Compositions and methods for inhibiting angptl3 expression

Abstract

To provide oligonucleotides that inhibit the expression of angiopoietin-like protein-3 (ANGPTL3), as well as compositions comprising the oligonucleotides and uses thereof, particularly for the treatment of diseases, disorders, and / or conditions associated with the expression of ANGPTL3.SOLUTION: Provided is an RNAi oligonucleotide for reducing expression of ANGPTL3, comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the antisense strand has a region of complementarity to a target sequence of ANGPTL3 comprising a specific sequence, and wherein the region of complementarity is at least 15 contiguous nucleotides in length.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 991,335, filed March 18, 2020, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE DISCLOSURE The present disclosure relates to oligonucleotides that inhibit the expression of angiopoietin-like protein 3 (ANGPTL3) and uses thereof, particularly in connection with the treatment of diseases, disorders and / or conditions associated with the expression of ANGPTL3.

[0003] Sequence Listing Reference This disclosure is filed in electronic format along with a Sequence Listing, which is provided as a file with the filename "400930_182359_SL.txt", created on March 18, 2021, and 371 kilobytes in size. The information in this electronic Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0004] Disorders of lipid metabolism can result in elevated serum lipid levels, such as triglycerides and / or cholesterol. Elevated serum lipids are strongly associated with hypertension, cardiovascular disease, diabetes, and other pathological conditions. Despite advances in treatment, there remains a significant and unmet medical need for therapies to treat cardiovascular and metabolic diseases.

[0005] Hypertriglyceridemia is a lipid metabolism disorder characterized by abnormally elevated blood triglyceride levels (e.g., >150 mg / dL). Hypertriglyceridemia is associated with the development of cardiovascular disease (e.g., atherosclerosis). Severe hypertriglyceridemia (e.g., >500 mg / dL) can cause pancreatitis, eruptive xanthomas, or lipemia retinalis. In some cases, extremely high levels of chylomicrons can cause chylomicronemia syndrome, which is characterized by recurrent abdominal pain, nausea, vomiting, and pancreatitis (Pejic & Lee (2006) J. Am. Board. Fam. Med. 19:310-316). Hyperlipidemia is another lipid metabolism disorder characterized by elevated levels of any one or all lipids and / or lipoproteins in the blood.

[0006] ANGPTL3 is a member of the angiopoietin-like family of secreted proteins that regulate lipid metabolism and are expressed primarily in the liver (Koishi et al. (2002) Nat. Genet. 30:151-157). ANGPTL3 inhibits lipoprotein lipase (LPL), which catalyzes the hydrolysis of triglycerides, and endothelial lipase (EL), which hydrolyzes high-density lipoprotein (HDL) phospholipids. Summary of the Invention

[0007] Aspects of the present disclosure relate to compositions and methods for treating diseases, disorders, and / or conditions associated with ANGPTL3 expression. The present disclosure is based, in part, on the discovery and development of oligonucleotides that selectively inhibit and / or reduce the expression of ANGPTL3.

[0008] In some embodiments, the present disclosure provides an oligonucleotide for reducing expression of ANGPTL3, comprising an antisense strand comprising a sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, and 116.

[0009] In some embodiments, the disclosure provides an oligonucleotide for reducing expression of ANGPTL3, comprising a sense strand comprising a sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115.

[0010] In some embodiments, the oligonucleotide that reduces the expression of ANGPTL3 comprises an antisense strand 15 to 30 nucleotides in length and a sense strand 15 to 40 nucleotides in length, wherein the antisense strand has a region complementary to a target sequence of ANGPTL3 set forth in any one of SEQ ID NOs: 125, 126, 127, 118, 119, 120, 121, 122, 123, 124, and 117, and the complementary region is at least 15 consecutive nucleotides in length.

[0011] In some embodiments, the antisense strand is 19 to 27 nucleotides in length or 21 to 27 nucleotides in length, hi some embodiments, the antisense strand is 22 nucleotides in length.

[0012] In some embodiments, the sense strand is 19 to 40 nucleotides in length, hi some embodiments, the sense strand is 36 nucleotides in length.

[0013] In some embodiments, the oligonucleotide that reduces the expression of ANGPTL3 has a duplex region that is at least 19 nucleotides or at least 21 nucleotides in length. In some embodiments, the duplex region is 20 nucleotides in length.

[0014] In some embodiments, the region of complementarity to ANGPTL3 is at least 19 contiguous nucleotides in length, or at least 21 contiguous nucleotides in length.

[0015] In some embodiments, the oligonucleotide that reduces ANGPTL3 expression comprises a stem-loop at the 3' end of the sense strand, shown as S1-L-S2, where S1 is complementary to S2 and L forms a loop of 3 to 5 nucleotides in length between S1 and S2.

[0016] In some embodiments, the oligonucleotide that reduces the expression of ANGPTL3 comprises an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides in length and has a region complementary to ANGPTL3, and the sense strand comprises a stem-loop at its 3' end shown as S1-L-S2, wherein S1 in S1-L-S2 is complementary to S2 and L forms a loop 3 to 5 nucleotides in length between S1 and S2, and the antisense strand and the sense strand form a duplex structure at least 19 nucleotides in length, but they are not linked by a covalent bond.

[0017] In some embodiments, the loop L is a tetraloop. In some embodiments, L is 4 nucleotides in length. In some embodiments, L comprises the sequence GAAA.

[0018] In some embodiments, the oligonucleotide that reduces expression of ANGPTL3 comprises an antisense strand that is 27 nucleotides in length and a sense strand that is 25 nucleotides in length, hi some embodiments, the oligonucleotide comprises an antisense strand that is 22 nucleotides in length and a sense strand that is 36 nucleotides in length.

[0019] In some embodiments, the oligonucleotide having a duplex region comprises a 3' overhang sequence on the antisense strand, hi some embodiments, the 3' overhang sequence on the antisense strand is 2 nucleotides in length.

[0020] In some embodiments, the oligonucleotide that reduces ANGTPL3 expression comprises an antisense strand and a sense strand, each ranging from 21 to 23 nucleotides in length. In some embodiments, the oligonucleotide has a duplex structure ranging from 19 to 21 nucleotides in length. In some embodiments, the oligonucleotide comprises a 3' overhang sequence of one or more nucleotides in length, the 3' overhang sequence being present in the antisense strand, the sense strand, or the antisense strand and the sense strand. In some embodiments, the 3' overhang sequence is two nucleotides in length, the 3' overhang sequence being present in the antisense strand, the sense strand being 21 nucleotides in length, and the antisense strand being 23 nucleotides in length, and the sense strand and the antisense strand form a duplex of 21 nucleotides in length.

[0021] In some embodiments, the oligonucleotide that reduces the expression of ANGTPL3 comprises at least one modified nucleotide. In some embodiments, the modified nucleotide comprises a 2' modification. In some embodiments, all nucleotides of the oligonucleotide are modified, for example, with a 2' modification.

[0022] In some embodiments, the oligonucleotide that reduces the expression of ANGPTL3 comprises at least one modified internucleotide linkage, preferably a phosphorothioate linkage.

