RNAi agents targeting LPA genes and uses thereof

Abstract

Provided are an RNAi agent targeting a lipoprotein (a) (LPA) gene and a use thereof, and more particularly, an RNAi agent comprising an antisense strand having sequence complementarity with an LPA mRNA sequence and a sense strand having sequence complementarity with the antisense strand, and a pharmaceutical composition for preventing or treating cardiovascular diseases comprising the RNAi agent.

Description

Technical Field

[0001] The present disclosure relates to RNAi agents targeting LPA gene and uses thereof, and more particularly, to RNAi agents comprising an antisense strand having sequence complementarity to an LPA mRNA sequence and a sense strand having sequence complementarity to the antisense strand, and pharmaceutical compositions for preventing or treating cardiovascular diseases comprising the RNAi agents. Background Art

[0002] In recent years, the incidence of cardiovascular diseases has been increasing due to changes in living environments and the increasing intake of animal-derived foods caused by Westernized eating habits that often consume meat, fast food, and convenience foods. According to the World Health Organization (WHO), cardiovascular diseases are the leading cause of morbidity and mortality worldwide, and it is reported that approximately 8.9 million people died worldwide in 2019 due to cardiovascular diseases. Cardiovascular diseases refer to diseases of the circulatory system (such as the heart and blood vessels), and atherosclerosis is considered to be its direct and main cause. Atherosclerosis is a disease in which cholesterol or triglycerides accumulate in blood vessels, causing them to narrow, harden, and block, and there is currently no definitive method to reduce or eliminate the above symptoms. Currently, when atherosclerosis already exists, the focus is on preventing further progression or preventing death or complications due to atherosclerosis, while in cases where atherosclerosis has not yet occurred, methods of prevention by controlling or removing atherosclerosis risk factors are used.

[0003] Meanwhile, LPA or Lp(a) (lipoprotein(a)) is mainly expressed in liver tissue and is known to be one of the genetic risk factors associated with the occurrence of cardiovascular disease. The European Society of Cardiology (ESC) and the Atherosclerosis Society (EAS) recommend measuring LPA at least once in a lifetime to determine cardiovascular disease risk. In addition, it has been determined that genetically high levels of LPA of 180 mg / dL or more have a similar cardiovascular disease risk as heterozygous familial hypercholesterolemia, and a treatment target of less than 50 mg / dL is recommended. That is, LPA-lowering therapies can be used to treat diseases such as stroke, atherosclerosis, thrombosis, and cardiovascular diseases such as coronary heart disease and aortic stenosis. However, since the variability of LPA levels due to diet, exercise, or other life cycle changes is very limited, a new technology that can control the expression level of the LPA gene is needed.

[0004] Accordingly, as a result of intensive research efforts to develop new drugs for treating cardiovascular diseases, the present inventors developed RNAi agents capable of specifically inhibiting LPA mRNA expression (by binding to LPA mRNA), thereby completing the present disclosure. Summary of the invention

[0005] An object of the present disclosure is to provide RNAi agents that specifically inhibit LPA expression.

[0006] Another object of the present disclosure is to provide a pharmaceutical composition (including an RNAi agent) for preventing or treating cardiovascular diseases, or a method for preventing or treating cardiovascular diseases (including administering the pharmaceutical composition to a subject).

[0007] To achieve the above objectives, an RNAi agent is provided, comprising: an antisense strand having sequence complementarity with an LPA mRNA sequence and having a length of 19 nt to 21 nt; and a sense strand having sequence complementarity with the antisense strand and having 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.

[0008] Another aspect provides a pharmaceutical composition comprising a RNAi agent as an active ingredient for preventing or treating cardiovascular disease.

[0009] Yet another aspect provides a method for preventing or treating cardiovascular disease, the method comprising administering the RNAi agent to a subject.

[0010] Yet another aspect provides use of the RNAi agent for manufacturing a pharmaceutical product for preventing or treating cardiovascular disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The above and other aspects, features and advantages of certain embodiments of the present disclosure will become more apparent from the following description in conjunction with the accompanying drawings, in which:

[0012] Figure 1 is a diagram schematically showing the structure of LPA asiRNA consisting of a 16-mer sense strand and a 21-mer antisense strand;

[0013] Figure 2a The results of confirming the expression level of LPA mRNA after Hep3B cells were treated with 55 LPA asiRNAs (asiLPA-001 to asiLPA-055) at a concentration of 10 nM according to one aspect are shown;

[0014] Figure 2b The results of confirming the expression level of LPA mRNA after Hep3B cells were treated with 54 LPA asiRNAs (asiLPA-056 to asiLPA-109) at a concentration of 10 nM according to one aspect are shown;

[0015] Figure 3aThe results of confirming the expression level of LPA mRNA after Hep3B cells were treated with 55 LPA asiRNAs (asiLPA-110 to asiLPA-164) at a concentration of 10 nM according to one aspect are shown;

[0016] Figure 3b The results of confirming the expression level of LPA mRNA after Hep3B cells were treated with 54 LPA asiRNAs (asiLPA-165 to asiLPA-218) at a concentration of 10 nM according to one aspect are shown;

[0017] Figure 4a The results of confirming the expression level of LPA mRNA after Hep3B cells were treated with 55 LPA asiRNAs (asiLPA-219 to asiLPA-273) at a concentration of 10 nM according to one aspect are shown;

[0018] Figure 4b The results of confirming the expression level of LPA mRNA after Hep3B cells were treated with 54 LPA asiRNAs (asiLPA-274 to asiLPA-327) at a concentration of 10 nM according to one aspect are shown;

[0019] Figure 5a The results of confirming the expression level of LPA mRNA after Hep3B cells were treated with 54 LPA asiRNAs (asiLPA-328 to asiLPA-381) at a concentration of 10 nM according to one aspect are shown;

[0020] Figure 5b The results of confirming the expression level of LPA mRNA after Hep3B cells were treated with 53 LPA asiRNAs (asiLPA-382 to asiLPA-434) at a concentration of 10 nM according to one aspect are shown;

[0021] Figure 6 The results of confirming the expression level of LPA mRNA after Hep3B cells were treated with 65 LPA asiRNAs at a concentration of 1 nM are shown;

[0022] Figure 7 The results of confirming the expression level of LPA mRNA after Hep3B cells were treated with 65 LPA asiRNAs at a concentration of 1 nM are shown;

[0023] Figure 8 The results of confirming the expression level of LPA mRNA after Hep3B cells were treated with 40 LPA asiRNAs according to one aspect at a concentration of 0.1 nM respectively;

[0024] Fig. 9 The results of confirming the expression level of LPA mRNA after treating primary human hepatocytes with 40 LPA asiRNAs according to one aspect at a concentration of 10 nM respectively are shown;

[0025] Fig.10 The results of confirming the expression level of LPA mRNA after treating primary cynomolgus monkey hepatocytes with 40 LPA asiRNAs according to one aspect at a concentration of 10 nM respectively are shown;

[0026] Fig.11 The results of confirming the change of LPA expression level over time after subcutaneous administration of 6 LPA GalNAc-asiRNAs (OLX706A-006-1, OLX706A-032-1, OLX706A-033-1, OLX706A-034-1, OLX706A-036-1, OLX706A-043-1) (in which chemical modifications were introduced) according to one aspect at a single dose of 8 mg / kg (mpk) to SEAP-LPA (P1) HDI mice are shown;

[0027] Fig.12 The results of confirming the changes in LPA expression levels over time after subcutaneous administration of 9 LPA GalNAc-asiRNAs (OLX706A-006-1, OLX706A-007-1, OLX706A-033-1, OLX706A-043-1, OLX706A-062-1, OLX706A-064-1, OLX706A-092-1, OLX706A-108-1, OLX706A-369-1) according to one aspect to SEAP-LPA (P2) HDI mice at a single dose of 8 mg / kg (mpk) are shown;

