RNAi AGENT TARGETING APOC3 GENE AND USE THEREOF

Abstract

The disclosure relates to an RNAi agent targeting an apolipoprotein C-III (APOC3) gene and use thereof, and more specifically, to an RNAi agent including an antisense strand that has sequence complementarity to an APOC3 mRNA sequence and a sense strand that has sequence complementarity to the antisense strand, and a pharmaceutical composition for preventing or treating a lipid metabolism-related disease, including the RNAi agent.

Description

RNAi AGENT TARGETING APOC3 GENE AND USE THEREOF

[0001] The disclosure relates to an RNAi agent targeting an apolipoprotein C-III (APOC3) gene and use thereof, and more specifically, to an RNAi agent including an antisense strand that has sequence complementarity to an APOC3 mRNA sequence and a sense strand that has sequence complementarity to the antisense strand, and a pharmaceutical composition for preventing or treating a lipid metabolism-related disease, including the RNAi agent.

[0002] Adipose tissue or body fat is a loose connective tissue consisting of mostly adipocytes. Adipose tissue may include, in addition to adipocytes, stromal vascular fractions (SVFs) of cells such as preadipocytes, fibroblasts, vascular endothelial cells, and various immune cells. Adipose tissue is derived from preadipocytes. The main role of adipose tissue is not only to protect and block the body from shock, but also to store energy in the form of lipids. However, excessive accumulation of fat in adipose tissue is undesirable, and may cause pathological conditions such as obesity, lipid storage disease, and hyperlipidemia.

[0003] Typically, when the cholesterol level in the body increases, abnormalities in the metabolic regulation of cholesterol occur, causing quantitative changes in lipoprotein components such as low-density lipoprotein-cholesterol (LDL-cholesterol) and high-density lipoprotein-cholesterol (HDL-cholesterol). In particular, among blood cholesterol, LDL-cholesterol is known as a risk factor for cardiovascular diseases, and an increase in the concentration of blood triglycerides is known to be a main cause of coronary artery disease by lowering the concentration of HDL-cholesterol and increasing an amount of chylomicron remnants which are lipoprotein particles consisting mainly of triglycerides. In addition, since blood lipids pass through vascular endothelial cells better than other lipids, blood lipids are regarded as a main cause of arteriosclerosis, and a rapid increase in triglyceride levels after a meal is known to be closely related to the occurrence of stroke.

[0004] Meanwhile, apolipoprotein C-III (APOC3) is mainly synthesized in the liver and plays an important role in production, metabolism, and removal of triglyceride (TG)-rich lipoproteins from plasma.　In detail, increased APOC3 in the liver promotes secretion of TG-rich very-low-density lipoprotein-C (VLDL-C). In addition, excess APOC3 inhibits the activity of lipoprotein lipases and hepatic lipases, and contributes to increasing serum TG levels by delaying catabolism of TG-rich lipoproteins. Moreover, increased APOC3 may delay the removal of TG-rich lipoproteins and remnant particles thereof by interfering with binding to liver receptors. Therefore, increased APOC3 levels lead to the development of hypertriglyceridemia or result in high TG levels in blood. In addition, elevated TG levels may lead to the development of various diseases including cardiovascular disease, atherosclerosis, non-alcoholic fatty liver disease, polycystic ovarian syndrome, renal disease, obesity, type 2 diabetes (insulin resistance), hypertension, and the like (see KR 10-2020-0074975).

[0005] Accordingly, the present inventors, as a result of intensive research efforts to develop a new drug for treating a lipid metabolism-related disease, developed an RNAi agent capable of binding to APOC3 mRNA to specifically inhibit expression thereof, thereby completing the disclosure.

[0006] Provided is an RNAi agent that specifically inhibits expression of apolipoprotein C-III (APOC3).

[0007] Provided are a pharmaceutical composition for preventing or treating a lipid metabolism-related disease, the pharmaceutical composition including the RNAi agent, and a method of preventing or treating a lipid metabolism-related disease, the method including administering the pharmaceutical composition to a subject.

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

[0009] According to an aspect of the disclosure, an RNAi agent includes an antisense strand that has sequence complementarity to an APOC3 mRNA sequence and is 19 nucleotides (nt) to 21 nt in length, and a sense strand that has sequence complementarity to the antisense strand and is 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.

[0010] According to another aspect of the disclosure, a pharmaceutical composition for preventing or treating a lipid metabolism-related disease includes the RNAi agent as an active ingredient.

[0011] According to another aspect of the disclosure, a method of preventing or treating a lipid metabolism-related disease includes administering the RNAi agent to a subject.

[0012] According to another aspect of the disclosure, provided is use of the RNAi agent in preparation of a pharmaceutical product for preventing or treating a lipid metabolism-related disease.

[0013] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Expressions such as "at least one of," when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0014] Each description and embodiment disclosed in the present application may also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in the application fall within the scope of the application. In addition, it should not be construed that the scope of the present application is limited by the detailed description described below.

[0015] An aspect provides an RNAi agent including: an antisense strand that has sequence complementarity to an apolipoprotein C-III (APOC3) mRNA sequence and is 19 nucleotides (nt) to 21 nt in length; and a sense strand that has sequence complementarity to the antisense strand and is 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.

[0016]

[0017] RNAi agent

[0018] The term "RNA interference (RNAi)" refers to a mechanism for inhibiting expression of a target gene by inducing degradation of mRNA of the target gene by introduction of double-stranded RNA (dsRNA) composed of a strand having a sequence homology to mRNA of a target gene and a strand having a sequence complementary to the aforementioned strand.

[0019] The term "RNAi agent" or "nucleic acid molecules inducing RNAi" as used in the present specification refers to any agent or nucleic acid molecules capable of inhibiting or downregulating gene expression or viral replication by mediating the RNA interference in a sequence-specific manner. The term may refer to both an individual nucleic acid molecule, a plurality of the nucleic acid molecules, or a pool of the nucleic acid molecules. In an embodiment, the RNAi agent may be siRNA.

[0020] The term "small interfering RNA (siRNA; short interfering RNA)", used herein, refers to a short double-stranded RNA (dsRNA) that sequence-specifically mediates efficient gene silencing.

[0021] The term "gene" as used in the present specification should be considered in its broadest sense, and may encode a structural protein or a regulatory protein. Here, the regulatory protein includes a transcription factor, a heat shock protein, or a protein involved in DNA / RNA replication, transcription, and / or translation. In the disclosure, a target gene subject to expression suppression is inherent in the viral genome, and may be integrated into an animal genome or exist as an extrachromosomal component.

[0022] The term "antisense strand", used herein, refers to a polynucleotide that is substantially or 100 % complementary to a target nucleic acid of interest, and may be complementary in whole or in part with, for example, messenger RNA (mRNA), non-mRNA RNA sequences (e.g., microRNA, piwiRNA, tRNA, rRNA and hnRNA), or coding or non-coding DNA sequences.

[0023] The term "sense strand", used herein, refers to a polynucleotide having the same nucleic acid sequence as a target nucleic acid, or a polynucleotide in whole or in part the same with, for example, messenger RNA (mRNA), non-mRNA RNA sequences (e.g., microRNA, piwiRNA, tRNA, rRNA and hnRNA), or coding or non-coding DNA sequences.

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

[0025] The term "blunt end" as used in the present specification refers to a generally accepted meaning in the art. Regarding the RNAi agent or nucleic acid molecule as used in the present specification, the term may refer to an end of a double-stranded siRNA molecule that lacks overhanging nucleotides. The siRNA molecules described herein may be such that the 5'-end of an antisense strand and the 3'-end of a sense strand form a blunt end.

