 2'-H, and the remaining positions are modified with 2'-OMe; the 3' end has six thiophosphate bonds, and the 5' end has a 5'(E)-vinylphosphonate nucleotide.

[0088] In the embodiments, the RNAi agent may have the following modifications: in the sense strand, positions 5' to 3', 4, and 6 are modified with 2'-fluorine, the remaining positions are modified with 2'-OMe, the 5' end has two thiophosphate bonds, and optionally, the 3' end has a nucleotide linked to a GalNAc derivative via a thiophosphate bond or a phosphodiester bond; in the antisense strand, positions 5' to 3', 2, 14, and 16 are modified with 2'-fluorine, the existing 2'-OH groups at positions 5 and 7 are replaced with 2'-H, the remaining positions are modified with 2'-OMe, the 3' end has six thiophosphate bonds, and the 5' end has a 5'(E)-vinylphosphonate nucleotide.

[0089] In the embodiments, the RNAi agent containing a GalNAc derivative as described above is selected from Formula 1 or Formula 2; preferably Formula 2.

[0090] In the embodiments, the sense strand of the RNAi agent has a chemical modification pattern: (m)*(f)*(m)(f)(f)(f)(m)(m)(m)(m)(m)(m)(m)(m)(f)(m)(f)*[linker 2] or (m)*(m)*(m)(f)(m)(m)(m)(m)(m)(m)(m)(m)(m)(m)(m)(m)(m)(m)(m)(m)(m)*[linker 2],

[0091] The antisense strand of the RNAi agent has a chemical modification pattern: [EVP](m)*(f)*(m)(m)(d)(m)(d)(m)(m)(m)(m)(m)(m)(m)(f)(m)*(m)*(m)*(m)*(m)*(m)*(m)*(m).

[0092] Wherein, * indicates a thiophosphate bond, m indicates a 2'-O-methyl modified nucleotide, f indicates a 2'-fluorine modified nucleotide, d indicates that the existing 2'-OH is replaced by -H, EVP indicates an E-vinylphosphonate, and linker 2 indicates a GalNAc derivative of formula 2.

[0093] In this embodiment, the RNAi agent provided is a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of the target gene. In this embodiment, the target gene is APOC3.

[0094] In embodiments, the sense strand may have a sequence selected from the sense strand sequences listed in Tables 5, 6, 12, 13, and 14, and the antisense strand may have a sequence selected from the antisense strand sequences listed in Tables 5, 6, 12, 13, and 14. In a preferred embodiment, the RNAi agent is selected from GalNAC-asiRNAs listed in Tables 12, 13, and 14.

[0095] In an embodiment, the sense chain may include any sequence selected from the group consisting of (A) to (F) in the table below, and the antisense chain may include any sequence selected from the group consisting of (a) to (f) in the table below.

[0096]

[0097]

[0098] In an embodiment, the sense chain may have the sequence (mG)*(fG)*(mG)(fA)(fC)(fA)(mG)(mU)(mA)(mU)(mU)(mC)(mU)(fC)(mA)(fA)*[connector 2], and the antisense chain may have the sequence [EVP](mU)*(fU)*(mG)(mA)(dG)(mA)(dA)(mU)(mA)(mC)(mU)(mG)(mU)(fC)(mC)*(fC)*(mU)*(mU)*(mU)*(mU)*(mA).

[0099] In an embodiment, the sense chain may have the sequence (mG)*(fG)*(mA)(fC)(fA)(fG)(mU)(mA)(mU)(mU)(mC)(mU)(mC)(fA)(mG)(fA)*[connector 2], and the antisense chain may have the sequence [EVP](mU)*(fC)*(mU)(mG)(dA)(mG)(dA)(mA)(mU)(mA)(mC)(mU)(mG)(fU)(mC)*(fC)*(mC)*(mU)*(mU)*(mU)*(mU).

[0100] In an embodiment, the sense chain may have the sequence (mA)*(mC)*(mA)(fG)(mU)(fA)(mU)(mU)(mC)(mU)(mC)(mA)(mG)(mU)(mG)(mA)*[connector 2], and the antisense chain may have the sequence [EVP](mU)*(fC)*(mA)(mC)(dT)(mG)(dA)(mG)(mA)(mA)(mU)(mA)(mC)(fU)(mG)*(fU)*(mC)*(mC)*(mU)*(mU).

[0101] In these sequences, * indicates a thiophosphate bond, m indicates 2'-O-methyl, f indicates 2'-fluorine, d indicates that the existing 2'-OH is replaced by -H, EVP indicates E-vinylphosphonate, connector 1 indicates a GalNAc derivative of formula 1, and connector 2 indicates a GalNAc derivative of formula 2.

