Novel RNA therapies and their use

Abstract

The present disclosure relates to a novel RNAi agent designed to reduce the expression of ANGPTL8 in the liver, the RNAi comprising a delivery moiety optionally conjugated to an oligonucleotide via a linker.The RNAi agent is useful for treating diseases involving the regulation of ANGPTL8 expression.

Description

[Technical Field]

[0001] This disclosure relates to a novel RNAi agent designed to reduce ANGPTL8 expression in the liver, the RNAi agent comprising a delivery portion optionally conjugated to an oligonucleotide via a linker. The RNAi agent is useful for treating diseases in which the regulation of ANGPTL8 expression is involved. [Background technology]

[0002] Angiopoietin-like protein 8 (ANGPTL8) is primarily expressed in the liver and adipose tissue and plays a crucial role in triglyceride metabolism. ANGPTL8, along with ANGPTL3 or ANGPTL4, is thought to regulate triglyceride levels by inhibiting the enzymatic activity of lipoprotein lipase (LPL), which, when active, hydrolyzes triglycerides and reduces circulating plasma triglycerides. Elevated levels of ANGPTL8 are observed in or associated with cardiovascular disease, diabetes, dyslipidemia (including high triglyceride levels), abnormal renal function, hypertension, non-alcoholic steatohepatitis (NASH) and other non-alcoholic fatty liver diseases, and obesity.

[0003] RNAi agents, such as those disclosed herein, enable the targeting of genes in a sequence-specific manner for the personalized treatment of many different types of diseases involving gene dysregulation. Compounds containing oligonucleotides, such as the RNAi agents disclosed herein, can act through different mechanisms depending on the specific type of oligonucleotide used. RNA interference molecules, including the RNAi agents disclosed herein, typically act to knock down or reduce the gene expression of a given target transcript, thereby reducing the level of the protein. By delivering RNAi molecules, such as the RNAi agents disclosed herein, to desired tissues in a patient, gene expression can be reduced in a tissue-specific manner.

[0004] One example is RNAi agents containing N-acetylgalactose (GalNAc) to target asialoglycoprotein receptors on hepatocytes. In particular, gibosilan sodium is an siRNA approved by the U.S. Food and Drug Administration (FDA) that targets the ALAS1 gene transcript to treat acute hepatic porphyria and uses a delivery portion containing GalNAc for entry into hepatocytes. Insclisiran is an siRNA approved by the FDA that targets the PCSK9 gene transcript to lower LDL cholesterol and also uses a delivery portion containing GalNAc for entry into hepatocytes. RNAi molecules containing siRNAs targeting ANGPTL8 are described, for example, in International Publication No. 2020104649. However, only four siRNA molecules have been approved for human use, and no therapeutic siRNAs targeting ANGPTL8 have yet been approved. Furthermore, preclinical and clinically available information is limited regarding the ideal attributes of in vivo therapeutic siRNAs, particularly for cardiovascular diseases, dyslipidemia (e.g., high triglycerides), and liver diseases or liver-related diseases, such as inflammatory liver disease.

[0005] There remains a need to provide alternative RNAi agents comprising a delivery moiety containing GalNAc and one or more oligonucleotides to reduce ANGPTL8 expression. More specifically, there is a need to provide RNAi agents comprising a novel GalNAc delivery moiety and a sense strand and an antisense strand, wherein the antisense strand is complementary to ANGPTL8 mRNA, and such RNAi agents exhibit one or more of the following: improved tissue exposure, preferably improved liver exposure, improved liver-to-kidney exposure ratio, improved knockdown, improved endurance response, improved pharmacokinetic profile, fewer off-target effects, improved toxicity profile, improved safety profile, fewer side effects, improved tolerability, improved control of cholesterol and / or triglyceride levels in patients, improved cardiovascular risk profile in patients, improved and / or simplified synthesis, a synthetic process with fewer degradation products, or any combination of these.

[0006] In one embodiment of this disclosure, formula I: [ka] [In the formula, R comprises a sense strand and an antisense strand, the antisense strand comprising at least 15 consecutive nucleotides of a sequence complementary to the mRNA transcript of ANGPTL8, the sense strand and antisense strand forming a complementary region of at least 15 nucleotides, the sense strand and antisense strand each independently being 15 to 30 nucleotides long, optionally each independently comprising one or more modified nucleotides, optionally each independently comprising one or more modified nucleotide interlinks, and R is optionally conjugated to formula I via a linker.] This is an RNA interference (RNAi) agent containing a delivery portion. In another embodiment, the antisense strand contains at least 15 consecutive nucleotides of a sequence complementary to SEQ ID NO: 1. In a further embodiment, the sense strand and the antisense strand are each independently 18 to 23 nucleotides long. In any further embodiment of these RNAi agents, the antisense strand forms a complementary region of at least 18 nucleotides to the mRNA transcript of ANGPTL8. In any different further embodiment of these RNAi agents, the antisense strand forms a complementary region of at least 18 nucleotides to SEQ ID NO: 1.

[0007] In a further embodiment, the RNAi agent is one in which the antisense strand contains at least 15 nucleotides of a sequence selected from the group consisting of SEQ ID NOs. 405 to 525. In yet another embodiment, the antisense strand contains at least 18 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs. 405 to 525.

[0008] In further embodiments of the RNAi agents disclosed herein, the antisense strand is SEQ ID NOs: 405, 408, 412, 413, 414, 415, 418, 420, 425, 426, 428, 429, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 448, 449, 451, 452, 454, 45 The sequence comprises at least 18 consecutive nucleotides selected from the group consisting of 7, 458, 459, 463, 464, 465, 466, 467, 468, 469, 471, 472, 473, 474, 475, 476, 479, 490, 491, 492, 493, 495, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, and 509. In another embodiment, the antisense strand is selected from the group of antisense strand sequences in Table 2.

[0009] In any embodiment of the RNAi agent disclosed herein, the RNAi agent of any antisense strand is 23 nucleotides long, or the sense strand is 21 nucleotides long, or both.

[0010] In another embodiment of the RNAi agent disclosed herein, the antisense strand is selected from the group consisting of SEQ ID NOs. 231-361 or sequences having at least 90% sequence identity thereto. In yet another embodiment of the RNAi agent disclosed herein, the sense strand is selected from the group consisting of SEQ ID NOs. 124-230 or sequences having at least 90% sequence identity thereto.

[0011] In any further embodiment of the RNAi agents described herein, the complementary region includes 0, 1, 2, or 3 mismatches between the sense strand and the antisense strand.

[0012] In further embodiments of the RNAi agent, the sense strand and the antisense strand each independently contain one or more modified nucleotides. In further embodiments, the one or more modified nucleotides are independently 2'-fluoro-modified nucleotides or 2'-O-methyl-modified nucleotides. In other embodiments, each nucleotide of the sense strand and each nucleotide of the antisense strand are modified nucleotides, and in further embodiments, each of the modified nucleotides is independently 2'-fluoro-modified nucleotide or 2'-O-methyl-modified nucleotide.

[0013] In other embodiments of the RNAi agent described herein, the sense strand and the antisense strand each independently contain one or more modified nucleotide bonds. In further embodiments, each modified nucleotide bond is a phosphorothioate bond. In other embodiments, the sense strand and the antisense strand each independently contain four phosphorothioate bonds. In further embodiments, the first two 5' nucleotides of the sense strand and the two terminal 3' nucleotides of the sense strand are phosphorothioate bonds. In further embodiments, the first two 5' nucleotides of the antisense strand and the two terminal 3' nucleotides of the antisense strand are phosphorothioate bonds.

[0014] In other embodiments of RNAi agents described herein, the 5' nucleotide of the antisense strand comprises a phosphate group or a phosphate analog.

[0015] In another embodiment, the present disclosure relates to formula I conjugated to R: [ka] [In the formula, R comprises a sense strand and an antisense strand, the antisense strand comprising at least 15 consecutive nucleotides of a sequence complementary to the mRNA transcript of ANGPTL8, the sense strand and antisense strand forming a complementary region of at least 15 nucleotides, each independently being 15 to 30 nucleotides long, optionally each independently comprising one or more modified nucleotides, optionally each independently comprising one or more modified nucleotide interbonds, and R is optionally conjugated to the delivery portion D of formula I via linker L. In another embodiment, the antisense strand comprises at least 15 consecutive nucleotides of a sequence complementary to sequence number 1. The present invention provides an RNAi agent comprising a delivery portion. In a further embodiment, the sense strand and the antisense strand are each independently 18 to 23 nucleotides long. In any further embodiment of these RNAi agents, the antisense strand forms a complementary region of at least 18 nucleotides to the mRNA transcript of ANGPTL8. In any different further embodiment of these RNAi agents, the antisense strand forms a complementary region of at least 18 nucleotides to SEQ ID NO: 1. In a further embodiment, the antisense strand contains at least 15 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 405 to 525.

[0016] Any of the compounds disclosed herein, including RNAi agents containing formula I, may have modifications or additions within formula I, or the compounds may contain an addition moiety. For example, one or more alkyl chains in formula I may be elongated or shortened, or the compounds containing formula I may further contain one or more oligonucleotides. The compounds disclosed herein, including RNAi agents containing formula I, are useful for delivering one or more oligonucleotides to cells having receptors for one or more N-acetylgalactose (GalNAc, or N-GalNAc, galnac, or Galnac) moieties, such as asialoglycoprotein receptor (ASPGR), which typically binds to three GalNAc moieties. Therefore, the compounds disclosed herein, including RNAi agents containing formula I, can be used to preferentially bind to hepatocytes expressing ASPGR, thereby promoting the entry of the compounds into hepatocytes. Furthermore, since ASPGR is also present in adipose tissue, the compounds containing formula I, including RNAi agents, can be used to deliver oligonucleotides to adipocytes expressing ASPGR.

[0017] In one embodiment, it is a compound or an RNAi agent comprising a delivery moiety and one or more oligonucleotides, the delivery moiety comprises Formula I, and the oligonucleotide is complementary to the ANGPTL8 gene (hereinafter, ANGPTL8 oligonucleotide). In a preferred further embodiment, the delivery moiety comprising Formula I binds to the extracellular receptor ASPGR and enables the entry of the oligonucleotide into cells including liver tissue, thereby delivering one or more ANGPTL8 oligonucleotides to the liver tissue. Also, the ANGPTL8 oligonucleotide is represented herein by R or the sense strand and / or antisense strand of this specification, and includes those shown in the sense sequence and antisense sequence shown in the sequence numbers of this specification.

[0018] The delivery moiety comprising Formula I can be used to deliver any number of ANGPTL8 oligonucleotides, such as an RNAi agent comprising R, and R comprises the sense strand and / or antisense strand disclosed herein for diagnostic purposes or preferably for therapeutic purposes. One or more oligonucleotides such as the sense strand and antisense strand disclosed herein may comprise DNA or RNA nucleotides or DNA or RNA nucleosides.

[0019] The oligonucleotides herein, including antisense strands, are designed to target intracellular ANGPTL8 sequences, i.e., to bind to or anneal to ANGPTL8 sequences, or to form complementary regions with ANGPTL8 sequences, thereby regulating gene expression, and preferably reducing ANGPTL8 gene expression. In one embodiment, a compound or RNAi agent comprising formula I disclosed herein for reducing the expression of an ANGPTL8 transcript. In a further embodiment, a compound or RNAi agent comprising formula I disclosed herein for reducing the expression of an ANGPTL8 transcript further reduces ANGPTL8 protein expression. In another embodiment, the reduction in the expression of the target transcript or target protein is about 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, 70, 65, 60, 55, or 50 percent. In further embodiments, the reduction in expression lasts for approximately 3 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0020] Those skilled in the art will recognize that one or more mismatches may exist between an ANGPTL8 oligonucleotide, such as an antisense nucleotide disclosed herein, and an ANGPTL8 target nucleotide sequence, and may still function to regulate gene expression. In another embodiment, the oligonucleotide has 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, or 70 percent sequence identity with the target sequence, or is complementary to the target sequence. The oligonucleotide may also have an overhang of 1 to 10, 1 to 5, or 1 to 3, or 3, 2, or 1 residue at either the 5' or 3' end. The 5' or 3' end may be further modified, for example, with debasic residues or phosphate groups.