[0023] In some embodiments, the 4' carbon of the sugar of the 5' nucleotide of the antisense strand comprises a phosphate analog, for example, oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

[0024] In some embodiments, at least one nucleotide of the oligonucleotide is bound to one or more targeting ligands, such as carbohydrates, amino sugars, cholesterol, polypeptides, or lipids. In some embodiments, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the GalNAc moiety comprises a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

[0025] In some embodiments, the targeting ligand is attached to one or more nucleotides of the stem-loop L. In some embodiments, up to four nucleotides of the stem-loop L are each attached to a monovalent GalNAc moiety.

[0026] In some embodiments, the oligonucleotide that reduces the expression of ANGPTL3 is an RNAi oligonucleotide.

[0027] In another aspect, the present disclosure provides a method for reducing the expression of ANGPTL3 in cells, cell populations, or subjects by administering the oligonucleotides described herein.In some embodiments, the method for reducing the expression of ANGPTL3 in cells, cell populations, or subjects comprises contacting cells or cell populations with an effective amount of the oligonucleotide described herein or a pharmaceutical composition thereof, or administering it to the subject.In some embodiments, the method for reducing ANGPTL3 expression comprises reducing the amount or level of ANGPTL3 mRNA, the amount or level of ANGPTL3 protein, or both.

[0028] In some embodiments, the present disclosure provides a method of reducing the amount or level of triglycerides (TG) in a subject by administering to the subject an effective amount of an oligonucleotide herein or a pharmaceutical composition thereof.

[0029] In some embodiments, the present disclosure provides a method of reducing the amount or level of cholesterol in a subject by administering to the subject an effective amount of an oligonucleotide herein or a pharmaceutical composition thereof.

[0030] In some embodiments, a subject treated with the oligonucleotide herein has a disease, disorder, or condition associated with expression of ANGPTL3. In some embodiments, a method for treating a subject having a disease, disorder, or condition associated with expression of ANGPTL3 comprises administering a therapeutically effective amount of the oligonucleotide herein or a pharmaceutical composition thereof to a subject in need thereof, thereby treating the subject.

[0031] In some embodiments, the oligonucleotides herein for administration comprise a sense strand 15 to 50 nucleotides in length and an antisense strand 15 to 30 nucleotides in length, wherein the sense strand forms a duplex region with the antisense strand, and the sense strand is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 240, 241, 242, 243, 244, 245, 246, 247, 249, 251 05, 107, 109, 111, 113, and 115, and the antisense strand comprises a complementary sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, and 116, or a pharmaceutical composition thereof, thereby treating a subject.

[0032] In some embodiments, a method for treating a subject having a disease, disorder, or condition associated with expression of ANGPTL3 comprises administering to a subject in need thereof a therapeutically effective amount of an oligonucleotide comprising a pair of sense and antisense strands selected from a row of Table 5, or a pharmaceutical composition thereof, thereby treating the subject.

[0033] In some embodiments, the disease, disorder, or condition associated with ANGPTL3 expression is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, dyslipidemia and / or cholesterol metabolism disorders, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), homozygous and heterozygous familial hypercholesterolemia, and statin-resistant hypercholesterolemia.

[0034] In some embodiments, the disease, disorder, or condition associated with expression of ANGPTL3 is cardiovascular disease, type II diabetes, hypertriglyceridemia, NASH, obesity, or a combination thereof.

[0035] In some embodiments, the oligonucleotide or pharmaceutical composition thereof is administered in combination with a second therapeutic agent or composition thereof.

[0036] In a further aspect, the present disclosure provides the use of any of the oligonucleotides of the present disclosure, or a pharmaceutical composition thereof, in the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with expression of ANGPTL3.

[0037] In some embodiments, the oligonucleotide of the present disclosure or the pharmaceutical composition of the present disclosure is for use or adapted for use in treating a disease, disorder, or condition associated with expression of ANGPTL3.

[0038] In a further aspect, the oligonucleotide of the present disclosure is provided in the form of a kit for treating diseases, disorders or conditions associated with the expression of ANGPTL3.In some embodiments, the kit comprises the oligonucleotide of the present disclosure and a pharmaceutically acceptable carrier.In some embodiments, the kit further comprises a package insert containing instructions for administration to a subject with diseases, disorders or conditions associated with the expression of ANGPTL3.

[0039] In some embodiments of the kit or use, the disease, disorder, or condition associated with ANGPTL3 expression is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, dyslipidemia and / or cholesterol metabolism disorders, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, NASH, NAFLD, homozygous and heterozygous familial hypercholesterolemia, and statin-resistant hypercholesterolemia.

[0040] In some embodiments of the kit or use, the disease, disorder, or condition associated with expression of ANGPTL3 is cardiovascular disease, type II diabetes, hypertriglyceridemia, NASH, obesity, or a combination thereof. [Brief explanation of the drawings]

[0041] [Figure 1-1] 1 provides a graph showing the percent (%) of ANGPTL3 mRNA in HuH-7 cells transfected with the indicated DsiRNAs relative to the percent of ANGPTL3 mRNA control mock-treated cells. [Figure 1-2] Same as above [Figure 2-1] 1 provides a graph showing the percent (%) of ANGPTL3 mRNA in HuH-7 cells transfected with the indicated DsiRNAs relative to the percent of ANGPTL3 mRNA control mock-treated cells. [Figure 2-2] Same as above [Figure 2-3] Same as above [Figure 3] 1 provides a schematic diagram showing the structure and chemical modification pattern of generic GalNAc-linked ANGPTL3 oligonucleotides. [Figure 4-1] A graph is provided showing the percent (%) of ANGPTL3 mRNA in liver samples obtained from mice treated with the indicated GalNAc-linked ANGPTL3 oligonucleotide relative to ANGPTL3 mRNA in liver samples obtained from mice treated with phosphate-buffered saline (PBS). [Figure 4-2] Same as above [Figure 4-3] Same as above [Figure 5A] 1 provides a graph depicting the percent (%) of ANGPTL3 mRNA in liver samples from non-human primates (NHPs) treated with the indicated GalNAc-linked ANGPTL3 oligonucleotides relative to PBS-treated NHPs at 28 days post-treatment. [Figure 5B]1 provides a graph depicting the percent (%) of ANGPTL3 mRNA in liver samples from non-human primates (NHPs) treated with the indicated GalNAc-linked ANGPTL3 oligonucleotides relative to PBS-treated NHPs at 56 days post-treatment. [Figure 5C] 1 provides a graph depicting the percent (%) of ANGPTL3 mRNA in liver samples from non-human primates (NHPs) treated with the indicated GalNAc-linked ANGPTL3 oligonucleotides relative to PBS-treated NHPs at 84 days post-treatment. [Figure 6] 1 provides a graph showing the average percent (%) of ANGPTL3 mRNA in liver samples from NHPs treated with the indicated GalNAc-linked ANGPTL3 oligonucleotides relative to ANGPTL3 mRNA in liver samples from NHPs treated with PBS over time. [Figure 7] 1 provides a graph showing the average percent (%) of ANGPTL3 protein in serum from NHPs treated with the indicated GalNAc-linked ANGPTL3 oligonucleotides relative to ANGPTL3 protein in serum from NHPs treated with PBS over time. DETAILED DESCRIPTION OF THE INVENTION

[0042] I. Definition

[0043] As used herein, "about" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In certain embodiments, "about" refers to a range of values ​​that is included within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (above or below) of the stated reference value, unless otherwise stated or otherwise apparent from the context (except where such number may exceed 100% of the possible values).