[0102] In detail, the sense and antisense strands of an RNAi agent can have sequences selected from any of the following sense and antisense sequences listed in the table above, and for example, an RNAi agent can have a combination of sense (A) and antisense (a); a combination of sense (B) and antisense (a); a combination of sense (B) and antisense (b); a combination of sense (C) and antisense (b); a combination of sense (C) and antisense (c); a combination of sense (D) and antisense (c); a combination of sense (D) and antisense (d); a combination of sense (E) and antisense (d); a combination of sense (E) and antisense (e); a combination of sense (F) and antisense (e); a combination of sense (F) and antisense (f); and a combination of sense (A) and antisense (f).

[0103] Another aspect of this disclosure provides a pharmaceutical composition for the prevention or treatment of lipid metabolism-related diseases, the pharmaceutical composition comprising an RNAi agent as an active ingredient.

[0104] Another aspect of this disclosure provides the use of RNAi agents in the preparation of pharmaceutical products for the prevention or treatment of lipid metabolism-related diseases.

[0105] Since the pharmaceutical composition contains or uses the aforementioned RNAi agent as is, its description is omitted to avoid making this specification overly complicated.

[0106] lipid metabolism-related diseases

[0107] The pharmaceutical composition can be used as the active ingredient in a pharmaceutical composition that prevents or treats lipid metabolism-related diseases by inhibiting the expression of the APOC3 gene.

[0108] Lipid metabolism-related diseases can be broadly defined as diseases caused by the synthesis and breakdown of lipids (including the synthesis of functional and structural lipids, such as those involved in cell membrane composition, and the breakdown of fats used for energy production). For example, lipid metabolism-related diseases can be caused by excessive accumulation of lipids in the body. Furthermore, lipid metabolism-related diseases are closely related to the expression level of the APOC3 gene and may include, for example, any of the following groups: hyperlipidemia, hypertriglyceridemia, obesity, atherosclerosis, diabetes, cardiovascular disease, coronary artery disease, familial chylomicronemia syndrome, dyslipidemia, stroke, liver inflammation, and hepatic steatosis. However, lipid metabolism-related diseases are not limited to these categories.

[0109] Pharmaceutical Composition

[0110] As used herein, the term "active ingredient" refers to an appropriate effective amount of an ingredient that produces a beneficial or desired clinical or biochemical outcome. In particular, the term can refer to an effective amount of a pharmaceutical agent, active agent, or RNAi agent.

[0111] An effective dose may be applied once or multiple times, and in unlimited circumstances may be an appropriate dose for preventing disease, relieving symptoms, reducing the severity of disease, stabilizing (i.e., not aggravating) the disease state, delaying or slowing the rate of disease progression, or improving or temporarily relieving and improving (partial or complete) the disease state.

[0112] As used herein, the term "prevention" refers to any action taken to prevent the onset of a disease, inhibit its development, or delay its progression. For example, the term refers to preventing or halting the development of lipid metabolism-related diseases or their characteristics, or to defending against or preventing the development of lipid metabolism-related diseases or their characteristics.

[0113] As used in this specification, the term "treatment" refers to both therapeutic treatment and preventive (or prophylactic) measures. Furthermore, treatment refers to any action that improves and beneficially alters the symptoms of a disease. For example, treatment means preventing, reducing, or improving lipid metabolism-related diseases or their characteristics in a subject, or delaying (weakening) the progression of lipid metabolism-related diseases or their characteristics.

[0114] As used in this specification, the term "effective amount" has the meaning generally accepted in the art. This term typically refers to the amount of a molecule, compound, or construct that researchers, veterinarians, physicians, or other clinicians seek to elicit a desired biological response (e.g., a beneficial response) in cells, tissues, systems, animals, or humans. In particular, a "therapeutic effective amount" refers to the amount of a molecule, compound, or construct capable of eliciting a desired medical response to the extent that a particular clinical treatment might be considered effective (due to, for example, a treatment-related change in a measurable parameter associated with a disease or disorder). A therapeutically effective amount of a drug used to treat a disease or disorder may be the amount necessary to elicit a treatment-related change in a parameter.

[0115] Those skilled in the art can determine the method of administering the pharmaceutical composition based on the patient's symptoms and the severity of the disease. Furthermore, the pharmaceutical composition can be formulated in various forms, such as powder, tablets, capsules, solutions, injections, ointments, syrups, etc., and can be provided in single-dose or multi-dose containers (e.g., sealed ampoules and vials).

[0116] The pharmaceutical compositions disclosed herein can be administered orally or parenterally. The routes of administration for the compositions disclosed herein may include, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, percutaneous, subcutaneous, intraperitoneal, intestinal, sublingual, or local administration, but are not limited thereto. The dosage of the compositions disclosed herein varies depending on the patient's weight, age, sex, health status, diet, time of administration, method of administration, excretion rate, or disease severity, and can be readily determined by those skilled in the art. Furthermore, the compositions disclosed herein can be formulated into suitable formulations for clinical use using known techniques.