[0028] Fig.13 The results of confirming the change of LPA expression level over time after subcutaneous administration of 5 LPA GalNAc-asiRNAs (OLX706A-006-1, OLX706A-064-1, OLX706A-170-1, OLX706A-171-1, OLX706A-187-1) (in which chemical modifications were introduced) according to one aspect to SEAP-LPA (P3) HDI mice at a single dose of 8 mg / kg (mpk) are shown;

[0029] Fig.14The results of confirming the expression level of LPA mRNA after treating primary human hepatocytes with 14 LPA GalNAc-asiRNAs (OLX706A-033-4, OLX706A-062-2, OLX706A-062-12, OLX706A-092-4, OLX706A-033-1, OLX706A-062-1, OLX706A-092-1, OLX706A-006-12, OLX706A-007-12, OLX706A-007-4, OLX706A-033-12, OLX706A-062-4, OLX706A-006-1, OLX706A-007-1) according to one aspect (chemical modifications introduced therein) at a concentration of 200 nM respectively are shown; and

[0030] Fig.15 The results of confirming changes in LPA expression levels after subcutaneous administration of 6 LPA GalNAc-asiRNAs (OLX706A-033-4, OLX706A-033-12, OLX706A-062-2, OLX706A-062-4, OLX706A-062-12, OLX706A-092-4) (in which chemical modifications were introduced) according to one aspect are shown;

[0031] Fig.16a The results of confirming the change of Apo(a) expression level over time after subcutaneous administration of 4 LPA GalNAc-asiRNAs (OLX706A-033-4, OLX706A-062-2, OLX706A-062-12, OLX706A-092-4) (in which chemical modifications were introduced) according to one aspect to Apo(a) TG mice at a single dose of 3 mg / kg (mpk) are shown;

[0032] Fig.16b The results of confirming the change of Apo(a) expression level over time after subcutaneous administration of three LPA GalNAc-asiRNAs (OLX706A-033-4, OLX706A-062-2, OLX706A-062-12) (in which chemical modifications were introduced) according to one aspect to Apo(a) TG mice at a single dose of 1 mg / kg (mpk) are shown;

[0033] Fig.17The results of confirming the changes in Lp(a) expression levels over time after subcutaneous administration of two LPAGaINAc-asiRNAs (OLX706A-033-4, OLX706A-062-12) (in which chemical modifications were introduced) according to one aspect to cynomolgus monkeys at a single dose of 3 mg / kg (mpk) are shown; DETAILED DESCRIPTION

[0034] Reference will now be made in detail to embodiments, which are illustrated in the accompanying drawings, wherein the same reference numerals refer to the same elements throughout. In this respect, embodiments of the present invention may have different forms and should not be construed as being limited to the descriptions listed herein. Therefore, embodiments are described only by reference to the figures as described below to explain the various aspects of this description. As used herein, the term "and / or" includes any and all combinations of at least one associated listed item. When an expression (e.g., "at least one,") is in front of a list of elements, it is to modify the entire list of elements, rather than a single element in the list of elements.

[0035] Each description and embodiment disclosed in this application can also be applied to other descriptions and embodiments. That is to say, all combinations of the various elements disclosed in this application are within the scope of this application. In addition, it should not be interpreted that the scope of this application is limited by the detailed description as described below.

[0036] In one aspect, a RNAi agent is provided, comprising: an antisense strand having sequence complementarity to an LPA mRNA sequence and having a length of 19 to 21 nt; and a sense strand having sequence complementarity to the antisense strand and having a length of 15 to 17 nt, wherein the 5′ end of the antisense strand and the 3′ end of the sense strand form a blunt end.

[0037] RNAi Agents

[0038] The term "RNA interference (RNAi)" refers to a mechanism for inhibiting the expression of a target gene by inducing degradation of the mRNA of the target gene by introducing double-stranded RNA (dsRNA) composed of a strand having sequence homology to the mRNA of the target gene and a strand having a sequence complementary to the above strand.

[0039] The term "RNAi agent" or "nucleic acid molecule that induces RNAi" as used herein refers to any agent or nucleic acid molecule that can inhibit or downregulate gene expression or viral replication by mediating RNA interference in a sequence-specific manner. The term can refer to a single nucleic acid molecule, a plurality of nucleic acid molecules, or a pool of nucleic acid molecules. In an embodiment, the RNAi agent can be siRNA.

[0040] As used herein, the term "small interfering RNA (siRNA; short interfering RNA)" refers to a short double-stranded RNA (dsRNA) that mediates efficient gene silencing with sequence specificity.

[0041] The term "gene" as used herein should be considered in its broadest sense and may encode a structural protein or a regulatory protein. In this regard, regulatory proteins include transcription factors, heat shock proteins, or proteins involved in DNA / RNA replication, transcription, and / or translation. In the present disclosure, the target gene to be inhibited is inherent to the viral genome and may be integrated into the animal genome or exist as an extrachromosomal component.

[0042] The term "antisense strand" as used herein refers to a polynucleotide that is substantially or 100% complementary to a target nucleic acid of interest and may 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.

[0043] The term "sense strand" as used herein refers to a polynucleotide having the same nucleic acid sequence as a target nucleic acid, or a polynucleotide that is completely or partially identical to, for example, a messenger RNA (mRNA), a non-mRNA RNA sequence (e.g., microRNA, piwiRNA, tRNA, rRNA, and hnRNA), or a coding or non-coding DNA sequence.

[0044] The term "complementarity" or "complementarity" used herein refers to the meaning generally accepted in the art. The term can generally refer to the formation or existence of hydrogen bonds between a nucleic acid sequence and another nucleic acid sequence by traditional Watson-Crick bonding or other non-traditional types as described herein. Complete complementarity can mean that all continuous residues of a nucleic acid sequence form hydrogen bonds with the same number of continuous residues in a second nucleic acid sequence. Partial complementarity within a nucleic acid molecule may include multiple mispairings or non-base pair nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more mispairings, e.g., 1 to 3 mispairings, non-nucleotide joints or non-base pair nucleotides). Partial complementarity may result in a bulge, loop, overhang or flush end between the sense strand and the antisense strand of a nucleic acid molecule or between the antisense strand of a nucleic acid molecule and the corresponding target nucleic acid molecule.

[0045] The term "blunt end" as used herein refers to the generally accepted meaning in the art. With respect to RNAi agents or nucleic acid molecules herein, the term may refer to the end of a double-stranded siRNA molecule lacking protruding nucleotides. The siRNA molecules described herein may form a blunt end at the 5'-end of the antisense strand and the 3'-end of the sense strand.

[0046] RNAi agents for silencing LPA expression

[0047] "Lipoprotein (a) (LPA)" is a heterogeneous low-density lipoprotein particle expressed mainly in the liver, composed of apolipoprotein (a) (Apo (a)) linked to low-density lipoprotein (LDL) via the ApoB polypeptide. It is reported that high levels of LPA in serum are not affected by diet or exercise, and are closely related to an increased risk of cardiovascular disease by increasing the likelihood of developing atherosclerosis. In addition, in terms of diagnostic and preventive medicine, high levels of LPA in patient serum are known to be a significant dominant, independent and genetic risk factor for coronary heart disease and aortic valve stenosis. Therefore, the LPA gene may be a potential therapeutic target for cardiovascular disease. The LPA protein can be interpreted as including naturally occurring wild-type LPA and its functional variants, and the sequence of the LPA protein or the gene encoding it can be obtained from known databases (such as GenBank of the National Institutes of Health).

[0048] The term "expression" as used herein refers to the generally accepted meaning in the art. The term generally refers to the 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 herein, expression levels can be determined or monitored by detecting mRNA levels or protein levels.

[0049] The term "inhibit" or "reduce" (as used in relation to LPA gene expression in a subject) refers to a statistically significant decrease compared to an untreated group or a normal control group. The decrease can be, for example, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, but depending on the detection or measurement method, it may be below the detection limit.