[0021] The term "percent sequence identity" with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleobases in a candidate sequence that are identical to the nucleotides, nucleosides, or nucleobases in the reference nucleic acid sequence, after optimally aligning the sequences and introducing gaps or overhangs as necessary to achieve the maximum percent sequence identity using the PID3 calculation, where the PID3 calculation is the number of identical nucleotide residues divided by the total number of nucleotides, nucleosides, or nucleobases in the shorter of the two sequences, times 100. See, e.g., Raghava, G., Barton, G.J. Quantification of the variation in percentage identity for protein sequence alignments. BMC Bioinformatics 7, 415 (2006). One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared.

[0022] One of ordinary skill in the art will recognize that modifications to an RNAi agent (or compound or RNAi molecule) that includes an ANGPTL8 oligonucleotide, such as the sense or antisense strand of the RNAi agent disclosed herein, can increase its stability and half-life. Modifications can be to the nucleotides or the phosphodiester backbone, i.e., the linkage between two nucleotide residues of the oligonucleotide, which is also referred to as the internucleotide linkage. For example, a 2'-modification on the sugar residue, preferably ribose, can increase its stability and half-life. These modifications include, but are not limited to, 2'-fluoro or 2'-methoxy modifications at the 2'-OH group of the unmodified sugar. Backbone modifications, also referred to herein as modified internucleotide linkages, include a change from a phosphodiester linkage to a phosphorothioate (PS) linkage.

[0023] Further embodiments of RNAi agents containing the delivery portion of formula I include such nucleotide and internucleotide bond modifications. Thus, in one embodiment, formula I: [ka] [In the formula, R includes a sense strand and an antisense strand. The RNAi agent comprises a delivery portion of formula I, where the antisense strand comprises at least 15 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 405-525, the sense strand and antisense strand form a complementary region of at least 15 nucleotides, the sense strand and antisense strand are each independently 18-23 nucleotides long, the sense strand and antisense strand each independently contain one or more modified nucleotides, optionally, the sense strand and antisense strand each independently contain one or more modified nucleotide interbonds, R is optionally conjugated to formula I via a linker, and one or more modified nucleotides are independently 2'-fluoromodified nucleotides or 2'-O-methyl modified nucleotides. In further embodiments, each nucleotide of the sense strand and each nucleotide of the antisense strand are modified nucleotides. In other embodiments, the sense strand and antisense strand each independently contain one or more modified nucleotide interbonds. In further embodiments, each modified nucleotide interbond is a phosphorothioate bond. In further embodiments, the sense strand and the antisense strand each independently contain four phosphorothioate bonds. In further embodiments, the first two nucleotides at the 5' end of the sense strand and the last two nucleotides at the 3' end of the sense strand contain phosphorothioate bonds, and the first two nucleotides at the 5' end of the antisense strand and the last two nucleotides at the 3' end of the antisense strand contain phosphorothioate bonds. In any embodiment of the RNAi agent disclosed herein, the 5' nucleotide of the antisense strand contains a phosphate group or a phosphate analog.

[0024] As used herein, “oligonucleotide” (or “multimer” or “oligomer” as interchangeably used herein), including the sense and antisense strands disclosed herein, means a chain of at least 10 nucleotides or nucleoside residues, and may include modified or unmodified bases, modified or unmodified sugars, and / or modified or unmodified bonds (also interchangeably referred herein as a skeleton, phosphodiester skeleton, or internucleotide bond). Nucleotide residues may be linked by phosphodiester bonds or modified bonds, also referred herein as phosphodiester internucleotide bonds or modified internucleotide bonds. Nucleotide residues may be modified by one or more atoms in a pyrimidine or purine ring of a nucleoside base, or by one or more atoms in a sugar residue, or by one or more atoms in a bond between a cyclic sugar and a nucleoside base. Modifications may also occur at the 5' or 3' end of the oligonucleotide strand, and such modified oligonucleotides or sense or antisense strands may be interchangeably referred to herein as oligonucleotides or sense or antisense strands unless otherwise evident from the context. In this specification, a nucleoside residue (i.e., a nucleotide lacking one or more phosphate groups) may be referred to as a modified nucleotide / nucleotide residue / nucleotide base, or simply a nucleotide / nucleotide residue / nucleotide base.

[0025] As used herein, “complementary” means a structural relationship between two nucleotides (e.g., on two opposing nucleic acids, or on a single nucleic acid strand, e.g., on opposing regions of a hairpin) that is expected to enable the two nucleotides to form base pairs with each other in a standard Watson-Crick pairing. For example, purine nucleotides of one nucleic acid that are complementary to a pyrimidine nucleotide of an opposing nucleic acid are complementary to each other. For example, they are expected to base pair together by forming hydrogen bonds with each other. Similarly, two antiparallel nucleic acids may have regions of multiple nucleotides that are complementary to each other in order to form complementary regions such as the sense and antisense strands of an RNAi agent described herein.

[0026] As used herein, “complementary region” means a nucleotide sequence of a nucleic acid (e.g., ds oligonucleotide) that is sufficiently complementary to an antiparallel nucleotide sequence to enable hybridization between two nucleotide sequences under appropriate hybridization conditions (e.g., in phosphate buffer, in a cell, etc.). In some embodiments, the oligonucleotides herein include a targeting sequence having a region complementary to the mRNA target sequence.

[0027] As used herein, “deoxyribonucleotide” means a nucleotide that, compared to a ribonucleotide, has a hydrogen atom instead of a hydroxyl group at the 2' position of its pentose sugar. Modified deoxyribonucleotides have one or more modifications or substitutions of atoms other than hydrogen at the 2' position of the sugar, and include modifications or substitutions of nucleic acid bases, sugars, or phosphate groups.

[0028] As used herein, “double-stranded oligonucleotide” or “ds oligonucleotide” means an oligonucleotide that is substantially in a double-stranded form. Complementary base pairing of the double-stranded region(s) of a ds oligonucleotide can be formed between antiparallel sequences of nucleotides of covalently separated nucleic acid strands. Similarly, complementary base pairing of the double-stranded region(s) of a ds oligonucleotide can be formed between antiparallel sequences of nucleotides of covalently linked nucleic acid strands. Furthermore, complementary base pairing of the double-stranded region(s) of a ds oligonucleotide can be formed from a single nucleic acid strand folded (e.g., via a hairpin) to provide a complementary antiparallel sequence of nucleotides to base pair together. A ds oligonucleotide can include two covalently separated nucleic acid strands that are completely double-stranded. However, a ds oligonucleotide can also include two covalently separated nucleic acid strands that are partially double-stranded (e.g., having overhangs at one or both ends). ds oligonucleotides may contain antiparallel sequences of partially complementary nucleotides and therefore may have one or more mismatches, which may include internal or terminal mismatches.

[0029] As used herein, “double helix” and “double helix region” with respect to nucleic acids (e.g., oligonucleotides) mean a double-stranded nucleic acid structure formed by complementary base pairing of two antiparallel sequences of nucleotides, whether formed by two covalently separated nucleic acid strands or by a single folded strand (e.g., via a hairpin), and which may be formed by annealing or hybridization under appropriate conditions.

[0030] As used herein, “linker” means a structure used to conjugate a nucleotide (e.g., oligonucleotide) to a delivery portion. A linker can be “unstable” or “cleavable,” meaning a linker that can be cleaved (e.g., by an acidic pH). Similarly, a linker can be “stable” or “incleavable,” meaning a linker that is not cleavable under physiological conditions.

[0031] As used herein, “modified nucleotide bond” means a nucleotide bond having one or more chemical modifications compared to a reference nucleotide bond having a phosphodiester bond. Modified nucleotide bonds may not exist in nature.

[0032] As used herein, “modified nucleotide” means a nucleotide having one or more chemical modifications compared to a corresponding reference nucleotide, selected from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. Modified nucleotides may be nucleotides that do not exist in nature. For example, a modified nucleotide may have one or more chemical modifications to its sugar, nucleic acid base, and / or phosphate group. In addition, or instead, a modified nucleotide may have one or more chemical moieties conjugated to a corresponding reference nucleotide.

[0033] As used herein, “nucleotide” means an organic compound having a nucleoside (e.g., nucleic acid bases such as adenine, cytosine, guanine, thymine, or uracil, and pentose sugars such as ribose or 2'-deoxyribose) and a phosphate group. A “nucleotide” can function as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0034] As used herein, “overhang” means a terminal nucleotide (or multiple nucleotides) arising from a single strand or region that extends beyond the end of the complementary strand forming a double helix. An overhang may include one or more unpaired nucleotides extending from the 5' or 3' terminal double helix region of a ds oligonucleotide. An overhang may be a 3' or 5' overhang on the antisense or sense strand of a ds oligonucleotide.

[0035] As used herein, “phosphate analog” or “phosphate mimetic” means a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is located at the 5'-terminal nucleotide of an oligonucleotide instead of 5'-phosphate. The 5'-phosphate analog may contain a phosphatase-resistant binding. Examples of phosphate analogs include, but are not limited to, 5'-phosphonates such as 5'-methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). Oligonucleotides may have a phosphate analog (referred to as a “4'-phosphate analog”) at the 4'-carbon position of the sugar of the 5'-terminal nucleotide. An example of a 4'-phosphate analog is an oxymethylphosphonate, in which the oxygen atom of an oxymethyl group is bonded to the sugar moiety (e.g., its 4'-carbon) or its analog. See, for example, International Publication No. 2018 / 045317.

[0036] Other modifications to the 5' end of oligonucleotides have also been developed (see, for example, International Publication No. 2011 / 133871, U.S. Patent No. 8,927,513, and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0037] "Percent complementarity" is calculated by dividing the number of nucleotides, nucleosides, or nucleic acid bases between two strands exhibiting standard pairing by the total number of nucleotides, nucleosides, or nucleic acid bases in the shorter of the two sequences, and multiplying by 100.

[0038] As used herein, "ribonucleotide" means a nucleotide having ribose as its pentose sugar and containing a hydroxyl group at the 2' position. Modified ribonucleotides, also referred to herein as modified nucleotides, are ribonucleotides having one or more modifications or substitutions of atoms other than hydrogen at the 2' position, and include modifications or substitutions of nucleic acid bases, sugars, or phosphate groups.

[0039] As used herein, “chain” refers to a single, continuous sequence of nucleotides joined together by internucleotide bonds (e.g., phosphodiester bonds or phosphorothioate bonds). A chain may have two free ends (e.g., a 5' end and a 3' end).

[0040] As used herein, “reduction in expression” with respect to a gene (e.g., ANGPTL8) means a reduction in the amount or level of RNA transcript (e.g., ANGPTL8 mRNA) or protein encoded by the gene, and / or a reduction in the amount or level of gene activity, in a cell, cell population, sample, or subject compared to a suitable reference (e.g., a reference cell, cell population, sample, or subject). For example, contacting cells with the oligonucleotides specified herein (e.g., oligonucleotides having an antisense strand having a nucleotide sequence complementary to the nucleotide sequence containing ANGPTL8 mRNA) may result in a reduction in the amount or level of mRNA, protein, and / or activity (e.g., via the degradation of ANGPTL8 mRNA by the RNAi pathway) compared to cells not treated with the ds oligonucleotide. Similarly, as used herein, “reduce expression” means an action that results in a reduction in the expression of a gene (e.g., ANGPTL8). Specifically, as used herein, “reduction in ANGPTL8 expression” means a reduction in the amount or level of ANGPTL8 mRNA, ANGPTL8 protein, and / or ANGPTL8 activity in cells, cell populations, samples, or subjects compared to a suitable reference (e.g., reference cells, cell populations, tissues, or subjects).