[0117] Another aspect of this disclosure provides a method for preventing or treating lipid metabolism-related diseases, the method comprising administering an RNAi agent to a subject.

[0118] Since the methods for treating lipid metabolism-related diseases include or use the aforementioned RNAi agents or pharmaceutical compositions as is, any content common to them is omitted to avoid making this specification overly complex.

[0119] As used herein, the term “subject” refers to a subject who requires treatment for a disease (in particular, a lipid metabolism-related disease), and more specifically, the term may include all mammals, such as humans or non-human primates, mice, dogs, cats, horses, cattle, sheep, pigs, goats, camels, and antelopes.

[0120] The disclosure will be described in more detail below with examples. However, these examples are intended to illustrate the disclosure, and the scope of the disclosure is not limited to these examples.

[0121] Example 1: Screening of nucleic acid molecules that induce RNAi targeting APOC3

[0122] In this example, 196 nucleic acid molecules that induce RNAi targeting APOC3 were prepared, and the inhibitory efficacy of these nucleic acid molecules on APOC3 expression was evaluated in order to screen APOC3 asiRNAs with effective RNAi efficacy according to the examples.

[0123] 1-1. Design and preparation of APOC3 asiRNA

[0124] To obtain a double-stranded nucleic acid molecule that induces RNAi targeting APOC3, the target sequence of the APOC3 gene is first selected, and then an APOC3 asymmetric siRNA (APOC3 asiRNA) is designed (sense strand (16-mer), antisense strand (21-mer), see [link to documentation]). Figure 1 Specifically, after obtaining APOC3 gene information by searching the NCBI database, and considering animal experiments, a total of 196 asiRNAs were designed and synthesized at a scale of 4 nmole by Olix US. Subsequently, the synthesized asiRNAs were subjected to 15% polyacrylamide gel electrophoresis (PAGE) and then quality control (QC) was performed using a ChemiDoc UV transilluminator (Biorad). The sequence information of the APOC3 asiRNAs designed according to the aforementioned method is shown in Tables 1 to 4 below.

[0125] [Table 1]

[0126]

[0127]

[0128]

[0129]

[0130] [Table 2]

[0131]

[0132]

[0133]

[0134]

[0135] [Table 3]

[0136]

[0137]

[0138]

[0139]

[0140] [Table 4]

[0141]

[0142]

[0143]

[0144]

[0145] 1-2. Screening of 196 APOC3 asiRNAs

[0146] Huh-7 cells (human hepatocellular carcinoma cells) were transfected with each of 196 APOC3 asiRNAs, and qRT-PCR was performed to measure the expression level of APOC3 mRNA. Specifically, Huh-7 cells were transfected at 8.0 × 10⁻⁶ mRNAs. 3 Cells were seeded at a concentration of 100 cells / well in 96-well plates and transfected with APOC3 asiRNA (1 nM) using 0.2 μl of lipofecamine RNAiMAX (Invitrogen, 13778150). 24 hours post-transfection, cell lysates were prepared using the SuperPrep™ Cell Lysis and 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, the synthesized cDNA was used as a template for quantitative PCR using the THUNDERBIRD probe qPCR MIX (TOYOBO, QPS-101), the APOC3 probe (Thermofisher, Hs00163644_m1), and the RNA18S5 probe (Thermofisher, Hs03928985_g1). Then, the expression level of APOC3 mRNA was identified using the CFX Connect real-time PCR system (Bio-Rad Laboratories). Additionally, after transfecting Huh-7 cells with 196 APOC3 asiRNAs at a concentration of 0.1 nM, the expression level of APOC3 mRNA was again measured by qRT-PCR using the same method described above. Meanwhile, in this example, the group treated only with the transfection reagent (simulated) was used as the control group.

[0147] The results are shown in Figures 2 to 5. Treatment of Huh-7 cells with APOC3 asiRNA (1 nM or 0.1 nM) confirmed the inhibitory effect on APOC3 mRNA expression in these cells. Furthermore, 40 APOC3 asiRNAs (asiAPOC3-001, 002, 023, 024, 083, 086, 094, 096, 097, 101, 104, 108, 118, 123, 128, 131, 132, 144, 168, 169, 170, 171, 172, 173, 175, 176, 177, 178, 179, 180, 181, 182, 183, 185, 190, 191, 192, 193, 194, and 195) exhibited relatively superior inhibitory efficiency against APOC3 mRNA expression.