[0050] 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 induce degradation of target genes through the antisense strand, which is one of the RNA double strands introduced into the cell and binds complementary to the mRNA of the target gene, and RNAi has recently become one of the most popular candidate technologies for the development of new drugs.

[0051] However, in contrast to this possibility, the side effects and shortcomings of siRNA are constantly reported. In order to develop a therapeutic agent based on RNAi, some obstacles must be overcome, such as 1) lack of an effective delivery system, 2) off-target effects, 3) induced immune response, and 4) saturation of the RNAi mechanism in the cell. Although siRNA is an effective method for directly regulating target gene expression, it is difficult to develop a therapeutic agent due to these problems. In this regard, asymmetric shorter duplex siRNA (asiRNA) is an asymmetric RNAi induction structure with a shorter double helix length compared to the 19+2 structure of siRNA in the art. The asiRNA technology overcomes the problems of off-target effects, RNAi mechanism saturation and TLR3 immune response present in the existing siRNA structural technology, and therefore it is possible to develop a new low-side effect RNAi drug.

[0052] Based on this, an asymmetric siRNA including a sense strand and an antisense strand complementary to the sense strand is presented in this example, and the siRNA according to the example does not cause problems such as off-target effects and saturation of the RNAi mechanism, and therefore can inhibit the expression of the LPA gene to a desired extent while stably maintaining a high delivery efficiency.

[0053] In one example, asymmetric siRNA (asiRNA) targeting LPA was designed and prepared, and after transfecting the asiRNA into cells expressing LPA, a nucleic acid molecule that induced RNAi with excellent knockdown efficiency, namely, LPA asiRNA, was selected.

[0054] In an embodiment, the RNAi agent is characterized by a sense strand length of 15 nt to 17 nt, and an antisense strand length of 19 nt to 21 nt. More preferably, the sense strand length may be 16 nt, and the antisense strand complementary thereto may be 19 nt, 20 nt or 21 nt in length, but is not limited thereto.

[0055] The 5'-end of the antisense strand and the 3'-end of the sense strand may form a blunt end. The 3'-end of the antisense strand may include an overhang of, for example, 2 nt to 6 nt.

[0056] In an embodiment, the sense strand can be a sequence selected from the sense strand sequences listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, and Table 9, and the antisense strand can include a sequence selected from the antisense strand sequences listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, and Table 9.

[0057] In an embodiment, the sense strand can be, for example, at least one selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 103, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 183, SEQ ID NO: 215, SEQ ID NO: 339, SEQ ID NO: 341, SEQ ID NO: 373, SEQ ID NO: 375, SEQ ID NO: 471, SEQ ID NO: 477, SEQ ID NO: 495, SEQ ID NO: 701, SEQ ID NO: 71 NO:709, SEQ ID NO:711, SEQ ID NO:713, SEQ ID NO:715, SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:743 and SEQ ID NO:775.

[0058] In an embodiment, the sense strand may be, for example, at least one selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 85, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 183, SEQ ID NO: 215, SEQ ID NO: 339, SEQ ID NO: 341, SEQ ID NO: 373, and SEQ ID NO: 737, for example, it may be any one selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 65, SEQ ID NO: 123, and SEQ ID NO: 183. Preferably, the sense strand may be any one selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 123, and SEQ ID NO: 183.

[0059] In an embodiment, the antisense strand can be, for example, at least one selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 104, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 184, SEQ ID NO: 216, SEQ ID NO: 340, SEQ ID NO: 342, SEQ ID NO: 374, SEQ ID NO: 376, SEQ ID NO: 472, SEQ ID NO: 478, SEQ ID NO: 496, SEQ ID NO: 702, SEQ ID NO: NO:710, SEQ ID NO:712, SEQ ID NO:714, SEQ ID NO:716, SEQ ID NO:736, SEQ ID NO:738, SEQ ID NO:740, SEQ ID NO:744 and SEQ ID NO:776.

[0060] In an embodiment, the antisense strand may be, for example, at least one selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 86, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 184, SEQ ID NO: 216, SEQ ID NO: 340, SEQ ID NO: 342, SEQ ID NO: 374, and SEQ ID NO: 738, for example, the antisense strand may be any one selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 66, SEQ ID NO: 124, and SEQ ID NO: 184. Preferably, the antisense strand may be any one selected from the group consisting of SEQ ID NO: 66, SEQ ID NO: 124, and SEQ ID NO: 184. In a preferred embodiment, the RNAi agent has a sense strand as shown in SEQ ID NO: 65 and an antisense strand as shown in SEQ ID NO: 66; the RNAi agent has a sense strand as shown in SEQ ID NO: 123 and an antisense strand as shown in SEQ ID NO: 124; or the RNAi agent has a sense strand as shown in SEQ ID NO: 183 and an antisense strand as shown in SEQ ID NO: 184.

[0061] Introduction of chemically modified RNAi agents

[0062] In a RNAi agent, the sense strand or the antisense strand may include one or more chemical modifications. In one aspect, the present disclosure provides new chemical modification patterns to improve the properties and effects of RNAi agents.

[0063] General siRNA cannot pass through the cell membrane due to its high negative charge and high molecular weight phosphate backbone structure, and is rapidly degraded and eliminated from the blood, making it difficult to deliver sufficient amounts to the actual target site for RNAi induction. Currently, for in vitro delivery, many efficient delivery methods using cationic lipids and cationic polymers have been developed, however, for in vivo, it is difficult to deliver siRNA as efficiently as in vitro, and there is a problem of reducing the efficiency of siRNA delivery due to interactions with various proteins present in the organism.

[0064] Therefore, in this example, an RNAi agent with improved hepatocyte-targeted delivery capability by introducing chemical modifications into the asymmetric siRNA structure is provided, and more particularly, an asymmetric siRNA construct (GalNAc asymmetric siRNA, GalNAc-asiRNA) capable of effective intracellular delivery without a separate carrier is provided.

[0065] In the present disclosure, chemical modifications in the sense strand or the antisense strand may include one or more selected from the following: conjugation with an N-acetylgalactosamine (GalNAc) derivative or a cell penetrating peptide; modification of nucleotide bonds with phosphorothioates, boranophosphates or methyl phosphonates; replacement of the -OH group at the 2′ carbon position of the sugar structure of the nucleotide with -H, -CH3(methyl), -OCH3(-O-methyl), -NH2, -F, -O-2-methoxyethyl-O-propyl, -O-2-methylmethylthioethyl, -O-3-aminopropyl or -O-3-dimethylaminopropyl; and replacement of the -OH group at the 5′ end with E-vinylphosphonate.

[0066] In an embodiment, the N-acetylgalactosamine (GalNAc) derivative may have a structure of Formula 1. The N-acetylgalactosamine (GalNAc) derivative recognizes the asialoglycoprotein (ASGPR) receptor on the surface of hepatocytes and helps the RNAi agent flow into the hepatocytes, i.e., acts as a part targeting ASGPR, and thus, the RNAi agent (in which the N-acetylgalactosamine (GalNAc) derivative is bound to its end) improves delivery to hepatocytes and may provide effective targeted treatment for liver diseases.

[0067] Formula 1

[0068]

[0069] In an embodiment, the sense strand may include one or more chemical modifications selected from the following: modification of 2 to 4 nucleotide bonds adjacent to the 5′ end with phosphorothioate, boranophosphate or methyl phosphonate; replacement of the -OH group at the 2′ carbon position of the sugar structure of at least one nucleotide with -H, -CH3(methyl), -OCH3(-O-methyl), -NH2, -F, -O-2-methoxyethyl-O-propyl, -O-2-methylmethylthioethyl, -O-3-aminopropyl or -O-3-dimethylaminopropyl; and conjugation of the 3′ end to an N-acetylgalactosamine (GalNAc) derivative or a cell penetrating peptide.