[0041] In certain embodiments, one or more oligonucleotides include small interfering RNA (siRNA), miRNA (microRNA), short hairpin RNA (shRNA), single guide RNA (sgRNA), or antisense oligonucleotide (ASO). In preferred embodiments, one or more ANGPTL8 oligonucleotides include siRNA. In another preferred embodiment, it is an RNAi agent comprising a sense strand and an antisense strand. In yet another preferred embodiment, one or more ANGPTL8 oligonucleotides are siRNAs comprising a sense strand and an antisense strand.

[0042] In some embodiments, the compound further comprises a linker for conjugating one or more ANGPTL8 oligonucleotides, such as R, where R comprises a sense strand and an antisense strand to formula I. In other embodiments, the RNAi agent disclosed herein comprises a linker for conjugating a double-stranded oligonucleotide comprising a sense strand or an antisense strand. In other embodiments, the RNAi agent comprises a sense strand conjugated to the delivery portion of formula I via the linker. Suitable linkers are known in the art. In one embodiment, the linker is an alkyl chain, preferably C 1~8 Includes. In further embodiments, the linker is an alkyl chain, preferably C 1~8 In a further embodiment, the linker includes linker 1 as shown below (formula II as specified herein, having linking points A and B). In a further embodiment, the linker is linker 1. In another embodiment, the linker includes a piperidine ring. In a further preferred embodiment, the linker includes linker 2 as shown below (formula III as specified herein, having linking points C and D). In a further preferred embodiment, the linker is linker 2.

[0043] [ka]

[0044] Those skilled in the art will recognize that the linker may be at the 5' or 3' end of an ANGPTL8 oligonucleotide containing R, where R contains the sense or antisense strand of the RNAi agent herein, or is bound to one of the internal nucleotides. Those skilled in the art will also recognize that the linker may be bound to or conjugated to the 5' or 3' end of an ANGPTL8 oligonucleotide containing the sense or antisense strand of the RNAi agent herein. Those skilled in the art will also recognize that, whether via a linker or not, the placement of a delivery portion, such as a delivery portion containing formula I, at the 5' end of an ANPTL8 oligonucleotide, such as the antisense strand of the RNAi agent herein, may need to overcome potential inefficient loading of Ago2 loading or other obstacles to RISC complex activity. For example, with respect to a delivery portion containing formula I bound to or conjugated to the sense or antisense strand of an RNAi agent herein, such as an siRNA containing a sense and antisense strand, the placement of the delivery portion at the 5' end of the antisense strand may create difficulties with Ago2 loading and hinder efficient knockdown. In preferred embodiments, one or more ANPTL8 oligonucleotides or RNAi agents comprise an siRNA including a sense strand and an antisense strand, wherein the delivery portion comprising formula I is located at the 3' end of the sense strand. In further embodiments, the delivery portion comprising formula I is conjugated to the 3' end of the sense strand via a linker. In even further embodiments, the linker comprises a ring structure, preferably a piperidine ring. In even further embodiments, the linker comprises linker 1 (formula II). In even further embodiments, the linker is linker 2 (formula III). In one embodiment, linker 1, linking point A, or linker 2, linking point C is conjugated to formula I. In one embodiment, linker 1, linking point A is conjugated to formula I, and linking point B is conjugated to R. In one embodiment, linker 2, linking point C is conjugated to formula I, and linking point D is conjugated to R. In one embodiment, linker 1 and linking point A are conjugated with formula I, and linking point B is conjugated with a phosphate group that is conjugated with R.In one embodiment, linker 2 and linking point C are conjugated with formula I, and linking point D is conjugated with a phosphate group that is conjugated with R.

[0045] In certain embodiments, the ANPTL8 oligonucleotide, such as an antisense chain, is complementary to the sequences in Table 1, which are complementary to the sequences represented by any one of SEQ ID NOs: 3 to 123. In a preferred embodiment, the ANPTL8 oligonucleotide is complementary to the sequences in Table 2, i.e., the sequences are complementary to the sequences represented by any one of SEQ ID NOs: 3, 6, 10, 11, 12, 13, 16, 18, 23, 24, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 46, 47, 49, 50, 52, 55, 56, 57, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74, 77, 88, 89, 90, 91, 93, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, or 107.

[0046] In further embodiments, the ANPTL8 oligonucleotide or RNAi agent described herein includes an siRNA comprising a sense strand and an antisense strand. In further embodiments, the siRNA is the sense strand shown in Table 2, i.e., SEQ ID NOs: 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 17 The sequence includes, or contains, an array represented by any one of 7, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, or 230.In other further embodiments, the ANPTL8 oligonucleotides or RNAi agents herein are the antisense strands of Table 2, i.e., SEQ ID NOs: 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 ,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293 ,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332 , includes a sequence represented by one of 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, or 360, or includes an antisense strand having this sequence. In further embodiments, the siRNA includes a sense strand and an antisense strand from one row of Table 11. In one of these further embodiments, the siRNA includes a sense strand and an antisense strand from row 1, i.e., the siRNA includes a sense strand containing SEQ ID NO: 124 and an antisense strand containing SEQ ID NO: 231. In another further embodiment, the siRNA includes a sense strand and an antisense strand from row 2, i.e., the siRNA includes a sense strand containing SEQ ID NO: 125 and an antisense strand containing SEQ ID NO: 232. In other further embodiments, the siRNA comprises a sense strand and an antisense strand from the same row in Table 11, the row being selected from the group consisting of rows 1 to 174 (rows labeled 1A to 174e5) in Table 11.

[0047] In further embodiments, the sense and antisense strands are modified. In further embodiments, the modification is made to the nucleotide, the backbone, i.e., the nucleoside bond (phosphodiester bond), or both. In further embodiments, one or more modified nucleotide bonds are phosphorothioate (PS) bonds. In other embodiments, the nucleotide bonds are modified nucleotide bonds that are phosphorothioate (PS) bonds. In further embodiments, the modification is a 2' fluoro group or a 2' methoxy group on the ribose, or a PS bond, or both. In further embodiments, one or more of all three of these enumerated modifications are present. In further embodiments, the siRNA contains 1 to 10 2' fluoro modifications on the ribose. In other embodiments, the siRNA contains 1 to 10 2' fluoro modifications on the ribose, and the rest of the nucleotide, i.e., nucleotides without 2' fluoro modifications, have 2' methoxy group modifications on the ribose.

[0048] In other embodiments of compounds such as RNAi agents disclosed herein, one or more oligonucleotides comprise an siRNA comprising a sense strand and an antisense strand. In further embodiments, the sense strand and antisense strand are each 15 to 40 nucleotides long. In preferred embodiments, the antisense strand is 23 nucleotides long. In preferred embodiments, the sense strand is 21 nucleotides long. In other preferred embodiments, the antisense strand is 23 nucleotides long and the sense strand is 21 nucleotides long. In yet another embodiment, the sense strand and antisense strand are annealed and optionally comprise one or more 5' nucleotide overhangs or 3' nucleotide overhangs, one or more 5' blunt ends or 3' blunt ends, or a combination thereof.

[0049] In another embodiment of the RNAi molecule comprising the RNAi agent disclosed herein, the 5' or 3' end of an ANGPTL8 oligonucleotide, such as one represented by R, where R comprises a sense strand and an antisense strand, is further modified. In a further embodiment, the 5' end of the antisense strand is optionally phosphorylated. In a further embodiment, the nucleotide at the 5' end of the antisense strand comprises a 5' vinyl phosphonate modification. In another embodiment, the nucleotide at the 5' end of the antisense strand comprises a phosphate group. In another embodiment, the nucleotide at the 5' end of the antisense strand comprises a phosphate analog.

[0050] In other embodiments, RNAi molecules such as the RNAi agents disclosed herein include siRNA comprising formula I and a sense strand or antisense strand of Table 6 or Table 8. In other embodiments, each RNAi molecule or RNAi agent disclosed herein includes formula I and a sense strand comprising one of the sequences of SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, or SEQ ID NO: 366. In other embodiments, each RNAi molecule or RNAi agent disclosed herein includes siRNA comprising formula I and an antisense strand comprising one of the sequences of SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, or SEQ ID NO: 372. In further embodiments, each RNAi molecule or RNAi agent disclosed herein includes formula I and the sequences described in a-f. a. Sequence IDs 361 and 367, b. Sequence IDs 362 and 368, c. Sequence IDs 363 and 369, d. Sequence ID 364 and Sequence ID 370, e. Sequence ID 365 and Sequence ID 371, or f. An siRNA comprising a sense strand and an antisense strand selected from a pair of sequences of sequence number 366 and sequence number 372.

[0051] In further embodiments, the RNAi molecule containing the RNAi agent disclosed herein is conjugated to formula I via a linker of formula III (i.e., linker 2). In further embodiments, the linker of formula III is conjugated to a nucleotide at the 3' end of the sense strand. In further embodiments, any 5' phosphate on the antisense strand is omitted, and one or more 2' fluoro residues in the ribose are removed. In further embodiments, any removed 2' fluoro residue is replaced by a 2' methoxy modification at the same position.

[0052] RNAi molecules herein, comprising one or more ANGPTL8 oligonucleotides, such as those represented by R, including Formula I, and those in which R comprises a sense strand and an antisense strand, are useful for the treatment of liver diseases or diseases involving adipose tissue. These are formulated into pharmaceutical compositions suitable for use in patients, preferably in humans. The pharmaceutical compositions disclosed herein comprise one or more carriers, diluents, and excipients that are compatible with the RNAi molecules or RNAi agents herein and other components of the composition or formulation, and are not harmful to the patient. Examples of pharmaceutical compositions and processes for their preparation can be found in "Remington: The Science and Practice of Pharmacy," Loyd, V., et al. Eds., 22. nd It can be found in Ed., Mack Publishing Co., 2012.

[0053] Accordingly, in one embodiment, the present invention relates to a pharmaceutical composition comprising one or more ANGPTL8 oligonucleotides, such as those represented by formula I, and R, wherein R comprises a sense strand and an antisense strand, and one or more pharmaceutically acceptable excipients. In further embodiments, RNA molecules, such as the RNAi molecules or RNAi agents disclosed herein, and the pharmaceutical compositions thereof, are intended for use in the treatment or therapy of a disease.

[0054] Another embodiment is an RNAi molecule or RNAi agent, or a pharmaceutical composition thereof, for therapeutic use, comprising one or more ANGPTL8 oligonucleotides, such as those represented by formula I, and in which R comprises a sense strand and an antisense strand. Another embodiment is an RNAi agent disclosed herein for therapeutic use. In a further embodiment, the treatment comprises reducing the level of ANGPTL8 expression, such as compared to untreated, a control, or a placebo. A further embodiment is for a liver disease or a liver-related disease. Another embodiment is a method for treating a liver disease, comprising administering an RNAi molecule, preferably administered in an effective amount, comprising one or more ANGPTL8 oligonucleotides comprising R, preferably formula I, and in which R comprises a sense strand and an antisense strand as disclosed herein, or any of the aforementioned pharmaceutical compositions. Another embodiment is a method for treating a liver disease in a patient who requires treatment of a liver disease, comprising administering an RNAi agent, preferably in an effective amount thereof, or a pharmaceutical composition comprising an RNAi agent and one or more pharmaceutically acceptable excipients.