[0148] Screening of 1-3.40 APOC3 asiRNAs

[0149] Primary human hepatocytes were transfected with each of the 40 selected APOC3 asiRNAs, and qRT-PCR was performed to measure the expression level of APOC3 mRNA. Specifically, primary human hepatocytes were transfected at a concentration of 1.0 × 10⁻⁶ mRNAs. 4 Cells were seeded at a concentration of 100 cells / well in 96-well plates and transfected with APOC3 asiRNA (1 nM) using lipofecamine RNAiMAX (Ingenieur, 13778150). Twenty-four hours post-transfection, cell lysates were prepared using the SuperPrep™ Cell Lysis and 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 with the THUNDERBIRD probe qPCRMIX (Toyobo, QPS-101), the APOC3 probe (Thermo Fisher Scientific, Hs00163644_m1), and the RNA18S5 probe (Thermo Fisher Scientific, Hs03928985_g1). The expression level of APOC3 mRNA was then identified using the CFXConnect Real-Time PCR System (Bio-Rayet). In addition, after transfecting primary human hepatocytes with 40 APOC3 asiRNAs at a concentration of 0.1 nM, the expression level of APOC3 mRNA was measured again by qRT-PCR using the same method as described above. Meanwhile, in this example, the group treated only with the transfection reagent (simulated) was used as the control group.

[0150] The results are as follows Figure 6 and Figure 7As shown, treatment with APOC3 asiRNA (1 nM or 0.1 nM) confirmed the inhibitory effect on APOC3 mRNA expression in primary human hepatocytes. Furthermore, 21 APOC3 asiRNAs (asiAPOC3-002, 023, 083, 086, 094, 096, 097, 101, 104, 128, 144, 168, 169, 170, 171, 172, 178, 179, 181, 182, and 194) exhibited relatively superior inhibitory efficiency against APOC3 mRNA expression.

[0151] Example 2: Screening of chemically modified nucleic acid molecules that induce RNAi

[0152] In this example, based on the experimental results of Example 1, 84 nucleic acid molecules with chemical modifications targeting APOC3 were prepared to induce RNAi, and the inhibitory efficacy of the nucleic acid molecules on APOC3 expression was evaluated in order to screen APOC3 GalNAc-asiRNAs with effective RNAi efficacy.

[0153] 2-1. Preparation of chemically modified nucleic acid molecules for inducing RNAi

[0154] A chemically modified APOC3 GalNAc-asiRNA was designed as a double-stranded nucleic acid molecule for highly efficient RNAi targeting APOC3. Specifically, considering the experimental results of Example 1, 21 APOC3 asiRNAs (asiAPOC3-002, 023, 083, 086, 094, 096, 097, 101, 104, 128, 144, 168, 169, 170, 171, 172, 178, 179, 181, 182, and 194) were selected as the basic backbone. Then, 84 APOC3 GalNAc-asiRNAs were designed based on each of the basic backbones, with the following modifications introduced: 1) replacing the -OH group at the 2' carbon position in the sugar structure of the sense and antisense nucleotides with -H, -OMe or -F; 2) introducing thiophosphate modification into the backbone at the 5' or 3' end of the antisense strand or the 5' end of the sense strand; 3) introducing phosphate ester at the 5' end of the antisense strand; and 4) introducing GalNAc derivative at the 3' end of the sense strand.

[0155] The sequence information of the chemically modified APOC3 GalNAc-asiRNA prepared in this example is shown in Tables 5 and 6. Specifically, in Tables 5 and 6, "*" indicates a form in which the existing phosphodiester bond is replaced by a thiophosphate bond, "d" indicates a form in which the existing 2'-OH group is replaced by a 2'-H group, where, for example, dT refers to the existing uridine 2'-OH group being replaced by a 2'-H group to modify it into thymine, and "m" indicates a form in which the existing 2'-OH group is replaced by a 2'-O-methyl group. In addition, "f" indicates (e.g., in the case of fG) a form in which the existing guanine 2'-OH group is replaced by a fluorine group, "Phos" indicates a form in which the existing 5'-OH group is replaced by a phosphate group, and "Linker 1" indicates that the trivalent GalNAc derivative of Formula 1 is bound to the 3' end of the sense chain (see Table 7).

[0156] [Table 5]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163] [Table 6]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169] [Table 7]

[0170] symbol Chemical modification * Thiophosphate bond d deoxidation m 2'-O-methyl f 2'-Fluorine Phos 5'-phosphate Connector 1 GalNAc derivatives of Formula 1

[0171] [Formula 1]

[0172]

[0173] Screening of 2-2.84 APOC3 GalNAc-asiRNAs

[0174] Primary human hepatocytes were treated with each of the 84 selected GalNAc-asiRNAs, and then qRT-PCR was performed to measure the expression level of APOC3 mRNA. Specifically, primary human hepatocytes were treated at a concentration of 3.0 × 10⁻⁶ mRNAs. 4 Cells were seeded per well in 96-well plates and treated with APOC3 GalNAc-asiRNA (200 nM). Twenty-four hours after treatment, cell lysates were prepared using the SuperPrep™ Cell Lysis and 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. The synthesized cDNA was then used as a template for quantitative PCR using the THUNDERBIRD probe qPCR MIX (Toyobo, QPS-101), the APOC3 probe (Thermofisher, Hs00163644_m1), and the RNA18S5 probe (Thermofisher, Hs03928985_g1). The expression level of APOC3 mRNA was then identified using the CFX Connect Real-Time PCR System (Bio-Rayet). In this example, the untreated group (NT) served as the control group.