[0044] As used herein, "administer," "administering," "administration," and the like refer to providing a substance (e.g., an oligonucleotide) to a subject in a pharmacologically useful form (e.g., to treat a condition in the subject).

[0045] As used herein, "ANGPTL3" refers to angiopoietin-like protein 3, a member of the angiopoietin-like family of secreted polypeptides. ANGPTL3 is primarily expressed in the mammalian liver, and the ANGPTL3 protein has the characteristic structure of angiopoietin, including a signal peptide, an N-terminal coiled-coil domain, and a C-terminal fibrinogen (FBN)-like domain. For purposes of this disclosure, "ANGPTL3" refers to ANGPTL3 from any vertebrate or mammal, including, but not limited to, human, mouse, primate, monkey, bovine, chicken, rodent, rat, pig, sheep, and guinea pig. ANGPTL3 refers to fragments and variants of native ANGPTL3 that maintain at least one in vivo or in vitro activity of native ANGPTL3. ANGPTL3 encompasses the full-length, unprocessed precursor form of ANGPTL3, as well as mature forms resulting from post-translational cleavage of the signal peptide and forms resulting from proteolytic processing of the FBN-like domain. An exemplary sequence of human ANGPTL3 mRNA transcript is publicly available (GenBank accession number GI:41327750 (NM_014495.2)) and disclosed herein (SEQ ID NO:128). An exemplary sequence of cynomolgus monkey ANGPTL3 mRNA is publicly available (GenBank accession number GI:102136264 (XM_005543185.2)) and disclosed herein (SEQ ID NO:129). An exemplary sequence of mouse ANGPTL3 mRNA is publicly available (GenBank accession number GI:142388354 (NM_013913.3)) and disclosed herein (SEQ ID NO:130). An exemplary sequence of rat ANGPTL3 is publicly available (GenBank accession number GI:68163568 (NM_001025065.1)) and disclosed herein (SEQ ID NO:131).

[0046] As used herein, "asialoglycoprotein receptor" or "ASGPR" refers to a bipartite C-type lectin formed by a major 48 kDa subunit (ASGPR-1) and a minor 40 kDa subunit (ASGPR-2). ASGPR is primarily expressed on the sinusoidal surface of hepatocytes and plays a primary role in the binding, internalization, and subsequent excretion of circulating glycoproteins containing terminal galactose or GalNAc residues (asialoglycoproteins).

[0047] As used herein, "attenuate," "attenuating," "attenuation," and the like refer to a decrease or effective halt. As a non-limiting example, one or more of the treatments herein can reduce or effectively halt the onset or progression of dyslipidemia, hypertriglyceridemia, and hyperlipidemia in a subject. This attenuation can be exemplified, for example, by a decrease in one or more aspects of dyslipidemia, hypertriglyceridemia, and hyperlipidemia (e.g., symptoms, tissue characteristics, cellular inflammatory or immune activity, etc.), or, if expected to be present, by undetectable progression (worsening) of one or more aspects of dyslipidemia, hypertriglyceridemia, and hyperlipidemia in the subject, or by undetectable aspects of dyslipidemia, hypertriglyceridemia, and hyperlipidemia in the subject.

[0048] As used herein, "complementary" refers to a structural relationship between two nucleotides (e.g., in two opposing nucleic acids or in opposing regions of a single nucleic acid strand) that allows the two nucleotides to base pair with each other. For example, purine nucleotides in one nucleic acid that are complementary to pyrimidine nucleotides in an opposing nucleic acid can base pair together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides may base pair in a Watson-Crick manner or any other manner that allows the formation of a stable duplex. In some embodiments, two nucleic acids may have a region of multiple nucleotides that are complementary to each other to form a complementary region, as described herein.

[0049] As used herein, "deoxyribonucleotide" refers to a nucleotide that, compared to a ribonucleotide, has a hydrogen atom instead of a hydroxyl atom at the 2' position of its pentose sugar. Modified deoxyribonucleotides are deoxyribonucleotides that have one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions of the sugar, phosphate group, or base.

[0050] As used herein, a "double-stranded oligonucleotide" or "ds oligonucleotide" refers to an oligonucleotide that is substantially in duplex form. In some embodiments, the complementary base pairing in the duplex region(s) of a ds oligonucleotide is formed between antiparallel sequences of nucleotides in covalently separate nucleic acid strands. In some embodiments, the complementary base pairing in the duplex region(s) of a ds oligonucleotide is formed between antiparallel sequences of nucleotides in covalently linked nucleic acid strands. In some embodiments, the complementary base pairing in the duplex region(s) of a ds oligonucleotide is formed in a single nucleic acid strand that is folded (e.g., by a hairpin) to provide a complementary antiparallel sequence of base-pairing nucleotides with each other. In some embodiments, a ds oligonucleotide comprises two covalently separate nucleic acid strands that are fully duplex with each other. However, in some embodiments, a ds oligonucleotide comprises two covalently separate nucleic acid strands that are partially duplex (e.g., have overhangs at one or both ends). In some embodiments, the ds oligonucleotides comprise antiparallel sequences of nucleotides that are partially complementary and therefore may have one or more mismatches, which may include internal or terminal mismatches.

[0051] As used herein, a "duplex" with respect to a nucleic acid (eg, an oligonucleotide) refers to the structure formed by complementary base-pairing of two antiparallel sequences of nucleotides.

[0052] As used herein, "excipient" refers to a non-therapeutic agent that may be included in the composition to, for example, provide or contribute a desired consistency or stabilizing effect.

[0053] As used herein, "hepatocyte" or "hepatocytes" refers to cells of the liver parenchyma. These cells account for approximately 70% to 85% of liver mass and produce serum albumin, FBN, and prothrombin group clotting factors (excluding factors 3 and 4). Markers of hepatocyte lineage cells include, but are not limited to, transthyretin (Ttr), glutamine synthetase (Glul), hepatocyte nuclear factor 1a (Hnf1a), and hepatocyte nuclear factor 4a (Hnf4a). Markers of mature hepatocytes include, but are not limited to, cytochrome P450 (Cyp3a11), fumarylacetoacetate hydrolase (Fah), glucose 6-phosphate (G6p), albumin (Alb), and OC2-2F8. See, e.g., Huch et al. (2013) Nature. 494:247-250.

[0054] As used herein, "hepatotoxic agent" refers to a chemical compound, virus, or other substance that is itself toxic to the liver or that can be processed to form metabolites that are toxic to the liver. Hepatotoxic agents include, but are not limited to, carbon tetrachloride (CCl4), acetaminophen (paracetamol), vinyl chloride, arsenic, chloroform, and nonsteroidal anti-inflammatory drugs (such as aspirin and phenylbutazone).

[0055] As used herein, a "labile linker" refers to a linker that can be cleaved (e.g., by acidic pH). A "fairly stable linker" refers to a linker that cannot be cleaved.

[0056] As used herein, "liver inflammation" or "hepatitis" refers to a physical condition in which the liver becomes enlarged, impaired, and / or distressed, particularly as a result of injury or infection, as may be caused by exposure to hepatotoxic agents. Symptoms may include jaundice (yellowing of the skin or eyes), fatigue, weakness, nausea, vomiting, loss of appetite, and weight loss. If left untreated, liver inflammation may progress to fibrosis, cirrhosis, liver failure, or liver cancer.