[0055] Another embodiment is an RNA interference (RNAi) agent comprising formula I,

[0056] [ka] The array also comprises a sense strand and an antisense strand, the antisense strand comprising at least 18 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs. 405 to 525, the sense strand and antisense strand forming a complementary region of at least 15 nucleotides, each independently being 18 to 23 nucleotides long, optionally each independently comprising one or more modified nucleotides, optionally one or more internucleotide bonds between the sense strand and antisense strand being modified internucleotide bonds, and optionally conjugated to formula I via a linker.

[0057] Oligonucleotides, such as sense and antisense strands, disclosed herein may also be conjugated to an alternative delivery portion to target the liver or other tissues to reduce ANGPTL8 expression. Oligonucleotides, such as sense and antisense strands, disclosed herein may also be delivered to the target tissue by encapsulation or by other means that do not require conjugation, in order to reduce ANGPTL8 expression.

[0058] Accordingly, other embodiments of the RNAi agent disclosed herein are RNAi agents comprising a delivery moiety having the formula RLD, optionally conjugated to R via a linker L, wherein R comprises an antisense strand, a sense strand, or both, the antisense strand comprising at least 15 consecutive nucleotides of a sequence complementary to the mRNA transcript of ANGPTL8, the sense strand and antisense strand forming a complementary region of at least 15 nucleotides, the sense strand and / or antisense strand being independently 15 to 30 nucleotides long if present and when present, optionally comprising one or more modified nucleotides if present and when present, and optionally comprising one or more modified nucleotide interlinks if present and when present. In another embodiment, the antisense strand comprising at least 15 consecutive nucleotides of a sequence complementary to Sequence ID No. 1. In a further embodiment, the sense strand and antisense strand are independently 18 to 23 nucleotides long. In a further embodiment of any of these RNAi agents, the antisense strand forms a complementary region of at least 18 nucleotides to the mRNA transcript of ANGPTL8. In a different further embodiment of any of these RNAi agents, the antisense strand forms a complementary region of at least 18 nucleotides to SEQ ID NO: 1.

[0059] In a further embodiment, the RNAi agent is one in which the antisense strand contains at least 15 nucleotides of a sequence selected from the group consisting of SEQ ID NOs. 405 to 525. In yet another embodiment, the antisense strand contains at least 18 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs. 405 to 525.

[0060] In further embodiments of the RNAi agents disclosed herein, the antisense strand is SEQ ID NOs: 405, 408, 412, 413, 414, 415, 418, 420, 425, 426, 428, 429, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 448, 449, 451, 452, 454, 45 The sequence comprises at least 15 consecutive nucleotides selected from the group consisting of 7, 458, 459, 463, 464, 465, 466, 467, 468, 469, 471, 472, 473, 474, 475, 476, 479, 490, 491, 492, 493, 495, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, and 509. In another embodiment, the antisense strand is selected from the group of antisense strand sequences in Table 2.

[0061] In another embodiment of the RNAi agent disclosed herein, the antisense strand is selected from the group consisting of SEQ ID NOs. 231-361 or sequences having at least 90% sequence identity thereto. In yet another embodiment of the RNAi agent disclosed herein, the sense strand is selected from the group consisting of SEQ ID NOs. 124-230 or sequences having at least 90% sequence identity thereto.

[0062] In any further embodiment of the RNAi agents described herein, the complementary region includes 0, 1, 2, or 3 mismatches between the sense strand and the antisense strand.

[0063] In further embodiments of the RNAi agent, the sense strand and the antisense strand each independently contain one or more modified nucleotides. In further embodiments, the one or more modified nucleotides are independently 2'-fluoro-modified nucleotides or 2'-O-methyl-modified nucleotides. In other embodiments, each nucleotide of the sense strand and each nucleotide of the antisense strand are modified nucleotides, and in further embodiments, each of the modified nucleotides is independently 2'-fluoro-modified nucleotide or 2'-O-methyl-modified nucleotide.

[0064] In other embodiments of the RNAi agent described herein, the sense strand and the antisense strand each independently contain one or more modified nucleotide bonds. In further embodiments, each modified nucleotide bond is a phosphorothioate bond. In other embodiments, the sense strand and the antisense strand each independently contain four phosphorothioate bonds. In further embodiments, the first two 5' nucleotides of the sense strand and the two terminal 3' nucleotides of the sense strand are phosphorothioate bonds. In further embodiments, the first two 5' nucleotides of the antisense strand and the two terminal 3' nucleotides of the antisense strand are phosphorothioate bonds.

[0065] In other embodiments of RNAi agents described herein, the 5' nucleotide of the antisense strand comprises a phosphate group or a phosphate analog.

[0066] Further embodiments of the RNAi agents disclosed herein include those in which the antisense strand is SEQ ID NOs: 405, 408, 412, 413, 414, 415, 418, 420, 425, 426, 428, 429, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 448, 449, 451, 452, 454, 45 It contains at least 18 consecutive nucleotides of a sequence selected from the group consisting of 7, 458, 459, 463, 464, 465, 466, 467, 468, 469, 471, 472, 473, 474, 475, 476, 479, 490, 491, 492, 493, 495, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, and 509.

[0067] In yet another further embodiment, the RNAi agent comprises an antisense strand having a length of 23 nucleotides. In yet another further embodiment, the sense strand of the RNAi agent has a length of 21 nucleotides. In a preferred embodiment, the antisense strand is 23 nucleotides long and the sense strand is 21 nucleotides long.

[0068] In another embodiment, the antisense strand of the RNAi agent is selected from the group consisting of SEQ ID NOs. 231-361 or sequences having at least 90% sequence identity thereto. In another embodiment, the sense strand of the RNAi agent is selected from the group consisting of SEQ ID NOs. 124-230 or sequences having at least 90% sequence identity thereto. The RNAi agent according to any one of the preceding claims, wherein the complementary region contains 0, 1, 2, or 3 mismatches between the sense strand and the antisense strand.

[0069] In further embodiments, the RNAi agent comprises a sense strand or an antisense strand containing one or more modified nucleotides. In further embodiments, the one or more modified nucleotides are independently nucleotides having a 2'-fluoro group on the ribose or a 2'-O-methyl group. In further embodiments, each nucleotide in the sense strand and the antisense strand is a modified nucleotide.

[0070] In another embodiment of the RNAi agent described herein, each of the two nucleotides at the 5' and 3' ends of the sense and antisense strands has a modified internucleotide bond. In a further embodiment, the modified internucleotide bond, if present, is a phosphorothiolate bond.

[0071] In another embodiment of the RNAi agent described herein, the nucleotide at the 5' end of the antisense strand has a modification or further modification which is a phosphate group or a phosphate analog.

[0072] In further embodiments of the RNAi agent described herein, the antisense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 367-372 and 389-404, or a sequence having at least 90% sequence identity to these.

[0073] In other further embodiments of RNAi agents described herein, the sense strand comprises a sequence selected from the group consisting of SEQ ID NOs. 361-366 and 373-388, or a sequence having at least 90% sequence identity to these.

[0074] In other further embodiments of the RNAi agents disclosed herein, the sense strand and the antisense strand are: a. A sense strand having the sequence described in Sequence ID No. 361, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 367, or a sequence having at least 90% sequence identity thereto. b. A sense strand having the sequence described in Sequence ID No. 362, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 368, or a sequence having at least 90% sequence identity thereto. c. A sense strand having the sequence described in Sequence ID No. 363, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 369, or a sequence having at least 90% sequence identity thereto. d. A sense strand having the sequence described in Sequence ID No. 364, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 370, or a sequence having at least 90% sequence identity thereto. e. A sense strand having the sequence described in Sequence ID No. 365, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 371, or a sequence having at least 90% sequence identity thereto. f. A sense strand having the sequence described in Sequence ID No. 366, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 372, or a sequence having at least 90% sequence identity thereto. g. A sense strand having the sequence described in Sequence ID No. 373, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 389, or a sequence having at least 90% sequence identity thereto. h. A sense strand having the sequence described in Sequence ID No. 374, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 390, or a sequence having at least 90% sequence identity thereto. i. A sense strand having the sequence described in Sequence ID No. 375, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 391, or a sequence having at least 90% sequence identity thereto. j. A sense strand having the sequence described in Sequence ID No. 376, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 392, or a sequence having at least 90% sequence identity thereto. k. A sense strand having the sequence described in Sequence ID No. 377, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 393, or a sequence having at least 90% sequence identity thereto. l. A sense strand having the sequence described in Sequence ID No. 378, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 394, or a sequence having at least 90% sequence identity thereto, m. A sense strand having the sequence described in Sequence ID No. 379, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 395, or a sequence having at least 90% sequence identity thereto, n. A sense strand having the sequence described in Sequence ID No. 380, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 396, or a sequence having at least 90% sequence identity thereto. o. A sense strand having the sequence described in Sequence ID No. 381, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 396, or a sequence having at least 90% sequence identity thereto. A sense strand having the sequence described in Sequence ID No. 382, ​​or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 397, or a sequence having at least 90% sequence identity thereto. q. A sense strand having the sequence described in Sequence ID No. 383, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 398, or a sequence having at least 90% sequence identity thereto, r. A sense strand having the sequence described in Sequence ID No. 384, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 399, or a sequence having at least 90% sequence identity thereto, s. A sense strand having the sequence described in Sequence ID No. 385, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 400, or a sequence having at least 90% sequence identity thereto. t. A sense strand having the sequence described in Sequence ID No. 386, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 401, or a sequence having at least 90% sequence identity thereto. u. A sense strand having the sequence described in Sequence ID No. 387, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 402, or a sequence having at least 90% sequence identity thereto. v. A sense strand having the sequence described in Sequence ID No. 388, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 403, or a sequence having at least 90% sequence identity thereto, w. A pair of oligonucleotide sequences selected from the group consisting of a sense strand having the sequence described in Sequence ID No. 389 or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 404 or a sequence having at least 90% sequence identity thereto. Preferably, the RNAi agent having the listed oligonucleotide sequence pairs comprises formula I. In a further preferred embodiment, the RNAi agent comprises formula I conjugated to a nucleotide at the 3' end of the sense strand. In yet another embodiment, formula I is conjugated to a nucleotide at the 3' end of the sense strand via a linker. In yet another embodiment, the linker is a linker of formula III.