[0175] The results are shown in Figure 8. Treatment of primary human hepatocytes with APOC3 GalNAc-asiRNA confirmed the inhibitory effect on APOC3 mRNA expression in these cells. Furthermore, 42 APOC3 GalNAc-asiRNAs exhibiting relatively superior inhibitory efficiency against APOC3 mRNA expression were identified (OLX706C-002-2, 083-2, 083-4, 094-1, 094-2, 094-3, 094-4, 096-3, 096-4, 101-2, 101-4, 104-1, 104-2, 104-3, 104-4, 128-1, 128-...). 2, 128-3, 128-4, 144-2, 144-3, 168-1, 168-2, 168-3, 168-4, 169-2, 169-3, 169-4, 170-1, 170-2, 170-3, 170-4, 171-2, 171-3, 171-4, 172-1, 172-2, 172-3, 172-4, 179-4, 181-1 and 181-3).

[0176] Screening of 2-3.42 APOC3 GalNAc-asiRNAs

[0177] Primary human hepatocytes were treated with each of the 42 selected GalNAc-asiRNAs at concentrations of 20 nM or 100 nM, and then qRT-PCR was performed to measure APOC3 mRNA expression levels in the same manner as in Example 2-2. Meanwhile, in this example, the untreated group (NT) was used as the control group.

[0178] The results are shown in Figure 9. Treatment of primary human hepatocytes with APOC3 GalNAc-asiRNA confirmed the inhibitory effect on APOC3 mRNA expression in these cells. Furthermore, ten APOC3 GalNAc-asiRNAs (OLX706C-104-3, 168-4, 169-3, 169-4, 170-1, 170-3, 170-4, 172-2, 172-3, and 172-4) exhibited relatively superior inhibitory efficiency against APOC3 mRNA expression.

[0179] Example 3: Screening of chemically modified induced RNAi nucleic acid molecules using animal models

[0180] In this example, based on the experimental results of Example 2, for 10 nucleic acid molecules with chemical modifications targeting APOC3 that induce RNAi (OLX706C-104-3, 168-4, 169-3, 169-4, 170-1, 170-3, 170-4, 172-2, 172-3, and 172-4), serum APOC3 levels and / or serum triglyceride and total cholesterol levels were assessed using transgenic mice expressing human APOC3 to screen for APOC3GalNAc-asiRNAs with effective RNAi efficacy.

[0181] 3-1.10 screening of APOC3 GalNAc-asiRNAs

[0182] APOC3 GalNAc-asiRNA was administered subcutaneously at a single dose of 1 mg / kg (mpk) to transgenic mice expressing human APOC3 (including a 2.5 kb 5' flanking sequence and a 1.1 kb 3' flanking sequence) in the C57BL / 6J genetic background. Serum APOC3 levels were measured using an apolipoprotein C-III ELISA kit (ab154131) on days 6 and 14 from the date of subcutaneous injection. The measured APOC3 values ​​were then normalized relative to serum APOC3 levels before APOC3 GalNAc-asiRNA injection and serum APOC3 levels in a control group treated with saline on the day of measurement to assess APOC3 levels in mouse-derived serum. In this example, the saline-treated group served as a control.

[0183] The results are shown in Table 8 and Figure 10 As shown, treatment with APOC3 GalNAc-asiRNA confirmed a decrease in serum APOC3 levels in the transgenic mouse model. Furthermore, eight APOC3 GalNAc-asiRNAs (OLX706C-169-3, 169-4, 170-1, 170-3, 170-4, 172-2, 172-3, and 172-4) exhibited relatively superior inhibitory efficiency against APOC3 expression.

[0184] [Table 8]

[0185]

[0186] Screening of 3-2.8 APOC3 GalNAc-asiRNAs

[0187] Eight selected APOC3 GalNAc-asiRNAs were subcutaneously injected into transgenic mice at a single dose of 1 mg / kg (mpk). Serum APOC3 levels were measured using an apolipoprotein C-III ELISA kit (ab154131) on days 6, 14, 21, and 28 after injection, and serum triglyceride and total cholesterol levels were measured using an automated chemistry analyzer (FDC NX600V). The measured values ​​of APOC3, triglycerides, or total cholesterol were then normalized relative to pre-injection serum levels of APOC3, triglycerides, or total cholesterol to assess the levels of APOC3, triglycerides, and total cholesterol in mouse-derived serum. In this example, a saline-treated group served as a control group.