[0057] As used herein, "liver fibrosis" or "liver fibrosis" refers to the excessive accumulation of extracellular matrix proteins in the liver, which may include collagen (I, III, and IV), FBN, urin, elastin, laminin, hyaluronan, and proteoglycans, resulting from inflammation and liver cell death. If left untreated, liver fibrosis can progress to cirrhosis, liver failure, or liver cancer.

[0058] As used herein, a "loop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) flanked by two antiparallel regions of nucleic acid that are sufficiently complementary to each other such that under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cell), the two antiparallel regions flanking the unpaired region hybridize to form a duplex (called a "stem").

[0059] As used herein, the term "modified internucleotide linkage" refers to an internucleotide linkage that has one or more chemical modifications compared to a reference internucleotide linkage containing a phosphodiester bond. In some embodiments, the modified nucleotide is a non-natural linkage. Typically, the modified internucleotide linkage imparts one or more desirable properties to the nucleic acid in which the modified internucleotide linkage is present. For example, the modified nucleotide can improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0060] As used herein, a "modified nucleotide" refers to a nucleotide that has one or more chemical modifications compared to a corresponding reference nucleotide selected from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymidine deoxyribonucleotides. In some embodiments, the modified nucleotide is a non-natural nucleotide. In some embodiments, the modified nucleotide has one or more chemical modifications in its sugar, nucleobase, and / or phosphate group. In some embodiments, the modified nucleotide has one or more chemical moieties attached to the corresponding reference nucleotide. Typically, the modified nucleotide confers one or more desirable properties to the nucleic acid in which the modified nucleotide is present. For example, the modified nucleotide can improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0061] As used herein, "nicked tetraloop structure" refers to the structure of an RNAi oligonucleotide characterized by separate sense (passenger) and antisense (guide) strands, where the sense strand has a region complementary to the antisense strand, and at least one of the strands generally has a tetraloop configured such that the sense strand stabilizes an adjacent stem region formed within the at least one strand.

[0062] As used herein, "oligonucleotide" refers to a short nucleic acid (e.g., less than about 100 oligonucleotides in length). An oligonucleotide may be single-stranded (ss) or ds. An oligonucleotide may or may not have a double-stranded region. As one set of non-limiting examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a dicer substrate interfering RNA (dsiRNA), an antisense oligonucleotide, a short siRNA, or a ss siRNA. In some embodiments, the ds oligonucleotide is an RNAi oligonucleotide.

[0063] As used herein, "overhang" refers to terminal non-base-paired nucleotide(s) resulting from the extension of one strand or region beyond the end of the complementary strand with which it forms a duplex. In some embodiments, the overhang comprises one or more non-base-paired nucleotides extending from the duplex region at the 5'-end or 3'-end of the ds oligonucleotide. In certain embodiments, the overhang is a 3' or 5' overhang on the antisense or sense strand of the ds oligonucleotide.

[0064] As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, a phosphate analog is placed on the 5'-terminal nucleotide of an oligonucleotide in place of the 5'-phosphate, which is often susceptible to enzymatic removal. In some embodiments, the 5' phosphate analog comprises a phosphatase-resistant linkage. Phosphate analogs include 5' phosphonates, such as, but not limited to, 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). In some embodiments, an oligonucleotide has a phosphate analog at the 4' carbon of the sugar of the 5'-terminal nucleotide (referred to as a "4'-phosphate analog"). 4'-phosphate analogs include oxymethyl phosphonates or analogs thereof, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., its 4' carbon). See, e.g., U.S. Provisional Patent Application No. 62 / 383,207, filed September 2, 2016, and U.S. Provisional Patent Application No. 62 / 393,401, filed September 12, 2016. Other modifications to the 5' end of oligonucleotides have been developed (see, e.g., International Patent Application No. WO2011 / 133871, U.S. Patent No. 8,927,513, and Prakash et al. (2015) Nucleic Acids Res. 43:2993-3011).

[0065] As used herein, "reducing expression" of a gene (e.g., ANGPTL3) refers to a decrease in the amount or level of an RNA transcript (e.g., ANGPTL3 mRNA) or protein encoded by that gene, and / or a decrease in the amount or level of activity of that gene in a cell, cell population, sample, or subject, compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject). For example, contacting a cell with an oligonucleotide herein (e.g., an oligonucleotide comprising an antisense strand having a nucleotide sequence complementary to a nucleotide sequence comprising ANGPTL3 mRNA) can result in a decrease in the amount or level of ANGPTL3 mRNA, protein, and / or activity (e.g., via degradation of ANGPTL3 mRNA by the RNAi pathway), compared to a cell not treated with the ds oligonucleotide. Similarly, as used herein, "reducing expression" refers to an act that results in a decrease in expression of a gene (e.g., ANGPTL3). As used herein, "reduced ANGPTL3 expression" refers to a decrease in the amount or level of ANGPTL3 mRNA, ANGPTL3 protein, and / or ANGPTL3 activity in a cell, cell population, sample, or subject compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject).

[0066] As used herein, a "complementary region" refers to a nucleotide sequence of a nucleic acid (e.g., a ds oligonucleotide) that is sufficiently complementary to an antiparallel sequence of nucleotides to allow hybridization between the two nucleotide sequences under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cell, etc.). In some embodiments, the oligonucleotides herein comprise a target sequence having a region complementary to an mRNA target sequence.

[0067] As used herein, "ribonucleotide" refers to a nucleotide having as its pentose sugar a ribose with a hydroxyl group at the 2' position. A modified ribonucleotide is a ribonucleotide with one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions of the ribose, phosphate group, or base.

[0068] As used herein, "RNAi oligonucleotide" refers to either (a) a ds oligonucleotide having a sense strand (passenger) and an antisense strand (guide), where the antisense strand or a portion of the antisense strand is used to cleave the target mRNA by Argonaute 2 (Ago2) endonuclease, or (b) a ss oligonucleotide having a single antisense strand, where the antisense strand (or a portion of the antisense strand) is used to cleave the target mRNA by Ago2 endonuclease.

[0069] As used herein, a "strand" refers to a continuous sequence of nucleotides linked together via internucleotide bonds (e.g., phosphodiester or phosphorothioate bonds). In some embodiments, the strand has two free ends (e.g., a 5' end and a 3' end).

[0070] As used herein, "subject" refers to any mammal, including mice, rabbits, and humans. In one embodiment, the subject is a human or NHP. Furthermore, "individual" or "patient" may be used interchangeably with "subject."

[0071] As used herein, "synthetic" refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine (e.g., a solid-state nucleic acid synthesizer)) or that is not otherwise derived from a natural source (e.g., a cell or organism) that normally produces that molecule.