[0026]

[0027] RNAi agent for inhibiting expression of APOC3

[0028] The term "apolipoprotein C-III (APOC3)" describes one of very-low-density lipoproteins (VLDLs), which play an important role in production, metabolization, and removal of triglyceride-rich lipoproteins from plasma.　In detail, APOC3 inhibits a lipoprotein lipase which hydrolyzes triglycerides into free fatty acids. In addition, APOC3 inhibits but also ApoE-mediated hepatic uptake of triglyceride-rich lipoproteins through low-density lipoprotein receptors (LDLRs) and LDL receptor-related proteins (LRP) as well as through receptor independent endocytosis, and promotes the secretion of VLDLs from the liver. Moreover, according to a recent study, it has been reported that at least one mutation in APOC3 genes is highly correlated with improvement in lipid profile. Therefore, the APOC3 gene may be a potential therapeutic target for lipid metabolism-related diseases. The APOC3 may be interpreted to include the naturally occurring wild-type APOC3 and functional variants thereof, and the sequence of the APOC3 or a gene encoding the same may be obtained from a known database such as GenBank of the US National Institutes of Health.

[0029] The term "expression" as used herein refers to any meaning generally accepted in the art. The term may generally refer to a process by which a gene ultimately produces a protein. The expression includes, but is not limited to, transcription, splicing, post-transcriptional modification, or translation. As used in the present specification, an expression level may be determined or monitored by detection of an mRNA level or a protein level.

[0030] The term "inhibition" or "reduction", as used in reference to the expression of APOC3 gene in a subject, refers to a statistically significant decrease compared to an untreated or normal control group. The reduction may be, for example, at least 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, or 95 % or more, but it may be below a detection limit depending on a detection or measurement method.

[0031] siRNA is a small interfering RNA and is involved in RNA interference (RNAi). RNAi is an intracellular gene regulation mechanism first discovered in 1998 in Caenorthabditis elegans, and its mechanism of action is known to induce target gene degradation by an antisense strand, which is one of RNA double strands which are introduced into the cell, that complementarily binds to mRNA of a target gene, and RNAi has recently been one of the most popular candidate technologies for developing new drugs.

[0032] However, contrary to this possibility, side effects and disadvantages of siRNA have been continuously reported. For the development of RNAi-based therapeutic agents, it is necessary to overcome barriers such as 1) absence of an effective delivery system, 2) off-target effect, 3) induction of immune response, and 4) saturation of RNAi machinery in cells. Although siRNA is an effective method for directly regulating expression of a target gene, it is difficult to develop a therapeutic agent due to these issues. In this regard, asymmetric shorter duplex siRNA (asiRNA) is an asymmetric RNAi-induced structure having a shorter double helix length compared to the 19+2 structure of siRNA in the art. asiRNA technology overcame issues such as off-target effect, saturation of RNAi mechanism, and immune response by TLR3, which are identified in the existing siRNA structure technology, and accordingly, it is possible to develop new RNAi drugs with low side effects.

[0033] Based on this, asymmetric siRNA including a sense strand and an antisense strand complementary to the sense strand is presented in this embodiment, and since siRNA according to an embodiment does not cause issues such as off-target effect, saturation of RNAi mechanism, etc., siRNA may efficiently inhibit the expression of APOC3 genes to a desired degree while stably maintaining high delivery efficiency.

[0034] In an embodiment, asymmetric siRNA (asiRNA) targeting APOC3 is designed and prepared, and nucleic acid molecules inducing RNAi with excellent knockdown efficiency, i.e., APOC3 asiRNAs, were screened by using a cell or animal model expressing APOC3.

[0035] In an embodiment, the RNAi agent may include the sense strand that is 15 nt to 17 nt in length and the antisense strand that is 19 nt to 21 nt in length. In an embodiment, the length of the sense strand may be 16 nt, and the length of the complementary antisense strand may be 19 nt, 20 nt, or 21 nt, but embodiments are not particularly limited thereto.

[0036] The 5'-end of the antisense strand and the 3'-end of the sense strand may form a blunt end. The 3' end of the antisense strand may include, for example, an overhang of 2 nt to 6 nt.

[0037] In an embodiment, the sense strand may have a sequence selected from the sense strand sequences listed in Tables 1 to 4, and the antisense strand may have a sequence selected from antisense strand sequences listed in Tables 1 to 4.

[0038] In an embodiment, the sense strand may have, for example, any one 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 strand has any one sequence selected from the group consisting of SEQ ID NO: 337, SEQ ID NO: 339 and SEQ ID NO: 343.

[0039] In an embodiment, the antisense strand may have, for example, any one 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 strand has any one sequence selected from the group consisting of SEQ ID NO: 338, SEQ ID NO: 340, and SEQ ID NO: 344.

[0040] In an embodiment, the sense strand has any one 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 any one sequence selected from the group consisting of SEQ ID NO: 338, SEQ ID NO: 340, and SEQ ID NO: 344.

[0041] In an embodiment, the sense strand has a sequence of SEQ ID NO: 337, and the antisense strand has a sequence of SEQ ID NO: 338. In an embodiment, the sense strand has a sequence of SEQ ID NO: 339, and the antisense strand has a sequence of SEQ ID NO: 340. In an embodiment, the sense strand has a sequence of SEQ ID NO: 343, and the antisense strand has a sequence of SEQ ID NO: 344.

[0042]

[0043] RNAi agent with chemical modifications introduced

[0044] In the RNAi agent, the sense strand or the antisense strand may include one or more chemical modifications.

[0045] General siRNA cannot pass through the cell membrane due to high negative charge and high molecular body weight of the phosphate backbone structure, and is rapidly degraded and eliminated from the blood, making it difficult to deliver a sufficient amount to an actual target site for RNAi induction. Currently, in case of in vitro delivery, many high-efficiency delivery methods using cationic lipids and cationic polymers have been developed, however, in case of in vivo, it is difficult to deliver siRNA with as high efficiency as in vitro, and there is an issue of reduced siRNA delivery efficiency due to interaction with various proteins existing in the living body.

[0046] Accordingly, provided in the example is an RNAi agent having improved hepatocyte targeted delivery ability by introduction of chemical modifications to the asymmetric siRNA structure, more specifically, provided is an asymmetric siRNA construct (GalNAc asymmetric siRNA, GalNAc-asiRNA) capable of effective intracellular delivery without a separate carrier.

[0047] In the disclosure, the chemical modification in the sense strand or the antisense strand may include one or more selected from: binding with an N-acetylgalactosamine (GalNAc) derivative, or a cell-penetrating peptide; modification of a nucleotide bond with phosphorothioate, boranophosphate, or methyl phosphonate; substitution of an -OH group at a 2' carbon position of a sugar structure of a nucleotide with -H, -CH3(methyl), -OCH3(-O-methyl), -NH2, -F, -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; substitution of any nucleotide with an inverted abasic residue; and substitution of an -OH group at the 5' end with E-vinylphosphonate.

[0048] In an embodiment, asymmetric siRNA (GalNAC-asiRNA) targeting APOC3 and having the aforementioned chemical modification is designed and prepared. Afterwards, by using a cell or animal model expressing APOC3, nucleic acid molecules inducing RNAi, i.e., APOC3 GalNAC-asiRNA, with excellent knockdown efficiency and an effective effect of lowering the serum triglyceride level and serum total cholesterol level was screened without application of a separate delivery vehicle.