[0188] The results are shown in Tables 9 to 11 and Figures 11 to 13 As shown, decreased serum APOC3 levels, as well as decreased serum triglyceride and total cholesterol levels, were confirmed in transgenic mouse models treated with APOC3 GalNAc-asiRNA. Furthermore, three APOC3 GalNAc-asiRNAs (OLX706C-169-4, OLX706C-170-4, and OLX706C-172-4) exhibited the highest inhibitory efficiency against APOC3 expression.

[0189] [Table 9]

[0190]

[0191] [Table 10]

[0192]

[0193] [Table 11]

[0194]

[0195] Example 4: Conformation of the inhibitory effect on the expression of chemically modified nucleic acid molecules that induce RNAi.

[0196] In this example, three nucleic acid molecules were used as the basic backbone, and 12 chemical modification patterns were introduced for each of the basic backbones to prepare 36 nucleic acid molecules with chemical modifications targeting APOC3 that induce RNAi. Then, the efficacy of these APOC3-targeting nucleic acid molecules in inhibiting APOC3 expression and the changes in serum factor levels after expression inhibition were evaluated to screen for APOC3 GalNAc-asiRNAs with effective RNAi efficacy.

[0197] 4-1. Preparation of chemically modified nucleic acid molecules that induce RNAi

[0198] Chemically modified APOC3 GalNAc-asiRNAs were designed as double-stranded nucleic acid molecules for highly efficient RNAi targeting APOC3. Specifically, based on the experimental results of Examples 2 and 3, three APOC3 asiRNAs (asiAPOC3-169, 170, and 172) were selected as basic backbones. Then, 36 APOC3 GalNAc-asiRNAs were designed according to each of the basic backbones, with the following modifications: 1) replacing the -OH group at the 2' carbon position in the sugar structure of the sense and antisense nucleotides with -H, -OMe, or -F; 2) introducing phosphate thioester modification into the backbone at the 5' or 3' end of the antisense strand or the 5' or 3' end of the sense strand; 3) introducing E-vinylphosphonate (EVP) into the 5' end of the antisense strand; 4) introducing a reverse abase-free residue into the 5' or 3' end of the sense strand; and 5) introducing a GalNAc derivative into the 3' end of the sense strand.

[0199] The sequence information of the chemically modified APOC3 GalNAc-asiRNA prepared in this example is shown in Tables 12 to 14. In particular, the chemical modifications indicated by “*”, “d”, “m”, “f”, “EVP”, “invAb”, “Adapter 1” and “Adapter 2” in Tables 12 to 14 are shown in Table 15.

[0200] [Table 12]

[0201]

[0202]

[0203]

[0204] [Table 13]

[0205]

[0206]

[0207] [Table 14]

[0208]

[0209]

[0210] [Table 15]

[0211]

[0212]

[0213] [Formula 1]

[0214]

[0215] [Equation 2]

[0216] It has (S)- and (R)- configurations at the solid center (1) and (2) respectively.

[0217] 4-2.36 APOC3 GalNAc-asiRNAs were screened.

[0218] Primary human hepatocytes were treated with each of the 36 selected GalNAc-asiRNAs, and then qRT-PCR was performed to measure the expression level of APOC3 mRNA. Specifically, primary human hepatocytes were treated at a concentration of 3.0 × 10⁻⁶ mRNAs. 4Cells were seeded per well in 96-well plates and treated with APOC3 GalNAc-asiRNA (200 nM, 20 nM, or 2 nM). Twenty-four hours after treatment, cell lysates were prepared using the SuperPrep™ Cell Lysis and 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. The synthesized cDNA was then used as a template for quantitative PCR using the THUNDERBIRD probe qPCR MIX (Toyobo, QPS-101), the APOC3 probe (Thermo Fisher Scientific, Hs00163644_m1), and the RNA18S5 probe (Thermo Fisher Scientific, Hs03928985_g1). The expression level of APOC3 mRNA was then identified using the CFX Connect Real-Time PCR System (Bio-Rayet). In this example, the untreated group (NT) served as the control group.

[0219] The results are shown in Figures 14 to 16. Treatment of primary human hepatocytes with APOC3 GalNAc-asiRNA confirmed the inhibitory effect on APOC3 mRNA expression in these cells. Furthermore, seven APOC3 GalNAc-asiRNAs (OLX706C-169-16, OLX706C-170-16, OLX706C-170-20, OLX706C-170-24, OLX706C-172-7, OLX706C-172-8, and OLX706C-172-22) exhibited relatively superior inhibitory effects on APOC3 mRNA expression.

[0220] 4-3. Screening of 7 APOC3 GalNAc-asiRNAs using an animal model

[0221] Seven selected APOC3 GalNAc-asiRNAs were subcutaneously injected into transgenic mice at a single dose of 0.25 mg / kg (mpk). Serum APOC3 levels were measured by immunoturbidimetry on days 7, 14, 21, and 28 from the date of subcutaneous injection, and serum triglyceride and total cholesterol levels were measured using an automated chemistry analyzer (FDC NX600V). The measured values ​​of APOC3, triglycerides, or total cholesterol were then normalized relative to the serum levels of APOC3, triglycerides, or total cholesterol before injection of APOC3 GalNAc-asiRNA to assess the levels of APOC3, triglycerides, and total cholesterol in mouse-derived serum. In this example, a group treated with saline served as a control group.