[0072] As used herein, a "targeting ligand" refers to a molecule (e.g., a carbohydrate, amino sugar, cholesterol, polypeptide, or lipid) that selectively binds to a cognate molecule (e.g., a receptor) in a tissue or cell of interest and can be attached to another substance to target the other substance to the tissue or cell of interest. For example, in some embodiments, a targeting ligand may be attached to an oligonucleotide to target the oligonucleotide to a specific tissue or cell of interest. In some embodiments, a targeting ligand selectively binds to a cell surface receptor. Thus, in some embodiments, a targeting ligand, when attached to an oligonucleotide, selectively binds to a receptor expressed on the surface of the cell, thereby facilitating delivery of the oligonucleotide into a specific cell by endosomal internalization of the complex comprising the oligonucleotide, targeting ligand, and receptor by the cell. In some embodiments, the targeting ligand is attached to the oligonucleotide via a linker that is cleaved after or during cellular internalization, such that the oligonucleotide is released from the targeting ligand within the cell.

[0073] As used herein, "tetraloop" refers to a loop that increases the stability of an adjacent duplex formed by hybridization of a flanking sequence of nucleotides. The increased stability is determined by the average T of adjacent stem duplexes expected from a set of loops of equivalent length consisting of randomly selected sequences of nucleotides. m The melting temperature (T m For example, a tetraloop can be detected as an increase in T of at least about 50°C, at least about 55°C, at least about 56°C, at least about 58°C, at least about 60°C, at least about 65°C, or at least about 75°C in 10 mM NaHPO4 in a hairpin containing a duplex of at least 2 base pairs (bp) in length. mIn some embodiments, the tetraloop can stabilize adjacent bp of the stem duplex through stacking interactions. In addition, interactions between nucleotides of the tetraloop can include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al. (1990) Nature 346:680-682; Heus & Pardi (1991) Science. 253:191-194). In some embodiments, the tetraloop comprises or consists of 3 to 6 nucleotides, typically 4 to 5 nucleotides. In certain embodiments, the tetraloop comprises or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., may or may not be attached to a targeting moiety). In one embodiment, the tetraloop consists of 4 nucleotides. Any nucleotide can be used in the tetraloop, and the standard IUPAC-IUB symbols for such nucleotides can be used as described in Cornish-Bowden (1985) Nucleic Acids Res. 13:3021-3030. For example, the letter "N" can be used to mean that any base can be at that position, the letter "R" can be used to indicate that A (adenine) or G (guanine) can be at that position, and "B" can be used to indicate that C (cytosine), G (guanine), or T (thymine) can be at that position. Examples of tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al. (1990) Proc. Natl. Acad. Sci. USA 87:8467-8471; Antao et al. (1991) Nucleic Acids Res. 19:5901-5905).Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA)), the d(GNRA) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). See, e.g., Nakano et al. (2002) Biochem. 41:4281-14292; Shinji et al. (2000) Nippon Kagakkai Koen Yokoshu 78:731. In some embodiments, the tetraloop is comprised within a nicked tetraloop structure.

[0074] As used herein, "treat" or "treating" refers to the act of providing care to a subject in need thereof, for example, by administering a therapeutic agent (e.g., an oligonucleotide herein) to the subject to improve the health and / or well-being of the subject with respect to an existing condition (e.g., disease, disorder) or to prevent or reduce the likelihood of a condition occurring. In some embodiments, treatment involves reducing the frequency or severity of at least one sign, symptom, or contributing factor of a condition (e.g., disease, disorder) experienced by the subject.

[0075] II. Oligonucleotide inhibitors of ANGPTL3 expression

[0076] The present disclosure provides, inter alia, oligonucleotides that inhibit ANGPTL3 expression. In some embodiments, the oligonucleotides that inhibit ANGPTL3 expression herein target ANGPTL3 mRNA.

[0077] i.ANGPTL3 target sequence

[0078] In some embodiments, the oligonucleotide targets a target sequence containing ANGPTL3 mRNA. In some embodiments, the oligonucleotide, or a portion, fragment, or strand of the oligonucleotide (e.g., the antisense strand or guide strand of a ds oligonucleotide) inhibits ANGPTL3 expression by binding or annealing to a target sequence containing ANGPTL3 mRNA. In some embodiments, the oligonucleotide targets the ANGPTL3 target sequence to inhibit ANGPTL3 expression in vivo. In some embodiments, the amount or degree of inhibition of ANGPTL3 expression by an oligonucleotide targeting an ANGPTL3 target sequence correlates with the efficacy of the oligonucleotide. In some embodiments, the amount or degree of inhibition of ANGPTL3 expression by an oligonucleotide targeting an ANGPTL3 target sequence correlates with the amount or degree of therapeutic effect in a subject or patient with a disease, disorder, or condition associated with ANGPTL3 expression who is treated with the oligonucleotide.

[0079] The nucleotide sequences of mRNAs encoding ANGPTL3, including mRNAs from several different species (e.g., human, cynomolgus monkey, mouse, and rat; see, e.g., Example 1), were examined, and in vitro and in vivo testing (see, e.g., Examples 2 and 3) revealed that certain nucleotide sequences of ANGPTL3 mRNA are more susceptible to oligonucleotide-based inhibition than other nucleotide sequences and are therefore useful as target sequences for the oligonucleotides described herein. In some embodiments, the sense strand of an oligonucleotide (e.g., a ds oligonucleotide) described herein (e.g., Table 5) comprises an ANGPTL3 target sequence. In some embodiments, a portion or region of the sense strand of a ds oligonucleotide described herein (e.g., Table 5) comprises an ANGPTL3 target sequence. In some embodiments, the ANGPTL3 target sequence comprises or consists of the sequence of any one of SEQ ID NOs: 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, and 127.

[0080] ii.ANGPTL3 - Target-directed allocation

[0081] In some embodiments, the oligonucleotides herein have a region complementary to ANGPTL3 mRNA (e.g., within the target sequence of ANGPTL3 mRNA) to target the mRNA in cells and inhibit its expression. In some embodiments, the oligonucleotides herein comprise an ANGPTL3 targeting sequence (e.g., the antisense strand or guide strand of a ds oligonucleotide) having a complementary region that binds or anneals to the ANGPTL3 target sequence by complementary (Watson-Crick) base pairing. The targeting sequence or complementary region is generally of a length and base composition suitable to enable the oligonucleotide (or strand thereof) to bind or anneal to ANGPTL3 mRNA to inhibit its expression. In some embodiments, for example, the targeting sequence or complementary region is at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 nucleotides in length. In some embodiments, the targeting sequence or complementary region is about 12 to about 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides in length. In some embodiments, the targeting sequence or complementary region is about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length. In some embodiments, the targeting sequence or complementary region is 18 nucleotides in length. In some embodiments, the targeting sequence or complementary region is 19 nucleotides in length.In some embodiments, the targeting sequence or complementary region is 20 nucleotides in length. In some embodiments, the targeting sequence or complementary region is 21 nucleotides in length. In some embodiments, the targeting sequence or complementary region is 22 nucleotides in length. In some embodiments, the targeting sequence or complementary region is 23 nucleotides in length. In some embodiments, the targeting sequence or complementary region is 24 nucleotides in length.

[0082] In some embodiments, the oligonucleotide herein comprises a targeting sequence or a complementary region (e.g., the antisense strand or guide strand of a double-stranded oligonucleotide) that is completely complementary to the ANGPTL3 target sequence. In some embodiments, the targeting sequence or the complementary region is completely complementary to the ANGPTL3 target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity that is fully complementary to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity that is partially complementary to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115.