[0049] In an embodiment, the RNAi agent containing the N-acetylgalactosamine (GalNAc) derivative may have a structure of Formula 1, or 2: The GalNAc derivative recognizes asialoglycoprotein (ASGPR) receptors on a surface of a hepatocyte to help the RNAi agent to flow into the hepatocyte. That is, the GaINAc derivative acts as an ASGPR-targeting moiety, and thus, the RNAi agent in which the GalNAc derivative is bound to an end may have improved delivery capability to the hepatocyte, thereby providing effective targeted treatment for target diseases.

[0050] [Formula 1]

[0051]

[0052] [Formula 2]

[0053]

[0054] The structure of Formula 2 may be a racemate or an isomer with RR, SS, RS, or SR configurations at the stereocenters.

[0055] In an embodiment, the sense strand may have one or more chemical modifications selected from: modification of 1 to 4 nucleotide bonds adjacent to the 3' end or the 5' end to phosphorothioate, boranophosphate, or methyl phosphonate; substitution of an -OH group at a 2' carbon position of a 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; substitution of any nucleotide at the 3' end or the 5' end with an inverted abasic residue; and binding with a GalNAc derivative or a cell-penetrating peptide at the 3' end.

[0056] In an embodiment, the antisense strand may have one or more chemical modifications selected from: modification of 2 to 7 nucleotide bonds adjacent to the 3' end or the 5' end to phosphorothioate, boranophosphate, or methyl phosphonate; substitution of an -OH group at a 2' carbon position of a 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 an OH group at the 5' end with phosphate or E-vinylphosphonate.

[0057] In one or more embodiments, the RNAi agent may have one or more modifications selected from the group consisting of: modification of 1 to 7 nucleotide bonds adjacent to the 3' end or the 5' end of the sense strand or antisense strand with phosphorothioate; substitution of an -OH group at a 2' carbon position of a sugar structure of at least one nucleotide with -H, -OCH3, or -F; substitution of a nucleotide at the 3' end and / or the 5' end of the sense strand with an inverted abasic residue; binding with an N-acetylgalactosamine derivative at the 3' end of the sense strand; and substitution of an OH group at the 5' end of the antisense strand with phosphate or E-vinylphosphonate.

[0058] In an embodiment, the RNAi agent may have modifications as follows: in the sense strand, from 5' to 3', positions 2, 4, 5, 6, 14 and 16 are modified by 2'-fluoro, the rest positions are modified by 2'-OMe, at the 5' end, there are two phosphorothioate linkages, and optionally, at the 3' end, there is a nucleotide linked with an GalNAc derivative through a phosphorothioate linkage or phosphodiester linkage; in the antisense strand, from 5' to 3', positions 2, 14 and 16 are modified by 2'-fluoro, an existing 2'-OH group at positions 5 and 7 is substituted with 2'-H, and the rest positions are modified by 2'-OMe, at the 3' end, there are six phosphorothioate linkages, at the 5' end, there is a 5' (E)-vinylphosphonate nucleotide.

[0059] In an embodiment, the RNAi agent may have modificaitons as follows: in the sense strand, from 5' to 3', positions 4 and 6 are modified by 2'-fluoro, the rest positions are modified by 2'-OMe, at the 5' end, there are two phosphorothioate linkages, and optionally, at the 3' end, there is a nucleotide linked an GalNAc derivative through a phosphorothioate linkage or phosphodiester linkage ; in the antisense strand, from 5' to 3', positions 2, 14 and 16 are modified by 2'-fluoro, an existing 2'-OH group at positions 5 and 7 is substituted with 2'-H, and the rest positions are modified by 2'-OMe, at the 3' end, there are six phosphorothioate linkages, at the 5' end, there is a 5' (E)-vinylphosphonate nucleotide.

[0060] In an embodiment, the RNAi agent as described containing an GalNAc derivative is selected from Formula 1 or Formula 2; preferably Formula 2.

[0061] In an embodiment, the sense strand of the RNAi agent has the chemical modification pattern: (m)*(f)*(m)(f)(f)(f)(m)(m)(m)(m)(m)(m)(m)(f)(m)(f)*[Linker 2] , or (m)*(m)*(m)(f)(m)(f)(m)(m)(m)(m)(m)(m)(m)(m)(m)(m)*[Linker 2],

[0062] the antisense strand of the RNAi agent has the chemical modification pattern:[EVP](m)*(f)*(m)(m)(d)(m)(d)(m)(m)(m)(m)(m)(m)(f)(m)*(f)*(m)*(m)*(m)*(m)*(m).

[0063] wherein, * indicates a phosphorothioated bond, m indicates 2'-O-methyl modified nucleotide, f indicates 2'-fluoro modified nucleotide, d indicates substitution of existing 2'-OH with -H, EVP indicates E-vinylphosphonate, and Linker 2 indicates a GalNAc derivative in Formula 2.

[0064] In an embodiment, the RNAi agent as described provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene. In an embodiment, the target gene is APOC3.

[0065] In an embodiment, 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 antisense strand sequences listed in Tables 5, 6, 12, 13, and 14. In a preferred embodiment, the RNAi agent is selected from the GalNAC-asiRNA listed in Table 12, 13 and 14.

[0066] In an embodiment, the sense strand may include any one sequence selected from the group consisting of: (A) to (F) in the table below, and the antisense strand may include any one sequence selected from the group consisting of: (a) to (f) in the table below.

[0067]

[0068] In an embodiment, the sense strand may have a sequence of (mG)*(fG)*(mG)(fA)(fC)(fA)(mG)(mU)(mA)(mU)(mU)(mC)(mU)(fC)(mA)(fA)*[Linker 2], and the antisense strand may have a sequence of [EVP](mU)*(fU)*(mG)(mA)(dG)(mA)(dA)(mU)(mA)(mC)(mU)(mG)(mU)(fC)(mC)*(fC)*(mU)*(mU)*(mU)*(mU)*(mA).

[0069] In an embodiment, the sense strand may have a sequence of (mG)*(fG)*(mA)(fC)(fA)(fG)(mU)(mA)(mU)(mU)(mC)(mU)(mC)(fA)(mG)(fA)*[Linker 2], and the antisense strand may have a sequence of [EVP](mU)*(fC)*(mU)(mG)(dA)(mG)(dA)(mA)(mU)(mA)(mC)(mU)(mG)(fU)(mC)*(fC)*(mC)*(mU)*(mU)*(mU)*(mU).

[0070] In an embodiment, the sense strand may have a sequence of (mA)*(mC)*(mA)(fG)(mU)(fA)(mU)(mU)(mC)(mU)(mC)(mA)(mG)(mU)(mG)(mA)*[Linker 2], and the antisense strand may have a sequence of [EVP](mU)*(fC)*(mA)(mC)(dT)(mG)(dA)(mG)(mA)(mA)(mU)(mA)(mC)(fU)(mG)*(fU)*(mC)*(mC)*(mC)*(mU)*(mU).

[0071] In these sequences, * indicates a phosphorothioated bond, m indicates 2'-O-methyl, f indicates 2'-fluoro, d indicates substitution of existing 2'-OH with -H, EVP indicates E-vinylphosphonate, Linker 1 indicates a GalNAc derivative of Formula 1, and Linker 2 indicates a GalNAc derivative of Formula 2.