[0222] The results are shown in Tables 16 to 18 and Figures 17 to 19 As shown, decreased serum APOC3 levels, as well as decreased serum triglyceride and total cholesterol levels, were confirmed in transgenic mouse models treated with APOC3 GalNAc-asiRNA. Furthermore, three APOC3 GalNAc-asiRNAs (OLX706C-169-16, OLX706C-170-16, and OLX706C-172-8) exhibited relatively superior inhibitory effects on APOC3 expression.

[0223] [Table 16]

[0224]

[0225] [Table 17]

[0226]

[0227] [Table 18]

[0228]

[0229] This concludes the detailed description of certain portions of this disclosure. However, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of this disclosure is not limited thereto. Therefore, it is intended that the substantive scope of this disclosure be defined by the appended claims and their equivalents.

[0230] One approach to RNAi agents is to inhibit APOC3 expression by binding to and degrading APOC3 mRNA, while mitigating side effects such as nonspecific immune responses and off-target effects.

[0231] In addition, RNAi agents can be effectively delivered to hepatocytes and can be used as targeted therapeutic agents for lipid metabolism-related diseases.

[0232] It should be understood that the embodiments described herein should be considered in a descriptive sense only and not for limiting purposes. Typically, the description of features or aspects within each embodiment should be considered applicable to other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the accompanying drawings, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of this disclosure as defined by the following claims.

Claims

1. An RNAi agent comprising: an antisense strand having sequence complementarity to an apolipoprotein C-III (APOC3) mRNA sequence and being 19 nucleotides (nt) to 21 nt in length; and a sense strand having sequence complementarity to the antisense strand and being 15 nt 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 agent of claim 1, wherein the sense strand has a sequence selected from the sense strand sequences listed in Tables 1-4.

3. The RNAi agent of claim 1, wherein the sense strand has a sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 45, SEQ ID NO: 165, SEQ ID NO: 171, SEQ ID NO: 187, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 201, SEQ ID NO: 207, SEQ ID NO: 255, SEQ ID NO: 287, SEQ ID NO: 335, SEQ ID NO: 337, SEQ ID NO: 339, SEQ ID NO: 341, SEQ ID NO: 343, SEQ ID NO: 355, SEQ ID NO: 357, SEQ ID NO: 361, SEQ ID NO: 363, and SEQ ID NO:

387.

4. The RNAi agent of claim 1, wherein the antisense strand has a sequence selected from the antisense strand sequences listed in Tables 1-4.

5. The RNAi agent of claim 1, wherein the antisense strand has a sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 46, SEQ ID NO: 166, SEQ ID NO: 172, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 202, SEQ ID NO: 208, SEQ ID NO: 256, SEQ ID NO: 288, SEQ ID NO: 336, SEQ ID NO: 338, SEQ ID NO: 340, SEQ ID NO: 342, SEQ ID NO: 344, SEQ ID NO: 356, SEQ ID NO: 358, SEQ ID NO: 362, SEQ ID NO: 364, and SEQ ID NO:

388.

6. The RNAi agent of claim 1, wherein the sense strand has a sequence selected from the group consisting of: SEQ ID NO:337, SEQ ID NO:339, and SEQ ID NO:343, and the antisense strand has a sequence selected from the group consisting of: SEQ ID NO:338, SEQ ID NO:340, and SEQ ID NO:

344.

7. The RNAi agent of claim 1, wherein the sense strand has the sequence of SEQ ID NO:337 and the antisense strand has the sequence of SEQ ID NO:338; the sense strand has the sequence of SEQ ID NO:339 and the antisense strand has the sequence of SEQ ID NO:340; or the sense strand has the sequence of SEQ ID NO:343 and the antisense strand has the sequence of SEQ ID NO:

344.

8. The RNAi agent of any one of claims 1, 2, 3, 6, or 7, wherein the 3’ end of the sense strand is conjugated to an N-acetylgalactosamine (GalNAc) derivative.

9. The RNAi agent of any one of claims 1-7, wherein the sense strand or the antisense strand comprises one or more chemical modifications.

10. The RNAi agent of claim 9, wherein the sense strand comprises one or more chemical modifications selected from: modification of 1 to 4 nucleotide linkages adjacent to the 3’ end or 5’ end with phosphorothioate, boranophosphonate, or methylphosphonate; substitution of the -OH group at the 2’ carbon position in the sugar structure of at least one nucleotide with methyl (-CH3), -O-methyl (-OCH3), -NH2, -F, -O-2-methoxyethyl -O- propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; substitution of the nucleotide at the 3’ end or 5’ end with an inverted abasic residue; and conjugation of the 3’ end to an N-acetylgalactosamine derivative or a cell-penetrating peptide.