[0070] In an embodiment, the antisense strand may include one or more chemical modifications selected from the following: modifying 2 to 7 nucleotide bonds adjacent to the 3′ or 5′ end with thiophosphate, boranophosphate, or methyl phosphonate; replacing the -OH group at the 2′ carbon position of the sugar structure of at least one nucleotide with -H, -CH3(methyl), -OCH3(-O-methyl), -NH2, -F, -O-2-methoxyethyl-O-propyl, -O-2-methylmethylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; and replacing the -OH group at the 5′ end with E-vinylphosphonate.

[0071] In another embodiment, the RNAi agent may include at least one modification selected from the following: modifying 2 to 7 nucleotide bonds adjacent to the 3′ end or 5′ end in the sense strand or antisense strand to phosphorothioate; modifying the -OH group at the 2′ carbon position of the sugar structure of at least one nucleotide in the sense strand or antisense strand to -H, -OCH3 (-O-methyl) or -F; conjugating the 3′ end of the sense strand to an N-acetylgalactosamine (GalNAc) derivative; and replacing the -OH group at the 5′ end of the antisense strand with E-vinylphosphonate.

[0072] In another embodiment, the RNAi agent as described above has an antisense strand and a sense strand, wherein in the antisense strand, from 5' to 3', positions 2, 14, and 16 are modified with 2'-fluorine, positions 5 and 7 are deoxynucleosides, and the remaining positions are modified with 2'-OMe, the 3' end has six phosphorothioate bonds, and the 5' end has a 5'(E)-vinylphosphonate nucleotide; in the sense strand, from 5' to 3', positions 2, 4, 5, and 6 are modified with 2'-fluorine, and optionally, the 3' end has a nucleotide linked to an N-acetylgalactosamine (GalNAc) derivative.

[0073] In an embodiment, the RNAi agent as described above has a sense strand, from 5' to 3': 2, 4, 5, 6, 14 and 16 are modified by 2'-fluorine, and the remaining positions are modified by 2'-OMe. In an embodiment, the RNAi agent as described above has a sense strand, from 5' to 3': 2, 4, 5, 6 and 12 are modified by 2'-fluorine, and the remaining positions are modified by 2'-OMe. In some embodiments, the chemical modification method is as follows: in the antisense strand of the RNAi agent, from 5' to 3', positions 2, 14, and 16 are modified with 2'-fluorine, positions 5 and 7 are modified to deoxynucleosides, and the remaining positions are modified with 2'-OMe, the 3' end is modified with six thiophosphate bonds, and the 5' end has a 5'(E)-vinylphosphonate nucleotide; in the sense strand of the RNAi agent, from 5' to 3', positions 2, 4, 5, 6, 14, and 16 are modified with 2'-fluorine, and the remaining positions are modified with 2'-OMe, and optionally, the 3' end has a nucleotide connected to an N-acetylgalactosamine (GalNAc) derivative. In some embodiments, the chemical modification method is as follows: in the antisense strand of the RNAi agent, from 5' to 3', positions 2, 14, and 16 are modified with 2'-fluorine, positions 5 and 7 are modified to deoxynucleosides, and the remaining positions are modified with 2'-OMe, the 3' end is modified with six thiophosphate bonds, and the 5' end has a 5'(E)-vinylphosphonate nucleotide; in the sense strand of the RNAi agent, from 5' to 3', positions 2, 4, 5, 6, and 12 are modified with 2'-fluorine, and the remaining positions are modified with 2'-OMe, and optionally, the 3' end has a nucleotide connected to an N-acetylgalactosamine (GalNAc) derivative. In an embodiment, in the sense strand of the RNAi agent as described above, from 5' to 3' can be modified as:

[0074] (m)*(f)*(m)(f)(f)(f)(m)(m)(m)(m)(m)(m)(m)(f)(m)(f)(GAL);

[0075] And in the antisense strand of the RNAi agent as described above, from 5' to 3' can be modified as:

[0076] [EVP](m)*(f)*(m)(m)(d)(m)(d)(m)(m)(m)(m)(m)(m)(f)(m)*(f)*(m)*(m)*(m)*(m)*(m);

[0077] Wherein, * represents a phosphorothioate bond, (m) represents 2′-O-methyl, (f) represents 2′-fluoro, (d) represents the existing form where 2′-OH is replaced by -H, [EVP] represents E-vinylphosphonate, and (GAL) represents a trivalent GalNAc derivative. Preferably, the trivalent GalNAc derivative is of Formula 1.

[0078] In an embodiment, in the sense strand of the RNAi agent as described above, from 5' to 3' can be modified as:

[0079] (m)*(f)*(m)(f)(f)(f)(m)(m)(m)(m)(m)(f)(m)(m)(m)(m)(GAL);

[0080] And in the antisense strand of the RNAi agent as described above, from 5' to 3' can be modified as:

[0081] [EVP](m)*(f)*(m)(m)(d)(m)(d)(m)(m)(m)(m)(m)(m)(f)(m)*(f)*(m)*(m)*(m)*(m)*(m);

[0082] Wherein, * represents a phosphorothioate bond, (m) represents 2′-O-methyl, (f) represents 2′-fluoro, (d) represents the existing form where 2′-OH is replaced by -H, [EVP] represents E-vinylphosphonate, and (GAL) represents a trivalent GalNAc derivative. Preferably, the trivalent GalNAc derivative is of Formula 1.

[0083] In an embodiment, the sense strand may be a sequence selected from the sense strand sequences listed in Table 15, and the antisense strand may include a sequence selected from the antisense strand sequences listed in Table 15. In a preferred embodiment, the RNAi agent may be selected from the GalNAc-asiRNA conjugates listed in Table 15.

[0084] In an embodiment, the sense strand may be any one selected from: (A) to (D) in the following table, and the antisense strand may be any one selected from: (a) to (d) in the following table.

[0085]

[0086] In the above sequence, * represents a phosphorothioate bond, m represents 2′-O-methyl, f represents 2′-fluoro, d represents a form in which the existing 2′-OH is replaced by -H, EVP represents E-vinylphosphonate, and GAL represents a trivalent GalNAc derivative having formula 1.

[0087] In particular, the sense strand and antisense strand of the RNAi agent can be sequences of the following sense strands and antisense strands selected from the above table, for example, the RNAi agent can have a combination of sense strand (A) and antisense strand (a); a combination of sense strand (B) and antisense strand (b); a combination of sense strand (C) and antisense strand (c); or a combination of sense strand (D) and antisense strand (d).

[0088] Another aspect provides a pharmaceutical composition comprising a RNAi agent as an active ingredient for preventing or treating cardiovascular disease.

[0089] Yet another aspect provides use of a RNAi agent for the manufacture of a medicament for preventing or treating cardiovascular disease.

[0090] Since the pharmaceutical composition contains or uses the above-mentioned RNAi agent as it is, the description thereof is omitted to avoid making this specification too complicated.

[0091] Cardiovascular disease

[0092] The pharmaceutical composition can be used as an active ingredient of a pharmaceutical composition for preventing or treating cardiovascular diseases by inhibiting the expression of LPA gene.

[0093] Cardiovascular disease can be collectively referred to as primary diseases directly caused by various reasons such as abnormalities, dysfunctions, and injuries of the circulatory system in the body, as well as secondary diseases derived from primary diseases. Cardiovascular disease is a disease closely related to the expression level of the LPA gene, for example, it can be any one selected from the following: Buerger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic valve stenosis, myocardial infarction, aortic valve regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperbetalipoproteinemia, cerebrovascular atherosclerosis and venous thrombosis, but not limited thereto.

[0094] Pharmaceutical composition

[0095] The term "active ingredient" as used herein means an appropriate effective amount of an ingredient that produces a beneficial or desired clinical or biochemical result. In particular, the term can refer to an effective amount of a pharmaceutical agent, an active agent, or an RNAi agent.