[0072] In detail, the sense strand and the antisense strand of the RNAi agent may have sequences of any one of the following sense strand sequences and antisense strand sequences selected from the tables above, and for example, the RNAi agent may have a combination of a sense strand (A) and an antisense strand (a); a combination of a sense strand (B) and an antisense strand (a); a combination of a sense strand (B) and an antisense strand (b); a combination of a sense strand (C) and an antisense strand (b); a combination of a sense strand (C) and an antisense strand (c); a combination of a sense strand (D) and an antisense strand (c); a combination of a sense strand (D) and an antisense strand (d); a combination of a sense strand (E) and an antisense strand (d); a combination of a sense strand (E) and an antisense strand (e); a combination of a sense strand (F) and an antisense strand (e); a combination of a sense strand (F) and an antisense strand (f); and a combination of a sense strand (A) and an antisense strand (f).

[0073]

[0074] Another aspect of the disclosure provides a pharmaceutical composition for preventing or treating lipid metabolism-related diseases the pharmaceutical composition including the RNAi agent as an active ingredient.

[0075] Another aspect of the disclosure provides use of the RNAi agent in preparation of a pharmaceutical product for preventing or treating lipid metabolism-related diseases.

[0076] Since the pharmaceutical composition contains or uses the above-described RNAi agent as it is, descriptions thereof are omitted in order to avoid excessive complexity of the present specification.

[0077]

[0078] lipid metabolism-related disease

[0079] The pharmaceutical composition may be utilized as an active ingredient of the pharmaceutical composition for preventing or treating lipid metabolism-related diseases by inhibiting expression of an APOC3 gene.

[0080] The lipid metabolism-related disease may collectively refer to diseases caused by synthesis and decomposition of lipids, including synthesis of functional and structural lipids, such as those involved in cell membrane composition, and decomposition of fat for energy production. For example, the lipid metabolism-related disease may be a disease caused by excessive lipid accumulation in the body. In addition, the lipid metabolism-related disease is a disease that is closely related to the expression level of the APOC3 gene, and for example, may include any one selected from the group consisting of hyperlipidemia, hypertriglyceridemia, obesity, atherosclerosis, diabetes, cardiovascular disease, coronary artery disease, familial chylomicronemia syndrome, dyslipidemia, stroke, inflammation of the liver, and hepatic steatosis. However, the lipid metabolism-related disease is not limited thereto.

[0081]

[0082] Pharmaceutical composition

[0083] The term "effective ingredient" as used herein refers to an appropriate effective amount of an ingredient that brings about a beneficial or desirable clinical or biochemical outcome. Specifically, the term may refer to an agent, an active agent, or an RNAi agent of an effective amount.

[0084] The effective amount may be administered one or more times, and unlimitedly may be an appropriate amount for preventing a disease, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., not exacerbating) the disease state, delaying or reducing the rate of disease progression, or improving or temporarily alleviating and ameliorating (partially or fully) the disease state.

[0085] The term "prevention" as used herein refers to any action that blocks an occurrence of a disease in advance, suppresses a disease, or delays progression thereof. For example, the term refers to preventing or interrupting the development of the lipid metabolism-related disease or characteristic features thereof, or defending or protecting against the development of the lipid metabolism-related disease or characteristic features thereof.

[0086] The term "treatment" as used in the present specification refers to both therapeutic treatment and preventive or prophylactic measures. Also, it refers to any action that ameliorates and beneficially changes the symptoms of a disease. For example, it refers to preventing, reducing, or ameliorating the lipid metabolism-related disease or characteristic features thereof, or to delaying (attenuating) progression of the lipid metabolism-related disease or characteristic features thereof in a subject.

[0087] The term "effective amount" as used in the present specification refers to a generally accepted meaning in the art. The term generally refers to an amount of a molecule, compound, or construct that elicits an intended biological response (e.g., a beneficial response) in a cell, tissue, a system, an animal, or a human, sought by researchers, veterinarians, physicians, or other clinicians, etc. Specifically, a "therapeutically effective amount" refers to an amount of a molecule, compound, or construct that is capable of eliciting a desirable medical response to the extent that a particular clinical treatment may be considered effective, due to, for example, a therapeutically relevant change in a measurable parameter associated with the disease or disorder. The therapeutically effective amount of a drug for treatment of a disease or a disorder may be an amount necessary to cause a therapeutically relevant change in the parameter.

[0088] A method of administering the pharmaceutical composition may be determined by a person skilled in the art based on the symptoms of the patient and the severity of the disease. In addition, the pharmaceutical composition may be formulated in various forms such as powders, tablets, capsules, solutions, injections, ointments, syrups, etc., and may be provided in unit-dose or multi-dose containers, for example, sealed ampoules and bottles.

[0089] The pharmaceutical composition of the disclosure may be administered orally or parenterally. Routes of administration of the composition according to the disclosure may be, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, sublingual, or topical, but are not limited thereto. Dosage of the composition according to the disclosure varies depending on the patient's body weight, age, sex, health status, diet, administration time, method, excretion rate, or severity of disease, etc. and may be readily determined by those of average skill in the art. In addition, the composition of the disclosure may be formulated into a suitable formulation for clinical administration using known techniques.

[0090]

[0091] Another aspect of the disclosure provides a method of preventing or treating the lipid metabolism-related disease, the method including administering the RNAi agent to a subject.

[0092] Since the method of treating the lipid metabolism-related disease includes or uses the aforementioned RNAi agent or pharmaceutical composition as it is, any content common thereto is omitted to avoid undue complexity of the present specification.

[0093] The term "subject" as used herein refers to a subject in need of treatment for a disease, specifically, lipid metabolism-related disease, and more specifically, the term may include all mammals such as a human or a non-human primate, a mouse, a dog, a cat, a horse, a cow, sheep, a pig, a goat, a camel, and an antelope.

[0094] An RNAi agent according to an aspect may inhibit expression of APOC3 by binding to and degrading APOC3 mRNA, while alleviating side effects such as non-specific immune responses and off-target effects.

[0095] In addition, the RNAi agent according to an aspect is effectively delivered to hepatocytes and may be used as a target therapeutic agent for lipid metabolism-related diseases.

[0096] The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0097] FIG. 1 is a schematic diagram of a structure of APOC3 asiRNA consisting of a 16-mer sense strand and a 21-mer antisense strand;

[0098] FIG. 2a shows results of confirming expression levels of APOC3 mRNA, after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-001 to asiAPOC3-032) according to an aspect, at a concentration of 1 nM;

[0099] FIG. 2b shows results of confirming expression levels of APOC3 mRNA, after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-033 to asiAPOC3-064) according to an aspect, at a concentration of 1 nM;

[0100] FIG. 2c shows results of confirming expression levels of APOC3 mRNA, after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-065 to asiAPOC3-096) according to an aspect, at a concentration of 1 nM;

[0101] FIG. 3a shows results of confirming expression levels of APOC3 mRNA, after treating Huh-7 cells with 34 APOC3 asiRNAs (asiAPOC3-097 to asiAPOC3-130) according to an aspect, at a concentration of 1 nM;

[0102] FIG. 3b shows results of confirming expression levels of APOC3 mRNA, after treating Huh-7 cells with 34 APOC3 asiRNAs (asiAPOC3-131 to asiAPOC3-164) according to an aspect, at a concentration of 1 nM;

[0103] FIG. 3c shows results of confirming expression levels of APOC3 mRNA, after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-165 to asiAPOC3-196) according to an aspect, at a concentration of 1 nM;