11. The RNAi agent of claim 9, wherein the antisense strand comprises one or more chemical modifications selected from: modification of 2 to 7 nucleotide linkages adjacent to the 3’ end or 5’ end with phosphorothioate, boranophosphonate, or methylphosphonate; substitution of the -OH group at the 2’ carbon position in the sugar structure of at least one nucleotide with -H, -CH3, -OCH3, -NH2, -F, -O-2-methoxyethyl -O-propyl, -O-2- methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; and substitution of the OH group at the 5’ end with phosphonate or E-vinylphosphonate.

12. The RNAi agent of claim 9, wherein the RNAi agent comprises one or more modifications selected from: modification of 1 to 7 nucleotide linkages adjacent to the 3’ end or 5’ end in the sense strand or the antisense strand with phosphorothioate; modification of the -OH group at the 2’ carbon position in the sugar structure of at least one nucleotide in the sense strand or the antisense strand to -H, -OCH3, or -F; and substitution of the OH group at the 5’ end with phosphonate or E-vinylphosphonate. replacing the 3' terminal and / or 5' terminal nucleotides with inverted abasic residues; the 3' terminal of the sense strand is conjugated to an N-acetylgalactosamine derivative; and replacing the OH group of the 5' terminal of the antisense strand with a phosphorothioate or E-ethylenephosphonate.

13. The RNAi agent of claim 1, wherein the sense strand has a sequence selected from the sense strand sequences listed in Table 5, Table 6, Table 12, Table 13, and Table 14.

14. The RNAi agent of claim 1, wherein the antisense strand has a sequence selected from the antisense strand sequences listed in Table 5, Table 6, Table 12, Table 13, and Table 14.

15. The RNAi agent of claim 9, wherein the RNAi agent comprises one modification selected from the group consisting of: in the sense strand, from 5' to 3', positions 2, 4, 5, 6, 14, and 16 are 2'-fluoro modified, the remaining positions are 2'-OMe modified, the 5' terminal has two phosphorothioate linkages, and optionally, the 3' terminal is conjugated to a GalNAc derivative through a phosphorothioate linkage or a phosphodiester linkage; in the antisense strand, from 5' to 3', positions 2, 14, and 16 are 2'-fluoro modified, the existing 2'-OH groups at positions 5 and 7 are replaced with 2'-H, and the remaining positions are 2'-OMe modified, the 3' terminal has six phosphorothioate linkages, the 5' terminal has one 5'(E)-ethylenephosphonate nucleotide; or in the sense strand, from 5' to 3', positions 4 and 6 are 2'-fluoro modified, the remaining positions are 2'-OMe modified, the 5' terminal has two phosphorothioate linkages, and optionally, the 3' terminal is conjugated to a GalNAc derivative through a phosphorothioate linkage or a phosphodiester linkage; in the antisense strand, from 5' to 3', positions 2, 14, and 16 are 2'-fluoro modified, the existing 2'-OH groups at positions 5 and 7 are replaced with 2'-H, and the remaining positions are 2'-OMe modified, the 3' terminal has six phosphorothioate linkages, the 5' terminal has one 5'(E)-ethylenephosphonate nucleotide.

16. The RNAi agent of claim 15, wherein the RNAi agent containing a GalNAc derivative is Formula 1 or Formula 2; preferably Formula 2 17. The RNAi agent of any one of claims 1 to 14, which is OLX706C-169-3, OLX706C-169-4, OLX706C-170-1, OLX706C-170-3, OLX706C-170-4, OLX706C-172-2, OLX706C-172-3, OLX706C-172-4, OLX706C-169-16, OLX706C-170-16, OLX706C-170-20, OLX706C-170-24, OLX706C-172-7, OLX706C-172-8, or OLX706C-172-22, preferably which is OLX706C-169-4, OLX706C-170-4, OLX706C-172-4, OLX706C-169-16, OLX706C-170-16, or OLX706C-172-8.

18. A pharmaceutical composition comprising as an active ingredient the RNAi agent of any one of claims 1 to 17, and a pharmaceutically acceptable carrier.

19. Use of the RNAi agent of any one of claims 1 to 17 or the pharmaceutical composition of claim 18 for the manufacture of a pharmaceutical product for the prevention or treatment of a lipid metabolism related disease.

20. The use of claim 19, wherein the lipid metabolism related disease is selected from the group consisting of hyperlipidemia, hypertriglyceridemia, obesity, atherosclerosis, diabetes, cardiovascular disease, coronary artery disease, familial chylomicronemia syndrome, dyslipidemia, stroke, liver inflammation, and liver steatosis.

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