[0096] An effective amount may be administered once or multiple times, and in unlimited cases may be an appropriate amount for preventing a disease, alleviating symptoms, reducing the extent of a disease, stabilizing (i.e., not worsening) a disease state, delaying or reducing the rate of disease progression, or improving or temporarily alleviating and improving (partially or completely) a disease state.

[0097] The term "prevention" as used herein refers to any action to prevent the occurrence of a disease, inhibit a disease, or delay its progression. For example, the term refers to preventing the occurrence of, preventing the occurrence of, or defending against or preventing the occurrence of, a cardiovascular disease or its characteristics.

[0098] The term "treatment" as used herein refers to therapeutic treatment and preventive or prophylactic measures. In addition, it refers to any action that causes an improvement or beneficial change in the symptoms of a disease. For example, the term refers to preventing, reducing or improving cardiovascular disease or its characteristics in a subject, or delaying (attenuating) the progression of cardiovascular disease or its characteristics.

[0099] The term "effective amount" as used herein refers to a generally accepted meaning in the art. The term generally refers to the amount of a molecule, compound or construct that causes an expected biological response (e.g., a beneficial response) in a cell, tissue, system, animal or human being sought by a researcher, veterinarian, doctor or other clinician. In particular, a "therapeutically effective amount" refers to the amount of a molecule, compound or construct that can cause a desired medical response (to the extent that a particular clinical treatment may be considered effective), such as due to a treatment-related change in a measurable parameter associated with a disease or disorder. The therapeutically effective amount of a drug for treating a disease or disorder can be the amount necessary to cause a treatment-related change in a parameter.

[0100] Those skilled in the art can determine the method for administering the pharmaceutical composition based on the patient's symptoms and the severity of the disease. In addition, the pharmaceutical composition can be formulated into various forms, such as powders, tablets, capsules, solutions, injections, ointments, syrups, etc., and can be provided in unit doses or multi-dose containers (e.g., sealed ampoules and bottles).

[0101] The pharmaceutical composition of the present disclosure can be administered orally or parenterally. The route of administration of the composition according to the present disclosure can be, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, percutaneous, subcutaneous, intraperitoneal, intestinal, sublingual or local, but is not limited thereto. The dosage of the composition according to the present disclosure varies according to the patient's weight, age, sex, health status, diet, application time, method, excretion rate or disease severity, and can be easily determined by those of ordinary skill in the art. In addition, the composition of the present disclosure can be formulated into a suitable formulation for clinical administration using known techniques.

[0102] Yet another aspect provides a method for preventing or treating cardiovascular disease, the method comprising administering the RNAi agent to a subject.

[0103] Since the method for treating cardiovascular disease includes or uses the above-mentioned pharmaceutical composition as it is, the description thereof is omitted to avoid making this specification too complicated.

[0104] The term "subject" as used herein refers to a subject in need of treatment for a disease (particularly, cardiovascular disease), and more particularly, the term can include all mammals, such as humans or non-human primates, mice, dogs, cats, horses, cows, sheep, pigs, goats, camels and antelopes.

[0105] Hereinafter, the present disclosure will be described in more detail by examples. However, these examples are intended to illustrate the present disclosure, and the scope of the present disclosure is not limited to these examples.

[0106] Example 1: Screening of nucleic acid molecules for inducing RNAi targeting LPA

[0107] 1-1. Design and preparation of LPA asiRNA

[0108] In this example, in order to obtain a double-stranded nucleic acid molecule that induces efficient RNA interference targeting LPA, after selecting the target sequence of the LPA gene, its LPA asymmetric siRNA (LPA asiRNA) (sense strand (16-mer), antisense strand (21-mer), see Figure 1 ).

[0109] In particular, after obtaining LPA gene information by searching the NCBI database, a total of 434 asiRNAs were designed in combination with animal experiments and synthesized by OliX US at a 4nmole scale. Thereafter, the synthesized asiRNAs were subjected to gel electrophoresis using 15% polyacrylamide gel, and then quality control (QC) was performed using a ChemiDoc UV transilluminator (Biorad). The sequence information of the LPAasiRNA prepared according to the above method is shown in Tables 1 to 9 below.

[0110] [Table 1]

[0111]

[0112]

[0113]

[0114]

[0115] [Table 2]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] [Table 3]

[0122]

[0123]

[0124]

[0125]

[0126] [Table 4]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132] [Table 5]

[0133]

[0134]

[0135]

[0136]

[0137] [Table 6]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143] [Table 7]

[0144]

[0145]

[0146]

[0147]

[0148] [Table 8]

[0149]

[0150]

[0151]

[0152]

[0153] [Table 9]

[0154]

[0155]

[0156]

[0157] 1-2. Selection of 130 LPA asiRNAs

[0158] Hep3B cells (human liver cancer cells) were transfected with 434 LPA asiRNAs at a concentration of 10 nM, and a dual luciferase reporter gene assay was performed to measure the expression level of LPA mRNA. In particular, LPA asiRNA (10 nM) and psiCHECK-2-LPA plasmid DNA (10 ng / well) were delivered to Hep3B cells using lipofectamine 2000 (Invitrogen, 11668019, 0.2 μl / well). Then, the intensity of Renilla luciferase (expressed by LPA mRNA) and firefly luciferase was measured by using a dual luciferase reporter gene assay system (Promega, catalog number E1980), and the expression level of LPA mRNA was normalized by the intensity level of the constitutively expressed firefly luciferase in the state of introducing psiCHECK-2-LPA plasmid DNA to evaluate the expression level of LPA mRNA.

[0159] As shown in Figures 2 to 5, the results confirmed the effect of suppressing the expression of Renilla luciferase by LPA asiRNA treatment. In addition, 130 LPA asiRNAs (asiLPA-001, 002, 003, 004, 005, 006, 007, 008, 009, 010, 013, 016, 017, 018, 019, 020, 021, 022, 023, 024, 025, 026, 027, 031, 033, 032, 034, 035, 036, 038, 040, 041, 042, 043, 044, 045, 046, 047, 048, 049, 050, 051, 052, 053, 054, 055, 056, 057, 058, 059, 060 、039、040、041、042、043、044、050、051、052、053、054、055、056、059、061、062、063、064、066、070、079、088、092、098、102、105、108、110、119、120、121、124、129、1 30, 131, 146, 147, 149, 152, 161, 167, 168, 170, 171, 178, 187, 188, 204, 213, 214, 216, 217, 221, 230, 236, 238, 239, 240, 244, 245, 248, 249, 250, 251, 258, 265, 275 , 334, 343, 349, 351, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 364, 365, 367, 368, 369, 370, 371, 372, 373, 374, 379, 388, 400, 406, 411, 418, 422, 431, 434).

[0160] 1-3. Selection of 40 LPA asiRNAs

[0161] In the same manner as in Example 1-2, Hep3B cells (human liver cancer cells) were transfected with 130 selected LPA asiRNAs at a concentration of 1 nM, and a dual-luciferase reporter gene assay was performed to measure the expression level of LPA mRNA.

[0162] The results are as follows Figure 6 and Figure 7As shown, the effect of suppressing LPA mRNA expression by LPA asiRNA treatment was confirmed. In addition, 40 LPA asiRNAs (asiLPA-002, 006, 007, 008, 016, 018, 020, 024, 026, 027, 032, 033, 034, 036, 039, 040, 042, 044, 052, 063, 064, 092, 102, 108, 170, 171, 187, 188, 213, 236, 351, 355, 356, 357, 365, 368, 369, 370, 372, 388) showing relatively good expression suppression efficiency were identified.

[0163] 1-4. Confirmation of the effects of 40 LPA asiRNAs on expression inhibition

[0164] Hep3B cells, primary human hepatocytes, or primary cynomolgus monkey hepatocytes were transfected with 40 LPA asiRNAs, and dual-luciferase reporter gene assay or qRT-PCR was performed to measure the expression level of LPA mRNA.