[0083] In some embodiments, the oligonucleotides herein comprise a targeting sequence or a complementary region complementary to a contiguous sequence of nucleotides comprising ANGPTL3 mRNA, the contiguous sequence of nucleotides being about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 20, 12 to 18, 12 to 16, 14 to 22, 16 to 20, 18 to 20, or 18 to 19 nucleotides in length). In some embodiments, the oligonucleotides herein comprise a targeting sequence or a complementary region complementary to a contiguous sequence of nucleotides comprising ANGPTL3 mRNA, the contiguous sequence of nucleotides being 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous sequence of nucleotides comprising ANGPTL3 mRNA, wherein the contiguous sequence of nucleotides is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115, optionally, the contiguous sequence of nucleotides is 19 nucleotides in length.

[0084] In some embodiments, the targeting sequence or complementary region of the oligonucleotide complementary to consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115 spans the entire length of the antisense strand. In some embodiments, the region of complementarity of the oligonucleotide complementary to consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115 spans a portion of the entire length of the antisense strand. In some embodiments, the oligonucleotides herein comprise a region (e.g., on the antisense strand of a ds oligonucleotide) that is at least partially (e.g., completely) complementary to a contiguous stretch of nucleotides spanning 1 to 20 nucleotides of a sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115.

[0085] In some embodiments, the oligonucleotides herein comprise a targeting sequence or complementary region having one or more bp mismatches with the corresponding ANGPTL3 target sequence. In some embodiments, the targeting sequence or complementary region may have up to about one, two, three, four, or five mismatches with the corresponding ANGPTL3 target sequence, provided that the ability of the targeting sequence or complementary region to bind to or anneal with ANGPTL3 mRNA and / or the ability of the oligonucleotide to inhibit ANGPTL3 expression under appropriate hybridization conditions is maintained. Alternatively, the targeting sequence or complementary region may have no more than one, two, three, four, or five mismatches with the corresponding ANGPTL3 target sequence, provided that the ability of the targeting sequence or complementary region to bind to or anneal with ANGPTL3 mRNA and / or the ability of the oligonucleotide to inhibit ANGPTL3 expression under appropriate hybridization conditions is maintained. In some embodiments, the oligonucleotides comprise a targeting sequence or complementary region having one mismatch with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region with two mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region with three mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region with four mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region with five mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide comprises a target sequence or complementary region with multiple mismatches to the corresponding target sequence (e.g., two, three, four, five, or more mismatches), where at least two (e.g., all) of the mismatches are located consecutively (e.g., two, three, four, five, or more consecutive mismatches) or the mismatches are scattered throughout the targeting sequence or complementary region.

[0086] iii. Types of oligonucleotides

[0087] A variety of oligonucleotide types and / or structures are useful for targeting ANGPTL3 in the methods herein, including, but not limited to, RNAi oligonucleotides, antisense oligonucleotides, miRNAs, etc. Any of the oligonucleotide types described herein or elsewhere are contemplated for use as a framework for incorporating the ANGPTL3 targeting sequences herein.

[0088] In some embodiments, the oligonucleotides herein inhibit ANGPTL3 expression by participating in the RNA interference (RNAi) pathway upstream or downstream of Dicer intervention. For example, RNAi oligonucleotides have been developed in which each strand has at least one 3' overhang of 1 to 5 nucleotides and is 19 to 25 nucleotides in size (see, e.g., U.S. Pat. No. 8,372,968). Longer oligonucleotides that are processed by Dicer to generate active RNAi products have also been developed (see, e.g., U.S. Pat. No. 8,883,996). Further research has led to the creation of extended ds oligonucleotides in which at least one end of at least one strand extends beyond the targeting region of the duplex, with one strand containing a thermodynamically stable tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705, and International Patent Application Publication No. WO2010 / 033225). Such structures may contain ss extensions as well as ds extensions (on one or both sides of the molecule).

[0089] In some embodiments, the oligonucleotides herein are involved in the RNAi interference pathway downstream of Dicer intervention (e.g., Dicer cleavage). In some embodiments, the oligonucleotides have an overhang (e.g., 1, 2, or 3 nucleotides in length) at the 3' end of the sense strand. In some embodiments, an oligonucleotide (e.g., siRNA) can comprise a 21-nucleotide guide strand that is antisense to the target RNA and a complementary passenger strand, with both strands annealing to form a 19-bp duplex with a 2-nucleotide overhang at one or both 3' ends. Longer oligonucleotide designs are also possible, including oligonucleotides with a 23-nucleotide guide strand and a 21-nucleotide passenger strand, with a blunt end on the right side of the molecule (3' end of the passenger strand / 5' end of the guide strand) and a 2-nucleotide 3' guide strand overhang on the left side of the molecule (5' end of the passenger strand / 3' end of the guide strand). Such molecules have a 21-bp duplex region. See, for example, U.S. Patent Nos. 9,012,138, 9,012,621, and 9,193,753.

[0090] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, both of which are in the range of about 17 to 26 nucleotides in length (e.g., 17 to 26, 20 to 25, or 21 to 23). In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, both of which are in the range of 19 to 22 nucleotides in length. In some embodiments, the sense strand and the antisense strand are equal in length. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and a 3' overhang is present on either the sense strand or the antisense strand, or on both the sense strand and the antisense strand. In some embodiments, when the oligonucleotides herein comprise a sense strand and an antisense strand, both of which are in the range of 21 to 23 nucleotides in length, the 3' overhang on the sense strand, the antisense strand, or both the sense strand and the antisense strand is 1 or 2 nucleotides in length. In some embodiments, the oligonucleotide has a 22-nucleotide guide strand and a 20-nucleotide passenger strand, with a blunt end on the right side of the molecule (3' end of the passenger strand / 5' end of the guide strand) and a 2-nucleotide 3' guide strand overhang on the left side of the molecule (5' end of the passenger strand / 3' end of the guide strand). Such molecules have a 20 bp duplex region.

[0091] Other oligonucleotide designs for use in the compositions and methods herein include 16-mer siRNAs (see, e.g., NUCLEIC ACIDS IN CHEMISTRY AND BIOLOGY. Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., those with 19 bp or shorter stems; see, e.g., Moore et al. (2010) Methods Mol. Biol. 629:141-158), blunt-ended siRNAs (e.g., those 19 bp in length; see, e.g., Kraynack & Baker (2006) RNA 12:163-176), asymmetric siRNAs (aiRNAs; see, e.g., Sun et al. (2008) Nat. Biotechnol. 26:1379-1382), asymmetric short duplex siRNAs (see, e.g., Chang et al. (2009) Mol. Ther. 17:725-732), forked siRNA (see, e.g., Hohjoh (2004) FEBS Lett. 557:193-198), ss siRNA (Elsner (2012) Nat. Biotechnol. 30:1063), dumbbell-shaped circular siRNA (see, e.g., Abe et al. (2007) J. Am. Chem. Soc. 129:15108-15109), and small internally segmented interfering RNA (siRNA; see, e.g., Bramsen et al. (2007) Nucleic Acids Res. 35:5886-5897). Further non-limiting examples of oligonucleotide structures that can be used in some embodiments to reduce or inhibit the expression of ANGPTL3 include microRNAs (miRNAs), short hairpin RNAs (shRNAs), and short siRNAs (see, e.g., Hamilton et al. (2002) EMBO J. 21:4671-4679; see also U.S. Patent Application Publication No. 2009 / 0099115).