[0075] In other embodiments of RNAi agents described herein, the sense strand and antisense strand are: a. A sense strand having the sequence described in Sequence ID No. 361, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 367, or a sequence having at least 95% sequence identity thereto. b. A sense strand having the sequence described in Sequence ID No. 362, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 368, or a sequence having at least 95% sequence identity thereto. c. A sense strand having the sequence described in Sequence ID No. 363, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 369, or a sequence having at least 95% sequence identity thereto. d. A sense strand having the sequence described in Sequence ID No. 364, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 370, or a sequence having at least 95% sequence identity thereto. e. A sense strand having the sequence described in Sequence ID No. 365, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 371, or a sequence having at least 95% sequence identity thereto. f. A sense strand having the sequence described in Sequence ID No. 366, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 372, or a sequence having at least 95% sequence identity thereto. g. A sense strand having the sequence described in Sequence ID No. 373, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 389, or a sequence having at least 95% sequence identity thereto. h. A sense strand having the sequence described in Sequence ID No. 374, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 390, or a sequence having at least 95% sequence identity thereto. i. A sense strand having the sequence described in Sequence ID No. 375, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 391, or a sequence having at least 95% sequence identity thereto. j. A sense strand having the sequence described in Sequence ID No. 376, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 392, or a sequence having at least 95% sequence identity thereto. k. A sense strand having the sequence described in Sequence ID No. 377, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 393, or a sequence having at least 95% sequence identity thereto. l. A sense strand having the sequence described in Sequence ID No. 378, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 394, or a sequence having at least 95% sequence identity thereto. m. A sense strand having the sequence described in Sequence ID No. 379, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 395, or a sequence having at least 95% sequence identity thereto. n. A sense strand having the sequence described in Sequence ID No. 380, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 396, or a sequence having at least 95% sequence identity thereto. o. A sense strand having the sequence described in Sequence ID No. 381, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 396, or a sequence having at least 95% sequence identity thereto. A sense strand having the sequence described in Sequence ID No. 382, ​​or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 397, or a sequence having at least 95% sequence identity thereto. q. A sense strand having the sequence described in Sequence ID No. 383, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 398, or a sequence having at least 95% sequence identity thereto. r. A sense strand having the sequence described in Sequence ID No. 384, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 399, or a sequence having at least 95% sequence identity thereto, s. A sense strand having the sequence described in Sequence ID No. 385, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 400, or a sequence having at least 95% sequence identity thereto. t. A sense strand having the sequence described in Sequence ID No. 386, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 401, or a sequence having at least 95% sequence identity thereto. u. A sense strand having the sequence described in Sequence ID No. 387, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 402, or a sequence having at least 95% sequence identity thereto. v. A sense strand having the sequence described in Sequence ID No. 388, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 403, or a sequence having at least 95% sequence identity thereto, w. A pair of oligonucleotide sequences selected from the group consisting of a sense strand having the sequence described in Sequence ID No. 389 or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 404 or a sequence having at least 95% sequence identity thereto. Preferably, the RNAi agent having the listed oligonucleotide sequence pairs comprises formula I. In a further preferred embodiment, the RNAi agent comprises formula I conjugated to a nucleotide at the 3' end of the sense strand. In yet another embodiment, formula I is conjugated to a nucleotide at the 3' end of the sense strand via a linker. In yet another embodiment, the linker is a linker of formula III.

[0076] In other embodiments of the RNAi agents disclosed herein, the sense strand and the antisense strand are a. A sense strand having the sequence described in Sequence ID No. 361, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 367, or a sequence having at least 90% sequence identity thereto. b. A sense strand having the sequence described in Sequence ID No. 362, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 368, or a sequence having at least 90% sequence identity thereto. c. A sense strand having the sequence described in Sequence ID No. 363, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 369, or a sequence having at least 90% sequence identity thereto. d. A sense strand having the sequence described in Sequence ID No. 364, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 370, or a sequence having at least 90% sequence identity thereto, e. A pair of oligonucleotide sequences selected from the group consisting of a sense strand having the sequence described in Sequence ID No. 365 or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 371 or a sequence having at least 90% sequence identity thereto. Preferably, the RNAi agent having the listed oligonucleotide sequence pairs comprises formula I. In a further preferred embodiment, the RNAi agent comprises formula I conjugated to a nucleotide at the 3' end of the sense strand. In yet another embodiment, formula I is conjugated to a nucleotide at the 3' end of the sense strand via a linker. In yet another embodiment, the linker is a linker of formula III.

[0077] In further embodiments of the RNAi agents disclosed herein, the sense strand and the antisense strand are: a. A sense strand having the sequence described in Sequence ID No. 361, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 367, or a sequence having at least 95% sequence identity thereto. b. A sense strand having the sequence described in Sequence ID No. 362, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 368, or a sequence having at least 95% sequence identity thereto. c. A sense strand having the sequence described in Sequence ID No. 363, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 369, or a sequence having at least 95% sequence identity thereto. d. A sense strand having the sequence described in Sequence ID No. 364, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 370, or a sequence having at least 95% sequence identity thereto, e. A pair of oligonucleotide sequences selected from the group consisting of a sense strand having the sequence described in Sequence ID No. 365 or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 371 or a sequence having at least 95% sequence identity thereto.

[0078] Preferably, the RNAi agent having the listed oligonucleotide sequence pairs comprises formula I. In a further preferred embodiment, the RNAi agent comprises formula I conjugated to a nucleotide at the 3' end of the sense strand. In yet another embodiment, formula I is conjugated to a nucleotide at the 3' end of the sense strand via a linker. In yet another embodiment, the linker is a linker of formula III.

[0079] In another embodiment of the RNAi agent disclosed herein, the sense strand and the antisense strand are a. A sense strand having the sequence described in SEQ ID NO: 361, or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in SEQ ID NO: 367, or a sequence having at least 90% sequence identity thereto, b. An RNA agent comprising a pair of oligonucleotide sequences selected from the group consisting of a sense strand having the sequence described in Sequence ID No. 365 or a sequence having at least 90% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 371 or a sequence having at least 90% sequence identity thereto.

[0080] Preferably, the RNAi agent having the listed oligonucleotide sequence pairs comprises formula I. In a further preferred embodiment, the RNAi agent comprises formula I conjugated to a nucleotide at the 3' end of the sense strand. In yet another embodiment, formula I is conjugated to a nucleotide at the 3' end of the sense strand via a linker. In yet another embodiment, the linker is a linker of formula III.

[0081] In other embodiments of the RNAi agents disclosed herein, the sense strand and the antisense strand are a. A sense strand having the sequence described in Sequence ID No. 361, or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 367, or a sequence having at least 95% sequence identity thereto, b. A pair of oligonucleotide sequences selected from the group consisting of a sense strand having the sequence described in Sequence ID No. 365 or a sequence having at least 95% sequence identity thereto, and an antisense strand having the sequence described in Sequence ID No. 371 or a sequence having at least 95% sequence identity thereto.

[0082] Preferably, the RNAi agent having the listed oligonucleotide sequence pairs comprises formula I. In a further preferred embodiment, the RNAi agent comprises formula I conjugated to a nucleotide at the 3' end of the sense strand. In yet another embodiment, formula I is conjugated to a nucleotide at the 3' end of the sense strand via a linker. In yet another embodiment, the linker is a linker of formula III.

[0083] In other embodiments described herein, RNAi agents can reduce the expression of the ANGPTL8 gene in hepatocytes.

[0084] In other embodiments, the RNAi agents disclosed herein are intended for therapeutic use.

[0085] Another embodiment is an RNAi molecule or RNAi agent or pharmaceutical composition thereof disclosed herein, preferably comprising one or more ANGPTL8 oligonucleotides containing formula I and R, such as R containing a sense strand and / or antisense strand as disclosed herein, for use in the manufacture of a drug suitable for the treatment of liver diseases or diseases involving the liver, including dyslipidemia, cardiovascular disease, and non-alcoholic fatty liver disease (NAFLD), respectively. Another embodiment is the use of an RNAi molecule or RNAi agent or pharmaceutical composition thereof disclosed herein, preferably comprising one or more ANGPTL8 oligonucleotides containing formula I and R, such as R containing a sense strand and / or antisense strand as disclosed herein, in the manufacture of a drug suitable for the treatment of liver diseases or diseases involving the liver, including dyslipidemia, cardiovascular disease, and non-alcoholic fatty liver disease (NAFLD), respectively. In a further embodiment, NAFLD is non-alcoholic steatohepatitis (NASH). Another embodiment is an RNAi agent or pharmaceutical composition thereof disclosed herein for use in the manufacture of agents suitable for the treatment of liver diseases or liver-related diseases, including dyslipidemia, cardiovascular disease, and non-alcoholic fatty liver disease (NAFLD), respectively. In a further embodiment, NAFLD is non-alcoholic steatohepatitis (NASH).

[0086] Another embodiment is a method for treating dyslipidemia, comprising administering an effective amount of the RNAi molecule or pharmaceutical composition thereof disclosed herein to a patient who needs to be treated for dyslipidemia. Another embodiment is the RNAi molecule or pharmaceutical composition thereof disclosed herein for use in treating dyslipidemia. Another embodiment is a method for treating dyslipidemia, comprising administering an effective amount of the RNAi agent or pharmaceutical composition thereof disclosed herein to a patient who needs to be treated for dyslipidemia. Another embodiment is the RNAi agent or pharmaceutical composition thereof disclosed herein for use in treating dyslipidemia.

[0087] In other embodiments, a method for treating cardiovascular disease is provided, comprising administering an effective amount of an RNAi molecule or a pharmaceutical composition thereof disclosed herein to a patient who requires treatment for cardiovascular disease. Another embodiment is an RNAi molecule or a pharmaceutical composition thereof disclosed herein for use in the treatment of cardiovascular disease, measured by a reduction in the risk of hospitalization and / or cardiovascular events and / or either or both of these. In other embodiments, a method for treating cardiovascular disease is provided, comprising administering an effective amount of an RNAi agent or a pharmaceutical composition thereof disclosed herein to a patient who requires treatment for cardiovascular disease. Another embodiment is an RNAi agent or a pharmaceutical composition thereof disclosed herein for use in the treatment of cardiovascular disease, measured by a reduction in the risk of hospitalization and / or cardiovascular events and / or either or both of these.

[0088] In other embodiments, the method for preventing a cardiovascular event comprises administering an effective amount of an RNAi molecule or a pharmaceutical composition thereof disclosed herein to a patient who requires prevention of a cardiovascular event. In other embodiments, the method for preventing a cardiovascular event comprises administering an effective amount of an RNAi agent or a pharmaceutical composition thereof disclosed herein to a patient who requires prevention of a cardiovascular event. In further embodiments, the cardiovascular event is myocardial infarction.

[0089] In another embodiment, the method for reducing hospitalizations related to cardiovascular disease or events comprises administering an effective amount of an RNAi molecule, such as an RNAi agent disclosed herein, or a pharmaceutical composition thereof, to a patient who needs to reduce hospitalizations related to cardiovascular disease or events.

[0090] Another embodiment is a method for treating non-alcoholic fatty liver disease (NAFLD), comprising administering an effective amount of an RNAi molecule, such as an RNAi agent disclosed herein, or a pharmaceutical composition thereof, to a patient who requires treatment for non-alcoholic fatty liver disease (NAFLD). Another embodiment is an RNAi molecule, such as an RNAi agent disclosed herein, or a pharmaceutical composition thereof, for use in treating NAFLD. In a further embodiment, NAFLD is NASH.

[0091] In another embodiment, the method for lowering triglyceride levels includes administering an effective amount of an RNAi molecule, such as an RNAi agent disclosed herein, or a pharmaceutical composition thereof, to a patient who requires a reduction in triglyceride levels.

[0092] In another embodiment, the method for inhibiting lipoprotein lipase (LPL) comprises administering an effective amount of an RNAi molecule, such as an RNAi agent disclosed herein, or a pharmaceutical composition thereof, to a patient who requires inhibition of LPL.

[0093] In another embodiment, the method for increasing high-triglyceride lipoprotein catabolism comprises administering an effective amount of an RNAi molecule, such as an RNAi agent disclosed herein, or a pharmaceutical composition thereof, to a patient who requires increased high-triglyceride lipoprotein catabolism.

[0094] In another embodiment, a method for treating liver disease in a patient who would benefit from reducing the expression level of ANGPTL8 is provided, comprising administering an effective amount of an RNAi molecule or a pharmaceutical composition thereof, such as an RNAi agent disclosed herein, to the patient who is in need of treatment for liver disease.

[0095] As used herein, the term “effective dose” means the amount that is effective in treating a disease or illness.