[0165] In particular, for the experiment on Hep3B cells, a total of 40 LPAasiRNAs were transfected into Hep3B cells at a concentration of 0.1 nM, and the expression level of LPA mRNA was measured (in the same manner as in Example 1-2). In addition, for the experiment on primary human hepatocytes, a total of 40 LPA asiRNAs were transfected into primary human hepatocytes at a concentration of 10 nM, and the expression level of LPA mRNA was measured by performing qRT-PCR. In particular, primary human hepatocytes were transfected with 1.0×10 4 Cells / well were seeded in a 96-well plate and transfected with LPA asiRNA (10 nM) by using lipofectamine 2000 (Invitrogen, 13778150). 24 hours after transfection, cell lysates were prepared by using SuperPrepTM Cell Lysis and RT Kit for qPCR (Kit II, TOYOBO, SCQ-401), and cDNA was synthesized by reverse transcription using the mRNA contained in the lysate as a template. Then, by using the synthesized cDNA as a template, cDNA was synthesized by using Gene expression assays and probes (Hs00916691_m1: human LPA, Hs03928985_g1: RNA18S5, Applied Biosystems) were used for quantitative PCR. Then, the expression level of LPA mRNA was identified by using the CFXOpus 384 Real-Time PCR System (Bio-Rad). In addition, for the experiment of primary cynomolgus monkey hepatocytes, primary cynomolgus monkey hepatocytes were transfected with 40 LPA asiRNAs at a concentration of 10 nM, and the expression level of LPA mRNA was measured by performing qRT-PCR. In particular, primary cynomolgus monkey hepatocytes were cultured at 4.5×10 4 Cells / well were seeded in a 96-well plate and transfected with LPAasiRNA (10 nM) by using lipofectamine 2000 (Invitrogen, 13778150). 24 hours after transfection, cell lysates were prepared using the SuperPrepTM Cell Lysis and RT Kit for qPCR (Kit II, Toyobo, SCQ-401), and cDNA was synthesized by reverse transcription using the mRNA contained in the lysate as a template. Then, by using the synthesized cDNA as a template, Gene expression assay and probes (Mf02789271_m1: cynomolgus monkey LPA, Mf04354341_g1: ACTB, Applied Biosystems) were used for quantitative PCR. Then, the expression level of LPA mRNA was identified by using the CFX Connect Real-Time PCR System (Bio-Rad).

[0166] The results are as follows Figures 8 to 10 As shown, the effect of suppressing LPA mRNA expression by LPA asiRNA treatment was confirmed in Hep3B cells, primary human hepatocytes, or primary cynomolgus monkey hepatocytes.

[0167] Based on all the experimental results of Example 1, 40 LPAasiRNAs (asiLPA-006, 007, 016, 018, 020, 024, 026, 027, 032, 033, 034, 036, 039, 040, 042, 043, 044, 052, 062, 063, 064, 092, 108, 170, 171, 187, 188, 236, 239, 248, 351, 355, 356, 357, 358, 368, 369, 370, 372, 388) showing relatively good expression inhibition efficiency were identified for further screening.

[0168] Example 2: Screening by using chemically modified RNAi-inducing nucleic acid molecules

[0169] 2-1. Introduction of chemically modified nucleic acid molecules for inducing RNAi

[0170] In this example, LPA GalNAc-asiRNA with chemical modification was designed as a double-stranded nucleic acid molecule for inducing efficient RNA interference targeting LPA. In particular, considering the experimental results of Example 1, 15 LPA asiRNAs (asiLPA-006, 007, 032, 033, 034, 036, 043, 062, 064, 092, 108, 170, 171, 187, 369) were selected as the basic skeleton. Then, 15 LPA GalNAc-asiRNAs were designed according to each basic skeleton, wherein 1) -OMe or -F was used to replace the -OH group at the 2' carbon position of the nucleotide sugar structure of the sense chain and the antisense chain, 2) thiophosphate modification was introduced in the skeleton at the 5' end or 3' end of the antisense chain and the 5' end of the sense chain, 3) phosphate was introduced at the 5' end of the antisense chain, and 4) GalNAc was introduced at the 3' end of the sense chain.

[0171] The sequence information of the chemically modified LPA GalNAc-asiRNA prepared in this example is shown in Table 10 below. In particular, in Table 10, "*" means a form in which an existing phosphodiester bond is replaced by a phosphorothioate bond, and "m" means a form in which an existing 2'-OH is replaced by a 2'-O-methyl group. In addition, "f" means (for example, in the case of fG) a form in which the 2'-OH of an existing guanine (G) is replaced by fluorine. In addition, "Phos" means a form in which 5'-OH is replaced by a phosphate, and "GAL" means a form in which a trivalent GalNAc derivative having Formula 1 is bonded to the 3' end of the sense chain (see Table 11).

[0172] [Table 10]

[0173]

[0174]

[0175]

[0176] [Table 11]

[0177] symbol Chemical modification * Phosphorothioate bond m 2′-O-methyl f 2′-Fluoro Phos Phosphate GAL A trivalent GalNAc derivative having formula 1

[0178] Formula 1

[0179]

[0180] 2-2. Confirmation of LPA expression inhibitory effect using animal models

[0181] The 15 LPA GalNAc-asiRNAs introduced with the above chemical modifications were respectively applied to SEAP-LPAHDI mice injected with 3 plasmids (P1, P2 and P3), and the changes in LPA levels were measured accordingly. In this regard, 3 plasmids were prepared by dividing the LPA from the Kringle IV site to the protease domain site into P1 (from Kringle IV-1 to Kringle IV-6), P2 (from Kringle IV-7 to Kringle IV-34) and P3 (from Kringle IV-35 to the protease domain). In particular, 3 plasmids (including LPA and secretory embryonic alkaline phosphatase (SEAP)) were injected into mice (Balb / c, female, 6 weeks old) by hydrodynamic injection (HDI). LPA GalNAc-asiRNA was subcutaneously injected into mice at a single dose of 8 mg / kg (mpk). On the 2nd, 7th and 21st days from the date of subcutaneous injection into mice, the levels of LPA were measured by using Phospha-Light TM SEAP reporter gene assay system (Invitrogen TM , T1015) to measure the SEAP level in serum, and the LPA level in serum derived from mice was confirmed by normalizing the measured value with the SEAP level in serum of a control group administered with saline on the day of measuring the LPA level and the SEAP level in serum before administration of LPA GalNAc-asiRNA.

[0182] The results are as follows Figures 11 to 13 As shown in Tables 12 to 14, the effect of suppressing SEAP reporter gene expression was confirmed by treatment with 15 types of LPA GalNAc-asiRNA (OLX706A-006-1, OLX706A-007-1, OLX706A-032-1, OLX706A-033-1, OLX706A-034-1, OLX706A-036-1, OLX706A-043-1, OLX706A-062-1, OLX706A-064-1, OLX706A-092-1, OLX706A-108-1, OLX706A-170-1, OLX706A-171-1, OLX706A-187-1, OLX706A-369-1). In particular, Fig.11 and Table 12 In mice introduced with P1 plasmid, Fig.12 and Table 13 In mice introduced with P2 plasmid, Fig.13 and Table 14 confirmed the effect of suppressing SEAP reporter gene expression by LPA GalNAc-asiRNA treatment in mice introduced with the P3 plasmid.