[0092] Furthermore, in some embodiments, the oligonucleotide herein that reduces or inhibits ANGPTL3 expression is ss. Such structures include, but are not limited to, ss RNAi molecules. Recent efforts have demonstrated the activity of ss RNAi molecules (see, for example, Matsui et al. (2016) Mol. Ther. 24:946-955). However, in some embodiments, the oligonucleotide herein is an antisense oligonucleotide (ASO). An antisense oligonucleotide is an ss oligonucleotide that, written from 5' to 3', comprises the reverse complement of a specific nucleic acid target segment and has a nucleobase sequence that is appropriately modified to induce RNase H-mediated cleavage of the target RNA in cells (e.g., as a gapmer) or inhibit the translation of target mRNA in cells (e.g., as a mixmer). As used herein, ASOs may be modified in any suitable manner known in the art, including, for example, those set forth in U.S. Patent No. 9,567,587 (including, e.g., modifications of length, sugar moieties (pyrimidines, purines), and heterocyclic moieties of nucleobases). Additionally, ASOs have been used for decades to reduce the expression of specific target genes (see, e.g., Bennett et al. (2017) Annu. Rev. Pharmacol. 57:81-105).

[0093] iv. Double-stranded oligonucleotides

[0094] The present disclosure provides ds oligonucleotides that target ANGPTL3 mRNA and inhibit the expression of ANGPTL3 (e.g., via the RNAi pathway), the ds oligonucleotides comprising a sense strand (also referred to herein as a passenger strand) and an antisense strand (also referred to herein as a guide strand). In some embodiments, the sense strand and the antisense strand are separate strands and are not covalently linked. In some embodiments, the sense strand and the antisense strand are covalently linked.

[0095] In some embodiments, the sense strand has a first region (R1) and a second region (R2), where R2 comprises a first subregion (S1), a tetraloop (L), or a triloop (triL), and a second subregion (S2). L or triL is located between S1 and S2, and S1 and S2 form a second duplex (D2). The length of D2 can vary. In some embodiments, D2 is about 1-6 bp in length. In some embodiments, D2 is 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, or 4-5 bp in length. In some embodiments, D2 is 1, 2, 3, 4, 5, or 6 bp in length. In some embodiments, D2 is 6 bp in length.

[0096] In some embodiments, R1 of the sense strand and the antisense strand form a first duplex (D1). In some embodiments, D1 is about 15 nucleotides in length (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21). In some embodiments, D1 is about 12-30 nucleotides in length (e.g., 12-30, 12-27, 15-22, 18-22, 18-25, 18-27, 18-30, or 21-30 nucleotides in length). In some embodiments, D1 is about 12 nucleotides in length (e.g., at least 12, at least 15, at least 20, at least 25, or at least 30). In some embodiments, D1 is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, D1 is 20 nucleotides in length. In some embodiments, D1, comprising a sense strand and an antisense strand, does not span the entire length of the sense strand and / or the antisense strand. In some embodiments, D1, comprising a sense strand and an antisense strand, spans the entire length of either the sense strand or the antisense strand and / or the entire length of both the sense strand and the antisense strand. In certain embodiments, D1, comprising a sense strand and an antisense strand, spans the entire length of both the sense strand and the antisense strand.

[0097] In some embodiments, the ds oligonucleotides herein are any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115, as arranged in Table 3. and an antisense strand comprising a complementary sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, and 116. In some embodiments, the sense strand comprises the sequence of SEQ ID NO: 99 and the antisense strand comprises the sequence of SEQ ID NO: 100.

[0098] In some embodiments, the ds oligonucleotides herein comprise a sense strand comprising the sequence of any one of SEQ ID NOs: 19, 25, 49, 71, 73, 75, 79, 99, 101, 103, and 113, and an antisense strand comprising a complementary sequence selected from SEQ ID NOs: 20, 26, 50, 72, 74, 76, 80, 100, 102, 104, and 114, as arranged in Table 4. In some embodiments, the sense strand comprises the sequence of SEQ ID NO: 99, and the antisense strand comprises the sequence of SEQ ID NO: 100.

[0099] It should be understood that in some embodiments, the structure of an oligonucleotide or other nucleic acid may be described with reference to a sequence set forth in a sequence listing. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., RNA counterparts of DNA nucleotides or DNA counterparts of RNA nucleotides) and / or one or more modified nucleotides and / or one or more modified internucleotide linkages and / or one or more other modifications compared to the specified sequence, while retaining essentially the same or similar complementary properties as the specified sequence.

[0100] In some embodiments, the ds oligonucleotide herein comprises a sense strand of 25 nucleotides and an antisense strand of 27 nucleotides, which generates the antisense strand that is incorporated into mature RISC when Dicer enzyme acts.In some embodiments, the sense strand of the ds oligonucleotide is longer than 27 nucleotides (for example, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides).In some embodiments, the sense strand of the ds oligonucleotide is longer than 25 nucleotides (for example, 26, 27, 28, 29, or 30 nucleotides).

[0101] In some embodiments, one 5' end of the oligonucleotide herein is thermodynamically unstable compared to the other 5' end. In some embodiments, asymmetric oligonucleotides are provided having a blunt end at the 3' end of the sense strand and an overhang at the 3' end of the antisense strand. In some embodiments, the 3' overhang of the antisense strand is about 1 to 8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length). Typically, RNAi oligonucleotides have a 2-nucleotide overhang at the 3' end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, the overhang is a 3' overhang comprising from 1 to 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides in length. However, in some embodiments, the overhang is a 5' overhang comprising from 1 to 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides in length.

[0102] In some embodiments, the two terminal nucleotides at the 3' end of the antisense strand are modified. In some embodiments, the two terminal nucleotides at the 3' end of the antisense strand are complementary to the target. In some embodiments, the two terminal nucleotides at the 3' end of the antisense strand are not complementary to the target. In some embodiments, the two terminal nucleotides at each 3' end of the oligonucleotide in the nicked tetraloop structure are GG. Typically, one or both of the two terminal GG nucleotides at each 3' end of the oligonucleotide are not complementary to the target.

[0103] In some embodiments, there are one or more (e.g., one, two, three, four, or five) mismatches between the sense strand and the antisense strand. When there are multiple mismatches between the sense strand and the antisense strand, they may be located consecutively (e.g., two, three, or more consecutively) or may be scattered throughout the complementary region. In some embodiments, the 3' end of the sense strand contains one or more mismatches. In one embodiment, two mismatches are incorporated into the 3' end of the sense strand. In some embodiments, base mismatches or destabilization of the segment at the 3' end of the sense strand of the oligonucleotide improve the efficacy of synthetic RNAi duplexes, possibly by promoting Dicer processing.

[0104] a. antisense strand

[0105] In some embodiments, oligonucleotides targeting ANGPTL3 disclosed herein comprise an antisense strand comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, and 116. In some embodiments, the oligonucleotide is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 , 110, 112, 114, and 116.