[0096] As used herein, the terms “to treat” or “to treat” mean the act of providing treatment to an individual in need by administering a therapeutic agent (e.g., an oligonucleotide as herein) to the individual for the purpose of improving the individual’s health and / or healthy state with respect to an existing condition (e.g., disease, illness) or to prevent or reduce the likelihood of the condition developing. Treatment may also include reducing the frequency or severity of at least one sign, symptom, or contributing factor of a condition (e.g., disease, illness) experienced by the individual.

[0097] RNAi agents are further described in the non-limiting examples herein.

[0098] Example 1

[0099] Table 1. Target and antisense sequences for designed siRNAs [Table 1-1]

[0100] (Continued from Table 1) [Table 1-2]

[0101] (Continued from Table 1) [Table 1-3]

[0102] As shown above in Table 1, siRNAs complementary to the 18mer region of the ANGPTL8 transcript NM_(SEQ ID NO: 1) are designed. The sense and antisense RNA oligonucleotide strands are 18–23 nucleotides long, have an arbitrary overhang of 1–5 ribonucleotides, have 1–10 fluoroadditions at the 2' position of the ribose, and the remaining residues are methylated at the 2' position of the ribose (producing 2'-methoxy, i.e., 2'O-methyl modification). Some antisense strands are phosphorylated at the 5' position. Each siRNA is conjugated to a delivery region containing three GalNAc groups; the selected delivery region contains formula I, and the other delivery regions contain a control region. One or more phosphodiester bonds are present at the 5' and 3' ends.

[0103] Knockdown of ANGPTL8 expression by these siRNAs is assayed using the following procedure: Mouse primary hepatocytes (MPHs) are freshly isolated from AAV-ANGPTL8 humanized mice and added to Corning plates at 15k per well. siRNAs are added directly to the wells. For single measurements, 1 μM (1000 nM) is used. To generate concentration / dose-response curves, final concentrations of GalNAc-conjugated siRNAs of 1000, 333, 111, 37, 12, 4, 1.37, 0.46, 0.15, 0.05, and 0.017 nM are used.

[0104] The treated cells are lysed, and RNA is directly isolated into a 96-well plate using Quick-RNA96Kit (Zymo Research). The eluted RNA is either used immediately or cryopreserved. cDNA is synthesized using Fast Advanced RT Master Mix (Invitrogen) in a thermocycler using the following steps: 37°C for 30 minutes, 95°C for 5 minutes, and then held at 4°C. Polymerase chain reaction (PCR) is performed via TaqMan RT PCR (Life Technologies) using the following cycle temperatures and times: 40 cycles of 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute.

[0105] Human ANGPTL8 levels were normalized to mouse Rplp0 (Life Technologies) and represent the relative knockdown of human ANGPTL8 mRNA expression compared to vehicle-treated control cells. IC50 values ​​were calculated using a four-parameter fitted model with XLFit.

[0106] Table 2 below shows the ANGPTL8 target regions of siRNAs that showed more than 50% knockdown.

[0107] Table 2. [Table 2-1]

[0108] (Continued from Table 2) [Table 2-2]

[0109] Example 2 ANGPTL8 siRNA conjugated to a control delivery region (cntrl-GalNAc) containing three GalNAc groups was assayed as described in Example 2, compared to the same siRNA conjugated to the delivery region of Formula I, and compared to siRNA lacking a 5' phosphate group on the antisense strand. The sense and antisense strands of the three siRNAs are shown in Table 3 below, and each of the three siRNAs has one of the sense-antisense strand pairs of SEQ ID NOs. 381 and 397, 382 and 398, or 366 and 372. Modifications are indicated immediately before the corresponding modified nucleotide. P represents phosphorylation, m represents methylation of an OH group that produces a 2' methoxy modification on ribose, and f represents a 2' fluoro modification of ribose. * (This represents the phosphorothioate modification of the phosphodiester bond in the skeleton.)

[0110] Table 3. [Table 3]

[0111] To compare the above siRNAs, three assays were performed to analyze the knockdown of ANGPTL8 expression, measured by ANGPTL3 / 8 levels, triglyceride levels, and mRNA levels (%KD).

[0112] Conjugated siRNAs will be tested in male transgenic mice for human cholesterol ester transfer protein (CETP) and apolipoprotein A1 (Taconic farms). Two adeno-associated virus (AAV) vectors will be administered to the mice by post-orbital injection. The first vector will contain an albumin promoter and a plasmid containing the coding sequence of human ANGPTL8 (NM_018687.7) SEQ ID NO: 1. The second vector will contain a mouse codon-optimized sequence of human ANGPTL3 (NP_055310.1) SEQ ID NO: 2. Three to five weeks after AAV administration, the animals' body weight will be measured and blood will be collected from the mice. This is considered pre-study blood collection. Serum will be prepared from the blood, triglycerides will be measured using a COBAS clinical chemistry analyzer (Roche), and ANGPTL3 / 8 protein levels will be measured by ELISA (Meso Scale Diagnostics). Mice were assigned to groups with similar body weight, serum triglycerides, and ANGPTL3 / 8 levels (n=6). Blood was collected on day 0 of the study and considered as baseline. Either PBS or test siRNA was administered subcutaneously to mice at doses of 3 and 10 mg / kg on day 0 of the study. Blood was collected from mice one and two weeks after siRNA administration under isoflurane anesthesia. Serum was prepared from the blood and triglycerides were measured using a COBAS clinical chemistry analyzer (Roche). Serum ANGPTL3 / 8 levels were measured by ELISA (Meso Scale Diagnostics). Triglycerides as a percentage change from baseline at one week were calculated as ((triglycerides at one week - baseline triglycerides) / (baseline triglycerides)). * It is calculated as 100. Triglycerides are similarly calculated as a percentage change from baseline after 2 weeks. ANGPTL3 / 8 as a percentage change from baseline after 1 week is ((ANGPTL3 / 8 after 1 week - ANGPTL3 / 8 at baseline) / (ANGPTL3 / 8 at baseline)). *It is calculated as 100. ANGPTL3 / 8 is similarly calculated as the percentage change from baseline at 2 weeks. Serum triglyceride and ANGPTL3 / 8 data were analyzed for statistical significance from the PBS group at the corresponding time points using ANOVA and Dunnett's method, with p<0.05 considered statistically significant (SAS Institute).

[0113] To measure in vitro knockdown, mice are euthanized under isoflurane anesthesia two weeks after subcutaneous injection. Liver tissue is collected from the mice and frozen in liquid nitrogen. The liver is homogenized with TriZol (Invitrogen) using Lysing Matrix D bead tubes with FastPrep-24 (MP Bio) to purify RNA, which is then resuspended in nuclease-free water. RNA is quantified using NanoDrop (ThermoFisher). Equal volumes of RNA are reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Life Technologies) with a ProFlex Thermocycler (Thermo Fisher). The thermocycler settings are 25°C for 10 minutes, 37°C for 2 hours, and then 85°C for 5 minutes. Template cDNA was combined with Taqman Universal Master Mix and Assays on Demand primer / probe sets, and RT-PCR was performed on a QuantStudio7 Flex Real-Time PCR system (Applied Biosystems) with the following parameters: 40 cycles of 2 minutes at 50°C, 10 minutes at 95°C, then 15 seconds at 95°C, and 1 minute at 60°C. Fold change (FC) was calculated by subtracting the CT value of mouse Rplp0 from the CT value of human ANGPTL8 to obtain the ΔCT value. The ΔCT value was calculated by subtracting the average ΔCT value of the untreated sample (PBS control) for each gene from the ΔCT value of each test sample. Fold change was calculated by taking the base-2 logarithm of the negative ΔCT value. Percent knockdown (%KD) was calculated by subtracting FC from 1 and multiplying by 100. The data are shown in Table 4. Two siRNAs containing the delivery portion of formula I performed better than the control delivery portion, showing lower levels of ANGPTL8, lower triglyceride levels, and a higher knockdown rate of ANGPTL8 mRNA.

[0114] Table 4. [Table 4] * This shows Dunnett's p<0.05 ANOVA.

[0115] Example 3 Exemplary siRNAs complementary to the 18mer region of the ANGPTL8 transcript NM_(SEQ ID NO: 1) (Table 1) were designed and are shown in the sequence listing below by their underlying nucleotide sequences, with each row representing an siRNA having a given sense and antisense strand. As shown, the underlying sense RNA oligonucleotide strands and antisense RNA oligonucleotide strands are 18–23 nucleotides long and have an arbitrary overhang of 1–5 ribonucleotides. The shown underlying nucleotide sequences are modified by 1–10 fluoroadditions at the 2' position of the ribose, and the remaining residues are methylated at the 2' position of the ribose (producing 2' methoxy modifications). Some antisense strands are phosphorylated at the 5' position. Each siRNA is conjugated to a delivery region containing three GalNAc groups; the selected delivery region contains formula I, and the other delivery regions contain a control region. One or more phosphodiester bonds are present at the 5' and 3' ends. The control GalNAc is bound to the 3' end of the sense strand. These subsets of siRNAs are tested in an in vivo knockdown assay.

[0116] For in vivo knockdown assays, selected siRNAs are tested in male C57bl / 6 mice (Taconic farms). Mice are administered an adeno-associated virus (AAV) vector containing an albumin promoter and a plasmid encoding human ANGPTL8 (SEQ ID NO: 1) (NCBI reference sequence NM_018687.7) (Vector BioLabs) by post-orbital injection. Two weeks after AAV administration, the body weight of the mice is measured. Mice are assigned to groups with similar body weights (n=5). Either PBS or the test siRNA is administered subcutaneously to the mice at a dose of 10 mg / kg. Seven days after subcutaneous injection, the mice are euthanized under isoflurane anesthesia. Liver tissue is collected from the mice and frozen in liquid nitrogen. RNA is isolated and purified from the collected liver tissue, used for cDNA synthesis, and quantified by RT-PCR.

[0117] The fold change (FC) is calculated by subtracting the CT value of mouse Rplp0 from the CT value of human ANGPTL8 to obtain the ΔCT value. The relative amount is calculated by taking the base-2 logarithm of the negative ΔΔCT value. The fold change is calculated by dividing the relative amount of each sample by the mean of the control group. The knockdown rate (Percent knock down, %KD) is calculated by subtracting FC from 1 and multiplying by 100. The data is shown in Table 5.

[0118] Table 5 [Table 5]

[0119] To measure the in vivo knockdown of the following siRNAs in Table 6, the same procedure described above for a 10 mg / kg dose was performed at a 3 mg / kg dose. (Each siRNA has a sense strand and an antisense strand in vertical order. The first siRNA tested includes the first two rows of the table, SEQ ID NOs. 373 and 389, the next siRNA includes the third and fourth rows of the table, SEQ ID NOs. 374 and 390, and so on. Control GalNAc is bound to the 3' end of the sense strand of each siRNA. Abbreviations for modifications are the same as those shown in Example 3 above. The results for the selected RNAs are shown below in Table 7.)

[0120] Table 6 [Table 6]

[0121] Table 7 [Table 7]

[0122] Example 5 GalNAc-siRNA will be tested in male transgenic mice using human cholesterol ester transfer protein (CETP) and apolipoprotein A1 (Taconic fars). The siRNA will be split and tested in three studies (n=2, n=2, and n=2). Mice will be administered two adeno-associated virus (AAV) vectors by post-orbital injection. One vector will contain an albumin promoter and a plasmid containing the coding sequence for human ANGPTL8 (SEQ ID NO: 1). The second vector will contain a mouse codon-optimized sequence for human ANGPTL3 (SEQ ID NO: 2) (NP_055310.1). Baseline blood samples will be collected from mice 4–6.5 weeks after AAV administration. Serum will be prepared from blood, triglycerides will be measured using a COBAS clinical chemistry analyzer (Roche), and ANGPTL3 / 8 will be measured by ELISA (Meso Scale Diagnostics). Mice were assigned to groups with similar serum triglyceride and ANGPTL3 / 8 levels. siRNAs were designed as shown in Table 8, with each siRNA conjugated to the delivery portion of Formula I. Either PBS or siRNA was administered subcutaneously to mice at doses of 0.3, 1, 3, and 10 mg / kg. Blood was collected from mice 3, 6, and 9 weeks after siRNA administration under isoflurane anesthesia. Serum was prepared from the blood, and triglycerides were measured using a COBAS clinical chemistry analyzer (Roche).