[0183] [Table 12]

[0184]

[0185] [Table 13]

[0186]

[0187] [Table 14]

[0188]

[0189]

[0190] Example 3: Preparation of chemically modified nucleic acid molecules for inducing RNAi

[0191] In this example, LPA GalNAc-asiRNA with chemical modification was designed as a double-stranded nucleic acid molecule for inducing efficient RNA interference targeting LPA. To this end, different types of LPA GalNAc-asiRNA were designed, wherein in LPA GalNAc-asiRNA, 1) the -OH group at the 2' carbon position of the sugar structure in the nucleotides of the sense and antisense chains was replaced by -OMe or -F, 2) phosphorothioate modification was introduced into the backbone of the 5' or 3' end of the antisense chain and the 5' end of the sense chain, 3) E-vinyl phosphonate was introduced at the 5' end of the antisense chain, and 4) GalNAc was introduced at the 3' end of the sense chain. In particular, the five LPA asiRNAs (asiLPA-006, asiLPA-007, asiLPA-033, asiLPA-062 and asiLPA-092) having effective inhibitory effects on LPA mRNA expression in Example 2 were used as the basic framework, and various modification patterns designed according to the chemical modification of Table 16 were introduced into the basic framework to obtain many different GalNAc-asiRNA conjugates, for example, OLX706A-006-12, OLX706A-007-12, OLX706A-007-4, OLX706A-033-4, OLX706A-033-12, OLX706A-062-2, OLX706A-062-4, OLX706A-062-12 and OLX706A-092-4 were prepared. Meanwhile, OLX706A-006-1, OLX706A-007-1, OLX706A-033-1, OLX706A-062-1 and OLX706A-092-1 in this example were prepared in the same manner as in Example 2.

[0192] The sequence information of the chemically modified LPA GalNAc-asiRNA prepared in this example is shown in Table 15 below. In particular, in Table 15, "*" means a form in which an existing phosphodiester bond is replaced by a phosphorothioate bond, and "m" means a form in which an existing 2'-OH is replaced by a 2'-O-methyl group. In addition, "f" means (for example, in the case of fG) a form in which the 2'-OH of an existing guanine (G) is replaced by fluorine. In addition, "d" means a form in which an existing 2'-OH is replaced by -H, "EVP" means a form in which an existing -OH group at the 5' end is replaced by an E-vinylphosphonate, and "GAL" means a form in which a trivalent GalNAc derivative having the following formula 1 is bonded to the 3' end of the sense chain (see Table 16).

[0193] [Table 15]

[0194]

[0195]

[0196] [Table 16]

[0197] symbol Chemical modification * Phosphorothioate bond m 2′-O-methyl f 2′-Fluoro d deoxidation EVP E-vinyl phosphonate GAL A trivalent GalNAc derivative having formula 1

[0198] Example 4: Confirmation of the effect of introducing chemically modified nucleic acid molecules for inducing RNAi on inhibiting LPA expression

[0199] 4-1. Confirmation of the inhibitory effect of LPA expression in vitro

[0200] After primary human hepatocytes were treated with the above-mentioned GalNAc-asiRNA, qRT-PCR was performed to measure the expression level of LPA mRNA. Specifically, primary human hepatocytes were cultured at 3.0×10 4 Cells / well were seeded in a 96-well plate and treated with LPAGaINAc-asiRNA (200 nM). 24 hours after transfection, cell lysates were prepared using the SuperPrepTM Cell Lysis and RT Kit for qPCR (Kit II, Toyobo Co., Ltd., SCQ-401), and cDNA was synthesized by reverse transcription using the mRNA contained in the lysate as a template. Then, by using the synthesized cDNA as a template, Gene expression assay and probes (Hs00916691_m1: human LPA, Hs03928985_g1: RNA18S5, Applied Biosystems) were used for quantitative PCR. Then, the expression level of LPA mRNA was identified by using CFXOpus 384 Real-Time PCR System (Bio-Rad).

[0201] The results are as follows Fig.14 As shown in Table 17, the effect of suppressing LPA mRNA expression was confirmed in primary human hepatocytes after treatment with OLX706A-006-12, OLX706A-007-12, OLX706A-007-4, OLX706A-033-4, OLX706A-033-12, OLX706A-062-2, OLX706A-062-4, OLX706A-062-12, and OLX706A-092-4.

[0202] [Table 17]

[0203]

[0204] 4-2. Confirmation of LPA expression inhibitory effect using animal models

[0205] The LPA GalNAc-asiRNA introduced with the above chemical modification was applied to HDI mice introduced with LPA, and the resulting LPA level changes were measured. In particular, plasmids (including LPA and secretory embryonic alkaline phosphatase (SEAP)) were injected into mice (Balb / c, female, 6 weeks old) by hydrodynamic injection (HDI). LPA GalNAc-asiRNA was injected subcutaneously into mice at a single dose of 5 mg / kg (mpk). On the 7th, 21st and 35th days from the date of subcutaneous injection into mice, the level of LPA was measured by using Phospha-Light TM SEAP reporter gene assay system (Invitrogen TM , T1015) measured the SEAP level in serum, and the measured value was normalized by the SEAP level in serum before administration of LPA GalNAc-asiRNA and the SEAP level in serum of a control group administered with saline to confirm the LPA level in serum derived from mice.

[0206] The results are as follows Fig.15 As shown in Table 18, the effect of suppressing SEAP reporter gene expression was confirmed in mice by treatment with OLX706A-033-4, OLX706A-033-12, OLX706A-062-2, OLX706A-062-4, OLX706A-062-12, and OLX706A-092-4.

[0207] [Table 18]

[0208]

[0209] Example 5. LPA knockdown efficacy study in Apo(a) transgenic model

[0210] Apo(a)8K TG mice were generated from frozen stocks of mice described in the literature (Merki et al., 2011, JACC [Journal of the American College of Cardiology], doi: 10.1016 / j.jacc.2010.10.052). Blood samples were collected on the first 2 days of the study (blood was collected before treatment) and serum apo(a) protein levels were determined by human lipoprotein A ELISA kit (Abcam, ab108878). Mice were grouped so that their initial apo(a) levels were uniform between experimental groups. Each cohort contained three animals (at least two female subjects). On study day 0, GalNAc-conjugated LPAasiRNA was administered subcutaneously at a single dose of 1 or 3 mpk. Blood samples were collected once a week after dosing. LPA expression inhibition was determined by measuring serum apo(a) protein in treated mice. For normalization, the values ​​of the pre-dose samples were averaged and set to 100%, and the data were analyzed as relative values ​​compared to pre-dose.

[0211] The results are as follows Fig.16a As shown in Table 19, OLX706A-062-12 showed 72% inhibition of LPA expression on day 7, 74% inhibition on day 14, and 72% inhibition on day 21. OLX706A-033-4 showed 74% inhibition of LPA expression on day 7, 61% inhibition on day 14, and 54% inhibition on day 21. OLX706A-092-4 showed 76% inhibition of LPA expression on day 7, 77% inhibition on day 14, and 76% inhibition on day 21.

[0212] like Fig.16b As shown in Table 20, OLX706A-062-12 showed 62% inhibition of LPA expression on day 7, 58% inhibition on day 14, and 57% inhibition on day 21. OLX706A-033-4 showed 41% inhibition of LPA expression on day 7, 28% inhibition on day 14, and 24% inhibition on day 21.

[0213] [Table 19]

[0214]

[0215] [Table 20]

[0216]

[0217] Example 6. Non-human primate GalNAc-asiLPA study to evaluate serum Lp(a) reduction

[0218] The cynomolgus monkey (Macaca fascicularis) was divided into groups so that its average body weight (about 2-4 kg) was comparable between the control group and the experimental group. Each cohort contained two animals. On the first day of the study, a single dose of 3 mpk of LPA asiRNA conjugated to GalNAc was administered subcutaneously. Blood samples were collected on the first 6 days and the first day of the study and collected once a week after administration. At the indicated time points, LPA expression inhibition was determined by measuring the Lp (a) serum concentration of the treated NHP using Lp (a) ELISA (Mercodia, 10-1106-01). The values ​​of the pre-dose samples were averaged and set to 100%, and the data were analyzed as relative values ​​compared to before administration.