[0104] FIG. 4a shows results of confirming expression levels of APOC3 mRNA, after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-001 to asiAPOC3-032) according to an aspect, at a concentration of 0.1 nM;

[0105] FIG. 4b shows results of confirming expression levels of APOC3 mRNA, after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-033 to asiAPOC3-064) according to an aspect, at a concentration of 0.1 nM;

[0106] FIG. 4c shows results of confirming expression levels of APOC3 mRNA, after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-065 to asiAPOC3-096) according to an aspect, at a concentration of 0.1 nM;

[0107] FIG. 5a shows results of confirming expression levels of APOC3 mRNA, after treating Huh-7 cells with 34 APOC3 asiRNAs (asiAPOC3-097 to asiAPOC3-130) according to an aspect, at a concentration of 0.1 nM;

[0108] FIG. 5b shows results of confirming expression levels of APOC3 mRNA, after treating Huh-7 cells with 34 APOC3 asiRNAs (asiAPOC3-131 to asiAPOC3-164) according to an aspect, at a concentration of 0.1 nM;

[0109] FIG. 5c shows results of confirming expression levels of APOC3 mRNA, after treating Huh-7 cells with 32 APOC3 asiRNAs (asiAPOC3-165 to asiAPOC3-196) according to an aspect, at a concentration of 0.1 nM;

[0110] FIG. 6 shows results of confirming expression levels of APOC3 mRNA, after treating primary human hepatocytes with 40 APOC3 asiRNAs according to an aspect, at a concentration of 1 nM;

[0111] FIG. 7 shows results of confirming expression levels of APOC3 mRNA, after treating primary human hepatocytes with 40 APOC3 asiRNAs according to an aspect, at a concentration of 0.1 nM;

[0112] FIG. 8a & 8b show results of confirming expression levels of APOC3 mRNA, after treating primary human hepatocytes with 40 & 44 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect, at a concentration of 200 nM;

[0113] FIGs. 9a & 9b show results of confirming expression levels of APOC3 mRNA, after treating primary human hepatocytes with 21 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect, at a concentration of 20 nM or 100 nM;

[0114] FIG. 10 shows results of confirming APOC3 levels in mouse-derived serum, after subcutaneously injecting 10 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect into transgenic mice expressing APOC3, at a single dose of 1 mg / kg(mpk);

[0115] FIG. 11 shows results of confirming APOC3 levels in mouse-derived serum, after subcutaneously injecting 8 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect into transgenic mice expressing APOC3, at a single dose of 1 mg / kg(mpk);

[0116] FIG. 12 shows results of confirming triglyceride levels in mouse-derived serum, after subcutaneously injecting 8 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect into transgenic mice expressing APOC3, at a single dose of 1 mg / kg(mpk);

[0117] FIG. 13 shows results of confirming total cholesterol levels in mouse-derived serum, after subcutaneously injecting 8 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect into transgenic mice expressing APOC3, at a single dose of 1 mg / kg(mpk);

[0118] FIG. 14a shows results of confirming expression levels of APOC3 mRNA, after treating primary human hepatocytes with 12 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect, at a concentration of 200 nM;

[0119] FIG. 14b shows results of confirming expression levels of APOC3 mRNA, after treating primary human hepatocytes with 12 APOC3 GalNAc-asiRNAshaving chemical modifications according to an aspect, at a concentration of 200 nM;

[0120] FIG. 14c shows results of confirming expression levels of APOC3 mRNA, after treating primary human hepatocytes with 12 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect, at a concentration of 200 nM;

[0121] FIG. 15a shows results of confirming expression levels of APOC3 mRNA, after treating primary human hepatocytes with 12 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect, at a concentration of 20 nM;

[0122] FIG. 15b shows results of confirming expression levels of APOC3 mRNA, after treating primary human hepatocytes with 12 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect, at a concentration of 20 nM;

[0123] FIG. 15c shows results of confirming expression levels of APOC3 mRNA, after treating primary human hepatocytes with 12 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect, at a concentration of 20 nM;

[0124] FIG. 16a shows results of confirming expression levels of APOC3 mRNA, after treating primary human hepatocytes with 12 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect, at a concentration of 2 nM;

[0125] FIG. 16b shows results of confirming expression levels of APOC3 mRNA, after treating primary human hepatocytes with 12 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect, at a concentration of 2 nM;

[0126] FIG. 16c shows results of confirming expression levels of APOC3 mRNA, after treating primary human hepatocytes with 12APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect, at a concentration of 2 nM;

[0127] FIG. 17 shows results of confirming APOC3 levels in mouse-derived serum, after subcutaneously injecting 7 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect into transgenic mice expressing APOC3, at a single dose of 0.25 mg / kg(mpk);

[0128] FIG. 18 shows results of confirming triglyceride levels in mouse-derived serum, after subcutaneously injecting 7 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect into transgenic mice expressing APOC3, at a single dose of 0.25 mg / kg(mpk); and

[0129] FIG. 19 shows results of confirming total cholesterol levels in mouse-derived serum, after subcutaneously injecting 7 APOC3 GalNAc-asiRNAs having chemical modifications according to an aspect into transgenic mice expressing APOC3, at a single dose of 0.25 mg / kg(mpk).

[0130] Hereinafter, the disclosure will be described in more detail through examples. However, these examples are intended to illustrate the disclosure, and the scope of the disclosure is not limited to these examples.

[0131]

[0132] Example 1: Screening of nucleic acid molecules inducing RNAi targeting APOC3

[0133] In this example, 196 nucleic acid molecules inducing RNAi targeting APOC3 were prepared, and inhibitory efficacy of the nucleic acid molecules on the expression of APOC3 was evaluated, so as to screen APOC3 asiRNA with effective RNAi efficacy according to an embodiment.

[0134]

[0135] 1-1. Design and preparation of APOC3 asiRNA

[0136] To obtain a double-stranded nucleic acid molecule that induces RNAi targeting the APOC3, target sequences for the APOC3 gene were selected first, and then, APOC3 asymmetric siRNAs (APOC3 asiRNAs) for the same were designed (sense strand (16-mer), antisense strand (21-mer), see FIG. 1). Specifically, after acquiring information of the APOC3 gene through searching the NCBI database, a total of 196 asiRNAs were designed in consideration of animal experiments, and synthesized at a 4-nmole scale by OliX US. Afterwards, the synthesized asiRNAs were subjected to 15 % polyacrylamide gel electrophoresis (PAGE), followed by quality control (QC) using a ChemiDoc UV transilluminator (Biorad). The sequence information of the APOC3 asiRNAs designed according to the aforementioned method is as shown in Tables 1 to 4 below.