[0123] Triglycerides as a percentage change from baseline after 3 weeks are calculated as ((triglycerides after 3 weeks - baseline triglycerides) / (baseline triglycerides)). * It is calculated as 100. Triglycerides are similarly calculated as a percentage change from baseline at 6 and 9 weeks. Triglyceride data were analyzed for statistical significance from the PBS group at the corresponding time points using ANOVA and Dunnett's method, with p<0.05 considered statistically significant (SAS Institute). The data are shown in Table 9.

[0124] Table 10 shows the corresponding in vitro knockdown rates at 1000 nM for each molecule.

[0125] Table 8 [Table 8]

[0126] Table 9 [Table 9]

[0127] Table 10 [Table 10]

[0128] Table 11 [Table 11-1]

[0129] (Continued from Table 11) [Table 11-2]

[0130] (Continued from Table 11) [Table 11-3]

[0131] Example 6 To quantify the level of the conjugated siRNA strands of Formula 1 in Table 13 in tissue samples, the tissue samples are homogenized in Clarity OTX cell lysis buffer (Phenomenex) to a final tissue concentration of 100 mg / mL. (For the sense strand, the OH metabolite is quantified due to rapid dephosphorylation in vivo). Plasma samples are diluted 1:10 (v / v) with Clarity OTX buffer. The samples are subjected to solid phase extraction using weak ion exchange resin (Waters, Oasis μElution SPE plate). The samples are eluted and subjected to liquid chromatography-high resolution mass spectrometry (LC-HRMS) as described in ASSAY and Drug Development Technologies, 10(3) pages 278-288 (2012).

[0132] Plasma exposure in mice or cynomolgus monkeys is measured at 6 and 24 hours post subcutaneous administration in monkeys. Liver exposure is determined in mice at 6, 24, 72, 168, 336, and 1343 hours. The results are subjected to non-compartmental analysis using the Phoenix software NCA package. C max , t 1 / 2 , and AUC are determined in plasma for both species, and liver C max , t 1 / 2 , and AUC are determined in mice, and liver t 1 / 2 and AUC are determined in monkeys.

[0133] Table 13 shows liver exposure of six conjugated siRNAs in cynomolgus monkeys. The liver was harvested at approximately 2 and 12 weeks after treatment by subcutaneous administration of 3 mg / kg of the listed ANGPTL8 siRNAs conjugated (at the 3'-terminal nucleotide of the sense strand) to the GalNAc-containing moiety of Formula I via Linker 2 (having Formula III) and subjected to the above detection method using LC / MS.

[0134] Tables 12a and 12b show two exemplary experiments of the knockdown rates of ANGPTL8 mRNA determined by RT-PCR of liver homogenates collected from cynomolgus monkeys before administration (one monkey) and after administration (several monkeys, 3 mg / kg each, as described below) of conjugated ANGPTL8 siRNA, in which the GalNAc-containing portion of formula I is conjugated to the 3' terminal nucleotide of the sense strand via linker 2 (containing formula III).

[0135] Table 12a Crab-eating macaque Efficacy and durability [Table 12] #n=4 * n=5

[0136] Table 12b Crab-eating macaque Efficacy and durability [Table 13] n=6 * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005 versus pre-treatment biopsy is statistically significant according to MMRM.

[0137] Table 13 Crab-eating macaque PK characteristics [Table 14]

[0138] The following numbered paragraphs provide additional embodiments of the RNAi agents and RNAi molecules disclosed herein.

[0139] 1. An RNA interference (RNAi) molecule or RNAi agent containing formula I, [ka] An RNA interference (RNAi) molecule or RNAi agent comprising one or more oligonucleotides containing 15-40 nucleotides bound to SEQ ID NO: 1, wherein R is optionally conjugated to the oligonucleotide via a linker. 2. One or more oligonucleotides are bound to one or more sequences listed in Table 1 or Table 2, and the RNAi molecule or RNAi agent is as described in paragraph 1. 3. One or more oligonucleotides comprising the sequences listed in Table 2, the RNAi molecules or RNAi agents described in paragraph 1. 4. One or more oligonucleotides comprising one or more modified nucleotides, an RNAi molecule or RNAi agent as described in any one of paragraphs 1 to 3. 5. One or more modified nucleotides are modified at the 2' position of a sugar, or at a pyrimidine or purine ring, or both, as described in paragraph 4 of the RNAi molecule or RNAi agent. 6. The RNAi molecule or RNAi agent described in paragraph 4 or 5, wherein one or more modified nucleotides are modified nucleotides containing a 2'-halogenated sugar group, a modified nucleotide containing a 2'-methylated sugar group, a modified nucleotide containing a 2'-methoxylated sugar group, a modified nucleotide containing a methylated purine, or a modified nucleotide containing a methylated pyrimidine ring, or any combination thereof. 7. One or more modified nucleotides are modified at the 2' position of a sugar, and the modification comprises one or more 2' fluoro groups, one or more 2' methoxy groups, or both, as described in any one of paragraphs 4 to 6, an RNAi molecule or RNAi agent. 8. One or more modified nucleotides are modified at the 2' position of a sugar, and the sugar is ribose, as described in any one of paragraphs 4-7, an RNAi molecule or RNAi agent. 9. An RNAi molecule or RNAi agent as described in any one of paragraphs 1 to 8, wherein one or more oligonucleotides include one or more modified bonds, and one or more of the modified bonds are phosphorothioate bonds. 10. One or more oligonucleotides comprising siRNA, wherein the siRNA comprises a sense strand and an antisense strand, as described in any one of paragraphs 1 to 9. 11. The RNAi molecule or RNAi agent described in paragraph 10, wherein the sense strand and antisense strand are each independently 15 to 40 nucleotides long. 12. An RNAi molecule or RNAi agent described in any one of paragraphs 1 to 11, further comprising a linker. 13. The linker is [ka] An RNAi molecule or RNAi agent as described in paragraph 12, comprising one of the above. 14. One or more oligonucleotides, including siRNA, are RNAi molecules or RNAi agents as described in any one of paragraphs 1 to 13. 15. siRNA is an RNAi molecule or RNAi agent as described in paragraph 14, comprising a sense strand and an antisense strand. 16. The RNAi molecule or RNAi agent described in paragraph 15, wherein the sense strand and antisense strand are each independently 15 to 40 nucleotides long. 17. An RNAi molecule or RNAi agent described in any one of paragraphs 14-16, wherein the sense strand and antisense strand are each independently 18-25 nucleotides long. 18. The RNAi molecule or RNAi agent described in paragraph 17, wherein the sense strand and antisense strand are annealed and optionally contain one or more 5' nucleotide overhangs or 3' nucleotide overhangs. 19. An RNAi molecule or RNAi agent described in any one of paragraphs 14-18, wherein the 5' end of the antisense strand is optionally phosphorylated. 20. An RNAi molecule or RNAi agent according to any one of paragraphs 1 to 19, comprising a compound of formula I and one or more oligonucleotides bound to any one of the sequences having sequence numbers 3 to 123 shown in Table 1. 21. An RNAi molecule or RNAi agent according to any one of paragraphs 1 to 20, comprising a compound of formula I and one or more oligonucleotides bound to any one of the sequences shown in Table 2. 22. An RNAi molecule or RNAi agent described in any of paragraphs 1 to 21, wherein one or more nucleotides are modified at the 2' position of ribose. 23. An RNAi molecule or RNAi agent as described in any of paragraphs 1 to 22, wherein at least one nucleotide ribose is modified with a 2' fluoro group or a 2' methoxy group. 24. siRNA is an RNAi molecule or RNAi agent described in any of paragraphs 1 to 23, comprising one or more modified linkages. 25. The RNAi molecule or RNAi agent described in paragraph 24, wherein one or more modifying bonds are phosphorothioate bonds. 26. The siRNA comprises a sense strand containing one of the sequences of SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, or SEQ ID NO: 366, as described in any one of paragraphs 1 to 25. 27. The RNAi molecule or RNAi agent described in any one of paragraphs 1 to 25, comprising an antisense strand containing one of the sequences of SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, or SEQ ID NO: 372, wherein the siRNA comprises an antisense strand. 28. siRNA is a~f: a. Sequence IDs 361 and 367, b. Sequence IDs 362 and 368, c. Sequence IDs 363 and 369, d. Sequence ID 364 and Sequence ID 370, e. Sequence ID 365 and Sequence ID 371, or f. An RNAi molecule or RNAi agent according to any one of paragraphs 1 to 27, comprising a sense strand and an antisense strand selected from a pair of sequences such as those described in SEQ ID NO: 366 and SEQ ID NO: 372. 29. A pharmaceutical composition comprising an RNAi molecule or RNAi agent described in any one of paragraphs 1 to 28, and at least one pharmaceutically acceptable excipient. 30. A method for treating dyslipidemia, comprising administering an RNAi molecule or RNAi agent, or a pharmaceutical composition thereof, described in any one of paragraphs 1 to 28, to a patient who is in need of treatment for dyslipidemia. 31. A method for treating a cardiovascular disease, comprising administering an effective amount of an RNAi molecule or RNAi agent, or a pharmaceutical composition thereof, described in any one of paragraphs 1 to 28, to a patient who is in need of treatment for a cardiovascular disease. 32. A method for preventing a cardiovascular event, comprising administering an effective amount of an RNAi molecule or RNAi agent, or a pharmaceutical composition thereof, described in any one of paragraphs 1 to 28, to a patient who is in need of prevention of a cardiovascular event. 33. The cardiovascular event is myocardial infarction, as described in paragraph 32. 34. A method for reducing hospitalization related to cardiovascular disease or an event, comprising administering an RNAi molecule or RNAi agent, or a pharmaceutical composition thereof, described in any one of paragraphs 1 to 28, to a patient who needs to reduce hospitalization related to cardiovascular disease or an event. 35. A method for treating non-alcoholic fatty liver disease (NAFLD), comprising administering an RNAi molecule or RNAi agent, or a pharmaceutical composition thereof, described in any one of paragraphs 1 to 28, to a patient who is in need of treatment for non-alcoholic fatty liver disease (NAFLD). 36. NAFLD is non-alcoholic steatohepatitis (NASH), as described in paragraph 35. 37. A method for lowering triglyceride levels, comprising administering an RNAi molecule or RNAi agent, or a pharmaceutical composition thereof, described in any one of paragraphs 1 to 28, to a patient who requires a reduction in triglyceride levels. 38. A method for reducing lipoprotein lipase (LPL) inhibition, comprising administering an RNAi molecule or RNAi agent, or a pharmaceutical composition thereof, described in any one of paragraphs 1 to 28, to a patient who requires a reduction in lipoprotein lipase (LPL) inhibition. 39. A method for increasing the catabolism of high-triglyceride lipoproteins, comprising administering an RNAi molecule or RNAi agent, or a pharmaceutical composition thereof, described in any one of paragraphs 1 to 28, to a patient who requires increased catabolism of high-triglyceride lipoproteins. 40. A method for treating a patient's liver disease that would benefit from reducing the expression level of ANGPTL8, comprising administering an RNAi molecule or RNAi agent, or a pharmaceutical composition thereof, described in any one of paragraphs 1 to 28, to a patient who is in need of treatment for a liver disease.