[0219] OLX706A-033-4 showed an inhibition of LPA expression of 89% on day 29, 80% on day 43, and 53% on day 64. OLX706A-062-12 showed an inhibition of LPA expression of 84% on day 29, 66% on day 43, and 43% on day 64. The experimental data are shown in Tables 21 and Fig.17 Shown in.

[0220] [Table 21]

[0221]

[0222] So far, specific parts of the present disclosure have been described in detail, but it is obvious to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present disclosure is not limited thereto. Therefore, it is intended that the essential scope of the present disclosure is defined by the appended claims and their equivalents.

[0223] According to one aspect, the RNAi agent can inhibit the expression of LPA protein by binding to and degrading LPA mRNA, while reducing side effects such as nonspecific immune response and off-target effects. A study was also conducted to evaluate the toxicity of the RNAi agent of the present disclosure after a single dose of subcutaneous administration in ICR mice over an 8-day observation period, and no significant signs of toxicity were found.

[0224] Furthermore, the RNAi agent according to one aspect is effectively delivered to hepatocytes and can be used as a targeted therapeutic agent for cardiovascular diseases.

[0225] It should be understood that the embodiments described herein should be considered only as descriptive and not for purposes of limitation. Typically, the description of features or aspects within each embodiment should be considered applicable to other similar features or aspects in other embodiments. Although one or more embodiments have been described with reference to the accompanying drawings, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present disclosure as defined by the following claims.

Claims

1. A RNAi agent comprising: an antisense strand having sequence complementarity with an LPA (lipoprotein (a)) mRNA sequence and having a length of 19 nt to 21 nt; and a sense strand having sequence complementarity with the antisense strand and having 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.

2. The RNAi agent of claim 1, wherein the sense strand has a sequence selected from the sense strand sequences listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, and Table 9.

3. The RNAi agent of claim 1, wherein the sense strand is any one selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 103, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 183, SEQ ID NO: 215, SEQ ID NO: 339, SEQ ID NO: 341, SEQ ID NO: 373, SEQ ID NO: 375, SEQ ID NO: 471, SEQ ID NO: 477, SEQ ID NO: 495, SEQ ID NO: ID NO:701, SEQ ID NO:709, SEQ ID NO:711, SEQ ID NO:713, SEQ ID NO:715, SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:743 and SEQ ID NO:

775.

4. The RNAi agent of claim 1, wherein the sense strand is any one selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 85, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 183, SEQ ID NO: 215, SEQ ID NO: 339, SEQ ID NO: 341, SEQ ID NO: 373, and SEQ ID NO: 737; and preferably, the sense strand is any one selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 65, SEQ ID NO: 123, and SEQ ID NO:

183.

5. The RNAi agent of claim 1, wherein the antisense strand has a sequence selected from the antisense strand sequences listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, and Table 9.

6. The RNAi agent of claim 1, wherein the antisense strand is any one selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 104, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 184, SEQ ID NO: 216, SEQ ID NO: 340, SEQ ID NO: 342, SEQ ID NO: 374, SEQ ID NO: 376, SEQ ID NO: 472, SEQ ID NO: 478, SEQ ID NO: 496, SEQ ID NO: ID NO:702, SEQ ID NO:710, SEQ ID NO:712, SEQ ID NO:714, SEQ ID NO:716, SEQ ID NO:736, SEQ ID NO:738, SEQ ID NO:740, SEQ ID NO:744 and SEQ ID NO:

776.

7. The RNAi agent of claim 1, wherein the antisense strand is any one selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 86, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 184, SEQ ID NO: 216, SEQ ID NO: 340, SEQ ID NO: 342, SEQ ID NO: 374, and SEQ ID NO: 738; and preferably, the antisense strand is any one selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 66, SEQ ID NO: 124, and SEQ ID NO: 184; more preferably, the RNAi agent has a sense strand as shown in SEQ ID NO: 65 and an antisense strand as shown in SEQ ID NO: 66; the RNAi agent has a sense strand as shown in SEQ ID NO: 123 and an antisense strand as shown in SEQ ID NO:

128. The antisense strand as shown in SEQ ID NO: 124; or the RNAi agent has the sense strand as shown in SEQ ID NO: 183 and the antisense strand as shown in SEQ ID NO:

184. The RNAi agent of claim 1 , wherein the 3′ end of the sense strand is bound to an N-acetylgalactosamine (GalNAc) derivative.

9. The RNAi agent of claim 1, wherein the sense strand or the antisense strand comprises at least one chemical modification.

10. The RNAi agent of claim 9, wherein the sense strand comprises at least one chemical modification selected from the group consisting of: Modification of 2 to 4 nucleotide bonds adjacent to the 5′ end with phosphorothioate, boranophosphate, or methylphosphonate; Substituting 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-methylmethylthioethyl, -O-3-aminopropyl or -O-3-dimethylaminopropyl; and The 3′ end is conjugated to an N-acetylgalactosamine (GalNAc) derivative or a cell-penetrating peptide.

11. The RNAi agent of claim 9, wherein the antisense strand comprises at least one chemical modification selected from the group consisting of: Modify 2 to 7 nucleotide linkages adjacent to the 3' or 5' terminus with phosphorothioate, boranophosphate, or methylphosphonate; and The -OH group at the 2′ carbon position of the sugar structure of at least one nucleotide is replaced with -H, -CH3(methyl), -OCH3(-O-methyl), -NH2, -F, -O-2-methoxyethyl-O-propyl, -O-2-methylmethylthioethyl, -O-3-aminopropyl or -O-3-dimethylaminopropyl; and the -OH group at the 5′ end is replaced with E-vinylphosphonate.

12. The RNAi agent of claim 9, comprising at least one modification selected from the group consisting of: modifying 2 to 7 nucleotide bonds adjacent to the 3′ end or the 5′ end in the sense strand or the antisense strand to phosphorothioate; Modifying the -OH group at the 2′ carbon position of the sugar structure of at least one nucleotide in the sense strand or the antisense strand to -H, -OCH3 (-O-methyl) or -F; The 3′ end of the sense strand is bound to an N-acetylgalactosamine (GalNAc) derivative; and The -OH group at the 5' end of the antisense strand was substituted with E-vinylphosphonate.

13. The RNAi agent of any one of claims 1-12, wherein in the antisense strand, from 5' to 3', positions 2, 14, and 16 are modified with 2'-fluorine, positions 5 and 7 are deoxynucleosides, and the remaining positions are modified with 2'-OMe, the 3' end has six phosphorothioate bonds, and the 5' end has a 5'(E)-vinylphosphonate nucleotide; in the sense strand, from 5' to 3', positions 2, 4, 5, and 6 are modified with 2'-fluorine, and optionally the 3' end has a nucleotide linked to an N-acetylgalactosamine (Ga1NAc) derivative.

14. The RNAi agent of claim 13, wherein in the sense strand, from 5' to 3': positions 2, 4, 5, 6, 14 and 16 are modified with 2'-fluorine, and the remaining positions are modified with 2'-OMe; or positions 2, 4, 5, 6 and 12 are modified with 2'-fluorine, and the remaining positions are modified with 2'-OMe.

15. The RNAi agent of claim 9, wherein the sense strand has a sequence selected from the sense strand sequences listed in Table 15.

16. The RNAi agent of claim 9, wherein the antisense strand has a sequence selected from the antisense strand sequences listed in Table 15, preferably, the RNAi agent is selected from the GalNAc-asiRNA conjugates in Table 15.

17. A pharmaceutical composition comprising the RNAi agent according to any one of claims 1 to 16 as an active ingredient and a pharmaceutically acceptable carrier.

18. Use of the RNAi agent according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 17 in the manufacture of a pharmaceutical product for preventing or treating cardiovascular diseases.

19. The method of claim 18, wherein the cardiovascular disease is selected from the group consisting of Buerger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, myocardial infarction, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperbetalipoproteinemia, cerebrovascular atherosclerosis, and venous thrombosis.

Images