[0137] [Table 1]

[0138]

[0139]

[0140] [Table 2]

[0141]

[0142]

[0143] [Table 3]

[0144]

[0145]

[0146] [Table 4]

[0147]

[0148]

[0149] 1-2. Screening of 196 APOC3 asiRNAs

[0150] After transfecting Huh-7 cells, the human liver cancer cells, with each of the 196 APOC3 asiRNAs, qRT-PCR was performed thereon to measure expression levels of APOC3 mRNA. Specifically, the Huh-7 cells were seeded in a 96 well plate at a concentration of 8.0Х103cells / well to be transfected with the APOC3 asiRNA (1 nM) by using 0.2 μl of lipofecamine RNAiMAX (Invitrogen, 13778150). 24 hours after the transfection, cell lysate was prepared by using a SuperPrepTM cell lysis & RT kit for qPCR kit II (TOYOBO, SCQ-401), and cDNA was synthesized through reverse transcription using the mRNA included in the lysate as a template. Afterwards, the synthesized cDNA was used as a template for quantitative PCR performed by using a THUNDERBIRD probe qPCR MIX (TOYOBO, QPS-101), an APOC3 probe (Thermofisher, Hs00163644_m1),and an RNA18S5 probe (Thermofisher, Hs03928985_g1). Then, the expression levels of APOC3 mRNA were identified by using a CFX Connect Real-Time PCR system (Bio-Rad). In addition, after transfection of Huh-7 cells with the 196 APOC3 asiRNAs at a concentration of 0.1 nM, the expression levels of APOC3 mRNA were measured again by qRT-PCR using the same method as described above. Meanwhile, in this example, a group (Mock) treated with only a transfection reagent was used as a control group.

[0151] As a result, as shown in FIGs. 2 to 5, the inhibitory effect on the expression of APOC3 mRNA was confirmed in the Huh-7 cells according to the treatment of the cells with the APOC3 asiRNA (1 nM or 0.1nM). Also, 40 APOC3 asiRNAs that exhibited relatively excellent inhibitory efficiency on the expression were identified (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).

[0152]

[0153] 1-3. Screening of 40 APOC3 asiRNAs

[0154] After transfecting primary human hepatocytes with each of the screened 40 APOC3 asiRNAs, qRT-PCR was performed thereon to measure expression levels of APOC3 mRNA. Specifically, the primary human hepatocytes were seeded in a 96 well plate at a concentration of 1.0Х104cells / well to be transfected with the APOC3 asiRNA (1 nM) by using lipofecamine RNAiMAX (Invitrogen, 13778150). 24 hours after the transfection, cell lysate was prepared by using a SuperPrepTM cell lysis & RT kit for qPCR kit II (TOYOBO, SCQ-401), and cDNA was synthesized through reverse transcription using the mRNA included in the lysate as a template. Afterwards, the synthesized cDNA was used as a template for quantitative PCR performed by using a THUNDERBIRD probe qPCR MIX (TOYOBO, QPS-101), an APOC3 probe (Thermofisher, Hs00163644_m1),and an RNA18S5 probe (Thermofisher, Hs03928985_g1). Then, the expression levels of APOC3 mRNA were identified by using a CFX Connect Real-Time PCR system (Bio-Rad). In addition, after transfection of primary human hepatocytes with the 40 APOC3 asiRNAs at a concentration of 0.1 nM, the expression levels of APOC3 mRNA were measured again by performing qRT-PCR using the same method as described above. Meanwhile, in this example, a group (Mock) treated with only a transfection reagent was used as a control group.

[0155] As a result, as shown in FIGs. 6 and 7, the inhibitory effect on the expression of APOC3 mRNA was confirmed in the primary human hepatocytes according to the treatment of the cells with the APOC3 asiRNA (1 nM or 0.1nM). Also, 21 APOC3 asiRNAs that exhibited relatively excellent inhibitory efficiency on the expression (asiAPOC3-002, 023, 083, 086, 094, 096, 097, 101, 104, 128, 144, 168, 169, 170, 171, 172, 178, 179, 181, 182, and 194).

[0156]

[0157] Example 2: Screening of nucleic acid molecules inducing RNAi having chemical modifications

[0158] In this example, based on the experimental results of Example 1, 84 nucleic acid molecules inducing RNAi having chemical modifications targeting APOC3 were introduced were prepared, and inhibitory efficacy of the nucleic acid molecules on the expression of APOC3 was evaluated, so as to screen APOC3 GalNAc-asiRNAs with effective RNAi efficacy.

[0159] 2-1. Preparation of nucleic acid molecules inducing RNAi having chemical modifications

[0160]

[0161] As a double-stranded nucleic acid molecule that induces highly efficient RNAi targeting APOC3, APOC3 GalNAc-asiRNA having chemical modifications introduced was designed. Specifically, in consideration of 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 basic skeletons. Then, 84 APOC3 GalNAc-asiRNAs were designed with each of the basic skeletons, wherein the following modifications were introduced: 1) substitution of an -OH group at a 2' carbon position of a sugar structure in the nucleotides of the sense strand and the antisense strand with -H, -OMe, or -F, 2) introduction of phosphorothioate modifications in the backbone of the 5' end or the 3' end of the antisense strand or the 5' end of the sense strand, 3) introduction of phosphate at the 5' end of the antisense strand, and 4) introduction of a GalNAc derivative at the 3' end of the sense strand.

[0162] The sequence information of the APOC3 GalNAc-asiRNA having chemical modifications introduced prepared in this example is shown in Tables 5 and 6. Specifically, in Tables 5 and 6, "*" indicates a form in which an existing phosphodiester bond is substituted with a phosphorothioate bond, "d" indicates a form in which an existing 2'-OH group is substituted with 2'-H, wherein, for example, dT refers to a form in which a 2'-OH group of existing uridine is substituted with 2'-H to be modified to thymine, and "m" indicates a form in which an existing 2'-OH group is substituted with 2'-O-methyl. In addition, "f" indicates, for example, in a case of fG, a form in which a 2'-OH group of existing guanine is substituted with fluoro, "Phos" indicates a form in which an existing 5'-OH group is substituted with phosphate, and "Linker 1" indicates a trivalent GalNAc derivative of Formula 1 is bound to the 3' end of the sense strand (see Table 7).

[0163] [Table 5]

[0164]

[0165]

[0166] [Table 6]

[0167]

[0168]

[0169] [Table 7]

[0170]

[0171] [Formula 1]

[0172]

[0173] 2-2. Screening of 84 APOC3 GalNAc-asiRNAs

[0174] After treating primary human hepatocytes with each of the screened 84 GalNAc-asiRNAs, qRT-PCR was performed thereon to measure expression levels of APOC3 mRNA. Specifically, the primary human hepatocytes were seeded in a 96 well plate at 3.0Х104cells / well, and treated with APOC3 GalNAc-asiRNA (200 nM). After 24 hours of the treatment, cell lysate was prepared by using a SuperPrepTM cell lysis & RT kit for qPCR kit II (TOYOBO, SCQ-401), and cDNA was synthesized through reverse transcription using the mRNA included in the lysate as a template. Afterwards, the synthesized cDNA was used as a template for quantitative PCR performed by using a THUNDERBIRD probe qPCR MIX (TOYOBO, QPS-101), an APOC3 probe (Thermofisher, Hs00163644_m1),and an RNA18S5 probe (Thermofisher, Hs03928985_g1). Then, the expression levels of APOC3 mRNA were identified by using a CFX Connect Real-Time PCR system (Bio-Rad). Meanwhile, in this example, a no treatment group (NT) was used as a control group.

[0175] As a result, as shown in FIG. 8, the inhibitory effect on the expression of APOC3 mRNA was confirmed in the primary human hepatocytes according to the treatment of the cells with the APOC3 GalNAc-asiRNA. Also, 42 APOC3 GalNAc-asiRNAs that exhibited relatively excellent inhibitory efficiency on the expression 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]

[0177] 2-3. Screening of 42 APOC3 GalNAc-asiRNAs

[0178] After treating primary human hepatocytes with each of the screened 42 GalNAc-asiRNAs at a concentration of 20 nM or 100 nM, qRT-PCR was performed thereon in the same manner as in Example 2-2 to measure expression levels of APOC3 mRNA. Meanwhile, in this example, a no treatment group (NT) was used as a control group.