[0140] Sequence List Sequence ID 1 Homo sapiens angiopoietin-like 8 (ANGPTL8) NCBI reference sequence: NM_018687.7 ATACCTTAGA CCCTCAGTCA TGCCAGTGCC TGCTCTGTGC CTGCTCTGGG CCCTGGCAAT GGTGACCCGG CCTGCCTCAG CGGCCCCCAT GGGCGGCCCA GAACTGGCAC AGCATGAGGA GCTGACCCTG CTCTTCCATG GGACCCTGCA GCTGGGCCAG GCCCTCAACG GTGTGTACAG GACCACGGAG GGACGGCTGA CAAAGGCCAG GAACAGCCTG GGTCTCTATG GCCGCACAAT AGAACTCCTG GGGCAGGAGG TCAGCCGGGG CCGGGATGCA GCCCAGGAAC TTCGGGCAAG CCTGTTGGAG ACTCAGATGG AGGAGATAT TCTGCAGCTG CAGGCAGAGG CCACAGCTGA GGTGCTGGGG GAGGTGGCCC AGGCACAGAA GGTGCTACGG GACAGCGTGC AGCGGCTAGA AGTCCAGCTG AGGAGCGCCT GGCTGGGCCC TGCCTACCGA GAATTTGAGG TCTTAAAGGC TCACGCTGAC AAGCAGAGCC ACATCCTATG GGCCCTCACA GGCCACGTGC AGCGGCAGAG GCGGGAGATG GTGGCACAGC AGCATCGGCT GCGACAGATC CAGGAGAC TCCACACAGC GGCGCTCCCA GCCTGAATCT GCCTGGATGG AACTGAGGAC CAATCATGCT GCAAGGAACA CTTCCACGCC CCGTGAGGCC CCTGTGCAGG GAGGAGCTGC CTGTTCACTG GGATCAGCCA GGGCGCCGGG CCCCACTTCT GAGCACAGAG CAGAGACAGA CGCAGGCGGG GACAAAAGGCA GAGAGAGTAG CCCCATTGGG GAAGGGTGGA GGAAGGACAT GTACCCTTTC ATGCCTACAC ACCCCTCATT AAAGCAGAGT CGTGGCATCT CA

[0141] Sequence ID 2 ANGPTL3 sequence used for expression in mice

[0142] Table 1. Target and antisense sequences for designed siRNAs [Table 15-1]

[0143] (Continued from Table 1) [Table 15-2]

[0144] (Continued from Table 1) [Table 15-3]

[0145] Table 2. [Table 16-1]

[0146] (Continued from Table 2) [Table 16-2]

[0147] Table 3. [Table 17]

[0148] Table 6 [Table 18]

[0149] Table 8 [Table 19]

[0150] Table 11 [Table 20-1]

[0151] (Continued from Table 11) [Table 20-2]

[0152] (Continued from Table 11) [Table 20-3]

Claims

1. Equation I conjugated to R: 【Chemistry 1】 An RNA interference (RNAi) agent comprising a delivery portion shown, R includes a sense strand and an antisense strand. The sense chain and the antisense chain are a. A sense strand comprising the sequence described in Sequence ID No. 124, and an antisense strand comprising the sequence described in Sequence ID No. 231, b. A sense strand comprising the sequence described in Sequence ID No. 126, and an antisense strand comprising the sequence described in Sequence ID No. 233, c. A sense strand consisting of the sequence described in Sequence ID No. 128, and an antisense strand consisting of the sequence described in Sequence ID No. 235, d. A sense strand consisting of the sequence described in Sequence ID No. 130, and an antisense strand consisting of the sequence described in Sequence ID No. 237, e. A sense strand consisting of the sequence described in Sequence ID No. 133, and an antisense strand consisting of the sequence described in Sequence ID No. 240, f. A sense strand consisting of the sequence described in Sequence ID No. 151, and an antisense strand consisting of the sequence described in Sequence ID No. 270, g. A sense strand consisting of the sequence described in Sequence ID No. 154, and an antisense strand consisting of the sequence described in Sequence ID No. 273, h. A sense strand consisting of the sequence described in Sequence ID No. 155, and an antisense strand consisting of the sequence described in Sequence ID No. 274, i. A sense strand consisting of the sequence described in Sequence ID No. 160, and an antisense strand consisting of the sequence described in Sequence ID No. 279, j. A sense strand consisting of the sequence described in Sequence ID No. 163, and an antisense strand consisting of the sequence described in Sequence ID No. 282, k. A sense strand consisting of the sequence described in Sequence ID No. 167, and an antisense strand consisting of the sequence described in Sequence ID No. 286, l. A sense strand consisting of the sequence described in Sequence ID No. 169, and an antisense strand consisting of the sequence described in Sequence ID No. 288, m. A sense strand consisting of the sequence described in Sequence ID No. 177, and an antisense strand consisting of the sequence described in Sequence ID No. 292, n. A sense strand consisting of the sequence described in Sequence ID No. 183, and an antisense strand consisting of the sequence described in Sequence ID No. 298, o. A sense strand consisting of the sequence described in Sequence ID No. 193, and an antisense strand consisting of the sequence described in Sequence ID No. 308, p. A sense strand consisting of the sequence described in Sequence ID No. 195, and an antisense strand consisting of the sequence described in Sequence ID No. 310, q. A sense strand consisting of the sequence described in Sequence ID No. 200, and an antisense strand consisting of the sequence described in Sequence ID No. 336, r. A sense strand consisting of the sequence described in Sequence ID No. 217, and an antisense strand consisting of the sequence described in Sequence ID No. 347, s. A sense strand consisting of the sequence described in Sequence ID No. 218, and an antisense strand consisting of the sequence described in Sequence ID No. 348, t. A sense strand consisting of the sequence described in Sequence ID No. 221, and an antisense strand consisting of the sequence described in Sequence ID No. 351, u. A sense strand consisting of the sequence described in Sequence ID No. 361, and an antisense strand consisting of the sequence described in Sequence ID No. 367, v. A sense strand consisting of the sequence described in Sequence ID No. 362, and an antisense strand consisting of the sequence described in Sequence ID No. 368, w. A sense strand consisting of the sequence described in Sequence ID No. 363, and an antisense strand consisting of the sequence described in Sequence ID No. 369, x. A sense strand consisting of the sequence described in Sequence ID No. 364, and an antisense strand consisting of the sequence described in Sequence ID No. 370, y. A sense strand consisting of the sequence described in Sequence ID No. 365, and an antisense strand consisting of the sequence described in Sequence ID No. 371, z. A sense strand consisting of the sequence described in Sequence ID No. 366, and an antisense strand consisting of the sequence described in Sequence ID No. 372, aa. A sense strand consisting of the sequence described in Sequence ID No. 373, and an antisense strand consisting of the sequence described in Sequence ID No. 389, bb. A sense strand consisting of the sequence described in Sequence ID No. 374, and an antisense strand consisting of the sequence described in Sequence ID No. 390, cc. A sense strand consisting of the sequence described in Sequence ID No. 375, and an antisense strand consisting of the sequence described in Sequence ID No. 391, dd. A sense strand consisting of the sequence described in Sequence ID No. 376, and an antisense strand consisting of the sequence described in Sequence ID No. 392, ee. A sense strand consisting of the sequence described in Sequence ID No. 377, and an antisense strand consisting of the sequence described in Sequence ID No. 393, ff. A sense strand consisting of the sequence described in Sequence ID No. 378, and an antisense strand consisting of the sequence described in Sequence ID No. 394, gg. A sense strand consisting of the sequence described in Sequence ID No. 379, and an antisense strand consisting of the sequence described in Sequence ID No. 395, hh. A sense strand consisting of the sequence described in Sequence ID No. 380, and an antisense strand consisting of the sequence described in Sequence ID No.

396. ii. A sense strand consisting of the sequence described in Sequence ID No. 381, and an antisense strand consisting of the sequence described in Sequence ID No.

397. jj. A sense strand consisting of the sequence described in Sequence ID No. 382, ​​and an antisense strand consisting of the sequence described in Sequence ID No. 398, kk. A sense strand consisting of the sequence described in Sequence ID No. 383, and an antisense strand consisting of the sequence described in Sequence ID No. 399, ll. A sense strand consisting of the sequence described in Sequence ID No. 384, and an antisense strand consisting of the sequence described in Sequence ID No. 400, mm. A sense strand consisting of the sequence described in Sequence ID No. 385, and an antisense strand consisting of the sequence described in Sequence ID No. 401, nn. A sense strand consisting of the sequence described in Sequence ID No. 386, and an antisense strand consisting of the sequence described in Sequence ID No. 402, oo. A sense strand consisting of the sequence described in Sequence ID No. 387, and an antisense strand consisting of the sequence described in Sequence ID No. 403, and pp. Sense strand consisting of the sequence described in Sequence ID No. 388, and antisense strand consisting of the sequence described in Sequence ID No.

404. An RNAi agent, which is a pair of oligonucleotide sequences selected from the group consisting of the following.

2. The sense chain and the antisense chain are a. A sense strand consisting of the sequence described in Sequence ID No. 361, and an antisense strand consisting of the sequence described in Sequence ID No. 367, b. A sense strand consisting of the sequence described in Sequence ID No. 362, and an antisense strand consisting of the sequence described in Sequence ID No. 368, c. A sense strand consisting of the sequence described in Sequence ID No. 363, and an antisense strand consisting of the sequence described in Sequence ID No. 369, d. A sense strand consisting of the sequence described in Sequence ID No. 364, and an antisense strand consisting of the sequence described in Sequence ID No. 370, and e. The RNAi agent according to claim 1, wherein the RNAi agent is a pair of oligonucleotide sequences selected from the group consisting of a sense strand consisting of the sequence described in Sequence ID No. 365 and an antisense strand consisting of the sequence described in Sequence ID No.

371.

3. The RNAi agent according to claim 1, wherein R is conjugated to formula I via a linker.

4. The RNAi agent according to claim 3, wherein R is conjugated to formula I via a linker, and the linker includes a linker of formula II having linkage points A and B, or the linker includes a linker of formula III having linkage points C and D, where linkage point A or C is conjugated to formula I, and linkage point B or D is conjugated to a phosphate group conjugated to R. 【Chemistry 2】

5. The RNAi agent according to claim 1, wherein R is conjugated to formula I via a linker, and the linker is a linker comprising formula III having linkage points C and D, where linkage point C is conjugated to formula I, and linkage point D is conjugated to a phosphate group conjugated to R. 【Transformation 3】

6. The sense chain and the antisense chain are a. A sense strand consisting of the sequence described in Sequence ID No. 361, and an antisense strand consisting of the sequence described in Sequence ID No. 367, and b. The RNAi agent according to claim 1, wherein the RNAi agent is a pair of oligonucleotide sequences selected from the group consisting of a sense strand consisting of the sequence described in Sequence ID No. 365 and an antisense strand consisting of the sequence described in Sequence ID No.

371.

7. The RNAi agent according to claim 1, wherein the RNAi agent can reduce the expression of the ANGPTL8 gene in hepatocytes.

8. A pharmaceutical composition comprising an RNAi agent according to any one of claims 1 to 7, for use in therapeutic purposes.

9. A pharmaceutical composition comprising an RNAi agent according to any one of claims 1 to 7, for use in the treatment of dyslipidemia.

10. A pharmaceutical composition comprising an RNAi agent according to any one of claims 1 to 7 and one or more pharmaceutically acceptable excipients.

11. Use of an RNAi agent according to any one of claims 1 to 7 for the manufacture of a therapeutic agent for dyslipidemia.

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