[0179] As a result, as shown in FIG. 9, the inhibitory effect on the expression of APOC3 mRNA was confirmed in the primary human hepatocytes according to the treatment of the cells with the APOC3 GalNAc-asiRNA. Also, 10 APOC3 GalNAc-asiRNAs that exhibited relatively excellent inhibitory efficiency on the expression were identified (OLX706C-104-3, 168-4, 169-3, 169-4, 170-1, 170-3, 170-4, 172-2, 172-3, and 172-4).

[0180]

[0181] Example 3: Screening of nucleic acid molecules inducing RNAi having chemical modifications, by using animal model

[0182] In this example, based on the experimental results of Example 2, for 10 nucleic acid molecules inducing RNAi (OLX706C-104-3, 168-4, 169-3, 169-4, 170-1, 170-3, 170-4, 172-2, 172-3, and 172-4) having chemical modifications targeting APOC3, the serum APOC3 level and / or the serum triglyceride level and serum total cholesterol level were evaluated by using a transgenic mouse expressing human APOC3, so as to screen APOC3 GalNAc-asiRNAs with effective RNAi efficacy.

[0183]

[0184] 3-1. Screening of 10 APOC3 GalNAc-asiRNAs

[0185] The APOC3 GalNAc-asiRNA was subcutaneously injected at a single dose of 1 mg / kg(mpk) into a transgenic mouse expressing human APOC3 (including 2.5 kb of 5' flanking sequence and 1.1 kb of 3' flanking sequence) under the C57BL / 6J genetic background. On Day 6 and Day 14 from the day of the subcutaneous injection into the mouse, the serum APOC3 level was measured by using an apolipoprotein C-III ELISA kit (ab154131). Then, the measured APOC3 value was normalized to the serum APOC3 level before the injection of the APOC3 GalNAc-asiRNA and to the serum APOC3 level of a control group that was administered with saline on the day of measurement, so as to evaluate the APOC3 level in the mouse-derived serum. Meanwhile, in this example, a group treated with saline was used as the control group.

[0186] As a result, as shown in Table 8 and FIG. 10, a decrease in the serum APOC3 level of the transgenic mouse model was confirmed according to the treatment of the APOC3 GalNAc-asiRNA. Also, 8 the APOC3 GalNAc-asiRNAs that exhibited relatively excellent inhibitory efficiency on the expression were identified (OLX706C-169-3, 169-4, 170-1, 170-3, 170-4, 172-2, 172-3, and 172-4).

[0187] [Table 8]

[0188]

[0189]

[0190] 3-2. Screening of 8 APOC3 GalNAc-asiRNAs

[0191] The screened 8 APOC3 GalNAc-asiRNAs were subcutaneously injected into a transgenic mouse at a single dose of 1 mg / kg(mpk). On Day 6, Day 14, Day 21, and Day 28 from the day of the subcutaneous injection into the mouse, an apolipoprotein C-III ELISA kit (ab154131) was used to measure the serum APOC3 level, and an automatic chemistry analyzer (FDC NX600V) was used to measure the serum triglyceride level and serum total cholesterol level. Then, the measured values of the APOC3, triglyceride, or total cholesterol were normalized to serum levels of the same before the injection of the APOC3 GalNAc-asiRNA, so as to evaluate the levels of APOC3, triglyceride, and total cholesterol in the mouse-derived serum. Meanwhile, in this example, a group treated with saline was used as a control group.

[0192] As a result, as shown in Tables 9 to 11 and FIGs. 11 to 13, a decrease in the serum APOC3 level and a decrease in the serum triglyceride level and serum total cholesterol level were confirmed in in the transgenic mouse models each treated with the APOC3 GalNAc-asiRNA. In addition, 3 APOC3 GalNAc-asiRNAs that exhibited the highest inhibitory efficiency on the expression were identified (OLX706C-169-4, OLX706C-170-4, and OLX706C-172-4).

[0193] [Table 9]

[0194]

[0195] [Table 10]

[0196]

[0197] [Table 11]

[0198]

[0199]

[0200] Example 4: Conformation of inhibitory effect on expression of nucleic acid molecules inducing RNAi having chemical modifications introduced

[0201] In this example, 3 nucleic acid molecules were used as the basic skeletons, and 12 chemically modified patterns were introduced for each of the basic skeletons, so as to prepare 36 nucleic acid molecules inducing RNAi having chemical modifications targeting APOC3. Then, regarding the nucleic acid molecules inducing RNAi having chemical modifications targeting APOC3, the efficacy of inhibiting APOC3 expression and the level of changes of serum factors upon the inhibition of expression were evaluated, so as to screen APOC3 GalNAc-asiRNA with effective RNAi efficacy.

[0202] 4-1. Preparation of nucleic acid molecules inducing RNAi having chemical modifications

[0203] As a double-stranded nucleic acid molecule that induces highly efficient RNAi targeting APOC3, APOC3 GalNAc-asiRNA having chemical modifications introduced was designed. Specifically, based on the experimental results of Example 2 and Example 3, 3 APOC3 asiRNAs (asiAPOC3-169, 170, and 172) were selected as basic skeletons. Then, 36 APOC3 GalNAc-asiRNAs were designed with each of the basic skeletons, wherein the following modifications were introduced: 1) substitution of an -OH group at a 2' carbon position of a sugar structure in the nucleotides of the sense strand and the antisense strand with -H, -OMe, or -F, 2) introduction of phosphorothioate modifications in the backbone of the 5' end or the 3' end of the antisense strand or the 5' end or the 3' end of the sense strand, 3) introduction of E-vinylphosphonate (EVP) at the 5' end of the antisense strand, 4) introduction of an inverted abasic residue at the 5' end or the 3' end of the sense strand, and 5) introduction of a GalNAc derivative at the 3' end of the sense strand.

[0204] The sequence information of the APOC3 GalNAc-asiRNA having chemical modifications introduced prepared in this example is shown in Tables 12 to 14. Specifically, in Tables 12 to 14, the chemical modifications indicated by "*", "d," "m," "f," "EVP," "invAb," "Linker 1," and "Linker 2" are as shown in Table 15.

[0205] [Table 12]

[0206]

[0207] [Table 13]

[0208]

[0209] [Table 14]

[0210]

[0211] [Table 15]

[0212]

[0213] [Formula 1]

[0214]

[0215] [Formula 2]

[0216] , wherein it possesses (S)- and (R)-configurations at stereocenters (1) and (2), respectively.

[0217]

[0218] 4-2. Screening of 36 APOC3 GalNAc-asiRNAs

[0219] After treating primary human hepatocytes with each of the screened 36 GalNAc-asiRNAs, qRT-PCR was performed thereon to measure expression levels of APOC3 mRNA. Specifically, the primary human hepatocytes were seeded in a 96 well plate at 3.0Х104cells / well, and treated with APOC3 GalNAc-asiRNA (200 nM, 20 nM, or 2 nM). After 24 hours of the treatment, cell lysate was prepared by using a SuperPrepTM cell lysis & RT kit for qPCR kit II (TOYOBO, SCQ-401), and cDNA was synthesized through reverse transcription using the mRNA included in the lysate as a template. Afterwards, the synthesized cDNA was used as a template for quantitative PCR performed by using a THUNDERBIRD probe qPCR MIX (TOYOBO, QPS-101), an APOC3 probe (Thermofisher, Hs00163644_m1), and an RNA18S5 probe (Thermofisher, Hs03928985_g1). Then, the expression levels of APOC3 mRNA were identified by using a CFX Connect Real-Time PCR system (Bio-Rad). Meanwhile, in this example, a no treatment group (NT) was used as a control group.

[0220] As a result, as shown in FIGs. 14 to 16, the inhibitory effect on the expression of APOC3 mRNA was confirmed in the primary human hepatocytes according to the treatment of the cells with the APOC3 GalNAc-asiRNA. In addition, 7 APOC3 GalNAc-asiRNAs that exhibited relatively excellent inhibitory efficacy on the expression were identified (OLX706C-169-16, OLX706C-170-16, OLX706C-170-20, OLX706C-170-24, OLX706C-172-7, OLX706C-172-8, and OLX706C-172-22).

[0221]

[0222] 4-3. Screening of 7 APOC3 GalNAc-asiRNAs by using animal model

[0223] The screened 7 APOC3 GalNAc-asiRNAs were subcutaneously injected into a transgenic mouse at a single dose of 0.25 mg / kg(mpk). On Day 7, Day 14, Day 21, and Day 28 from the day of the subcutaneous injection into the mouse, the serum APOC3 level was measured by using an immunoturbidimetry method, and the serum triglyceride level and serum total cholesterol level were used by using an automatic chemistry analyzer (FDC NX600V). Then, the measured values of the APOC3, triglyceride, or total cholesterol were normalized to serum levels of the same before the injection of the APOC3 GalNAc-asiRNA, so as to evaluate the levels of APOC3, triglyceride, and total cholesterol in the mouse-derived serum. Meanwhile, in this example, a group treated with saline was used as a control group.

[0224] As a result, as shown in Tables 16 to 18 and FIGs. 17 to 19, a decrease in the serum APOC3 level and a decrease in the serum triglyceride level and serum total cholesterol level were confirmed in in the transgenic mouse models each treated with the APOC3 GalNAc-asiRNA. In addition, 3 APOC3 GalNAc-asiRNAs that exhibited relatively excellent inhibitory efficacy on the expression were identified (OLX706C-169-16, OLX706C-170-16, and OLX706C-172-8).

[0225] [Table 16]

[0226]

[0227] [Table 17]

[0228]

[0229] [Table 18]

[0230]

[0231] Thus far, specific portions of the content of the disclosure have been described in detail, and it will be apparent to those of ordinary skill in the art that these specific descriptions are only preferred embodiments, and that the scope of the disclosure is not limited thereby. Accordingly, it is intended that the substantial scope of the disclosure is defined by the appended claims and their equivalents.

[0232] An RNAi agent according to an aspect may inhibit expression of APOC3 by binding to and degrading APOC3 mRNA, while alleviating side effects such as non-specific immune responses and off-target effects.

[0233] In addition, the RNAi agent according to an aspect is effectively delivered to hepatocytes and may be used as a target therapeutic agent for lipid metabolism-related diseases.

[0234]

[0235] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.

Claims

1.An RNAi agent comprising: an antisense strand that has sequence complementarity to an apolipoprotein C-III (APOC3) mRNA sequence and is 19 nucleotides (nt) to 21 nt in length; and a sense strand that has sequence complementarity to the antisense strand and is 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 sense strand sequences listed in Tables 1 to 4.3.The RNAi agent of claim 1, wherein the sense strand has any one 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 antisense strand sequences listed in Tables 1 to 4.5.The RNAi agent of claim 1, wherein the antisense strand has any one 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 any one 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 any one 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 a sequence of SEQ ID NO: 337, and the antisense strand has a sequence of SEQ ID NO: 338; the sense strand has a sequence of SEQ ID NO: 339, and the antisense strand has a sequence of SEQ ID NO: 340; or the sense strand has a sequence of SEQ ID NO: 343, and the antisense strand has a sequence of SEQ ID NO: 344.8.The RNAi agent of claim 1, wherein the sense strand is bound to an N-acetylgalactosamine (GalNAc) derivative at the 3' end.9.The RNAi agent of claim 1, 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 bonds adjacent to the 3' end or the 5' end with phosphorothioate, boranophosphate, or methyl phosphonate;substitution of an -OH group at a 2' carbon position of a 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 a nucleotide at the 3' end or the 5' end with an inverted abasic residue; andbinding with an N-acetylgalactosamine derivative or a cell-penetrating peptide at the 3' end.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 bonds adjacent to the 3' end or the 5' end with phosphorothioate, boranophosphate, or methyl phosphonate;substitution of an -OH group at a 2' carbon position of a 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; andsubstitution of an OH group at the 5' end with phosphate 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 bonds adjacent to the 3' end or the 5' end in the sense strand or the antisense strand with phosphorothioate;modification of an -OH group at a 2' carbon position of a sugar structure of at least one nucleotide in the sense strand or the antisense strand to -H, -OCH3, or -F;substitution of a nucleotide at the 3' end and / or the 5' end with an inverted abasic residue;binding with an N-acetylgalactosamine derivative at the 3' end of the sense strand; andsubstitution of an OH group at the 5' end of the antisense strand with phosphate or E-vinylphosphonate.13.The RNAi agent of claim 9, wherein the sense strand has a sequence selected from sense strand sequences listed in Tables 5, 6, 12, 13, and 14.14.The RNAi agent of claim 9, wherein the antisense strand has a sequence selected from antisense strand sequences listed in Tables 5, 6, 12, 13, and 14.15.The RNAi agent of claim 9, wherein the RNAi agent comprises modifications selected from one of the groups as follows:in the sense strand, from 5' to 3', positions 2, 4, 5, 6, 14 and 16 are modified by 2'-fluoro, the rest positions are modified by 2'-OMe, at the 5' end, there are two phosphorothioate linkages, and optionally, 3' end is bind with an GalNAc derivative through a phosphorothioate linkage or phosphodiester linkage; in the antisense strand, from 5' to 3', positions 2, 14 and 16 are modified by 2'-fluoro, an existing 2'-OH group at positions 5 and 7 is substituted with 2'-H, and the rest positions are modified by 2'-OMe, at the 3' end, there are six phosphorothioate linkages, at the 5' end, there is a 5' (E)-vinylphosphonate nucleotide; orin the sense strand, from 5' to 3', positions 4 and 6 are modified by 2'-fluoro, the rest positions are modified by 2'-OMe, at the 5' end, there are two phosphorothioate linkages, and optionally, 3' end is bind with an GalNAc of derivative through a phosphorothioate linkage or phosphodiester linkage; in the antisense strand, from 5' to 3', positions 2, 14 and 16 are modified by 2'-fluoro, an existing 2'-OH group at positions 5 and 7 is substituted with 2'-H, and the rest positions are modified by 2'-OMe, at the 3' end, there are six phosphorothioate linkages, at the 5' end, there is a 5' (E)-vinylphosphonate nucleotide.16.The RNAi agent of 15, wherein the RNAi agent containing an GalNAc derivative is Formula 1 or Formula 2; preferably Formula 2:[Formula 1],[Formula 2].17.The RNAi agent of claim 1 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, the RNAi agent 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 the RNAi agent according to any one of claims 1 to 17 as an active ingredient and pharmaceutically acceptable carriers.19.Use of the RNAi agent according to any one of claims 1 to 17 or the pharmaceutical composition of claim 18 in preparation of a pharmaceutical product for preventing or treating 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 hepatic steatosis.

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