[0106] In some embodiments, the ds oligonucleotide has an antisense strand that is up to 40 nucleotides in length (e.g., up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the oligonucleotide may have an antisense strand that is at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, oligonucleotides may have antisense strands ranging from about 12 to about 40 nucleotides in length (e.g., 12-40, 12-36, 12-32, 12-28, 15-40, 15-36, 15-32, 15-28, 17-22, 17-25, 19-27, 19-30, 20-40, 22-40, 25-40, or 32-40). In some embodiments, an oligonucleotide may have an antisense strand that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

[0107] In some embodiments, the antisense strand of oligonucleotide can be called " guide strand ".For example, if antisense strand can be incorporated into RNA-induced silencing complex (RISC) and bind to Argonaute protein, such as Ago2, or can be incorporated into or bind to one or more similar factors to induce target gene silencing, antisense strand can be called guide strand.In some embodiments, the sense strand that is complementary to guide strand can be called " passenger strand ".

[0108] B sense strand

[0109] In some embodiments, the oligonucleotides targeting ANGPTL3 herein comprise or consist of a sense strand sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115. In some embodiments, the oligonucleotide is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 , 107, 109, 111, 113, and 115.

[0110] In some embodiments, an oligonucleotide comprises a sense strand (or passenger strand) up to about 40 nucleotides in length (e.g., up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, an oligonucleotide may have a sense strand at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). In some embodiments, the oligonucleotides may have a sense strand ranging from about 12 to about 40 nucleotides in length (e.g., 12-40, 12-36, 12-32, 12-28, 15-40, 15-36, 15-32, 15-28, 17-21, 17-25, 19-27, 19-30, 20-40, 22-40, 25-40, or 32-40). In some embodiments, the oligonucleotide may have a sense strand that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

[0111] In some embodiments, the sense strand comprises a stem-loop structure at its 3'-end. In some embodiments, the sense strand comprises a stem-loop structure at its 5'-end. In some embodiments, the stem is a duplex of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 bp in length. In some embodiments, the stem-loop protects the molecule from degradation (e.g., enzymatic degradation) and promotes targeting properties for delivery to target cells. For example, in some embodiments, the loop provides additional nucleotides that can be modified without significantly affecting the gene expression inhibitory activity of the oligonucleotide. In certain embodiments, the oligonucleotides described herein are those in which the sense strand comprises a stem-loop (e.g., at its 3'-end) designated as S1-L-S2. where S1 is complementary to S2, and L forms a loop between S1 and S2 up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). Figure 3 shows non-limiting examples of such oligonucleotides.

[0112] In some embodiments, the loop (F) of the stem-loop is a tetraloop (e.g., in a nicked tetraloop structure). The tetraloop can contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, the tetraloop has 4-5 nucleotides.

[0113] v. Oligonucleotide Modification

[0114] Sugar modification

[0115] In some embodiments, modified sugars (also referred to herein as sugar analogs) comprise modified deoxyribose or ribose moieties, e.g., where one or more modifications occur at the 2', 3', 4', and / or 5' carbons of the sugar. In some embodiments, modified sugars may also comprise unnatural alternative carbon structures, such as those presented in locked nucleic acids ("LNAs," see e.g., Koshkin et al. (1998) Tetrahedon 54:3607-3630), unlocked nucleic acids ("UNAs," see e.g., Snead et al. (2013) Mol. Ther-Nucl. Acids 2:e103), and bridged nucleic acids ("BNAs," see e.g., Imanishi & Obika (2002) Chem Commun. (Camb) 21:1653-1659).

[0116] In some embodiments, the sugar nucleotide modification comprises a 2'-modification. In some embodiments, the 2'-modification may be 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-fluoro (2'-F), 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxyethyl] (2'-O-NMA), or 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA). In some embodiments, the modification is 2'-F, 2'-OMe, or 2'-MOE. In some embodiments, the sugar modification comprises a modification of the sugar ring, which may include modification of one or more carbons of the sugar ring. For example, modifications of the sugar of a nucleotide may include the 2'-oxygen of the sugar being linked to the 1'- or 4'-carbon of the sugar, or the 2'-oxygen being linked to the 1'- or 4'-carbon via an ethylene or methylene bridge. In some embodiments, modified nucleotides have acyclic sugars that lack a 2'-carbon to 3'-carbon linkage. In some embodiments, modified nucleotides have, for example, a thiol group at the 4'-position of the sugar.

[0117] In some embodiments, the oligonucleotides described herein comprise at least about one (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or more) modified nucleotide. In some embodiments, the sense strand of the oligonucleotide comprises at least about one (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or more) modified nucleotide. In some embodiments, the antisense strand of the oligonucleotide comprises at least about one (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more) modified nucleotide.

[0118] In some embodiments, all nucleotides in the sense strand of the oligonucleotide are modified. In some embodiments, all nucleotides in the antisense strand of the oligonucleotide are modified. In some embodiments, all nucleotides in the oligonucleotide (i.e., both the sense and antisense strands) are modified. In some embodiments, the modified nucleotides include a 2'-modification (e.g., 2'-F or 2'-OMe, 2'-MOE, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid). In some embodiments, the modified nucleotides include a 2'-modification (e.g., 2'-F or 2'-OMe).

[0119] The present disclosure provides oligonucleotides having different modification patterns. In some embodiments, the modified oligonucleotides comprise a sense strand sequence having a modification pattern as set forth in any one of Tables 3 and 4 (and FIG. 3), and an antisense strand having a modification pattern as set forth in any one of Tables 3 and 4 (and FIG. 3). In some embodiments, for these oligonucleotides, one or more of positions 8, 9, 10, or 11 of the sense strand are modified with a 2'-F group. In other embodiments, for these oligonucleotides, the sugar moieties of each of nucleotides at positions 1-7 and 12-20 of the sense strand are modified with a 2'-OMe.

[0120] In some embodiments, the present invention provides oligonucleotides that are or comprise a modified or unmodified sense strand selected from those listed in Table A. In some embodiments, the present invention provides oligonucleotides that are or comprise a modified or unmodified antisense strand selected from those listed in Table A. In some embodiments, the present invention provides modified or unmodified double-stranded oligonucleotides selected from those listed in Table A. In some embodiments, the present invention provides a sense strand modification pattern selected from those listed in Table A. In some embodiments, the present invention provides an antisense strand modification pattern selected from those listed in Table A.

[0121] In some embodiments, the antisense strand has three nucleotides modified with 2'-F at the 2' position of the sugar moiety. In some embodiments, the sugar moieties at positions 2, 5, and 14 of the antisense strand, and optionally up to three nucleotides at positions 1, 3, 7, and 10, are modified with 2'-F. In other embodiments, the sugar moieties at positions 2, 5, and 14 of the antisense strand are modified with 2'-F. In other embodiments, the sugar moieties at positions 1, 2, 5, and 14 of the antisense strand are modified with 2'-F. In yet other embodiments, the sugar moieties at positions 1, 2, 3, 5, 7, and 14 of the antisense strand are modified with 2'-F. In yet another embodiment, the sugar moieties at positions 1, 2, 3, 5, 10, and 14 of the antisense strand are modified with 2'-F. In another embodiment, the sugar moieties at positions 2, 3, 5, 7, 10, and 14 of the antisense strand are modified with 2'-F.

[0122] b. 5'-terminal phosphate

[0123] In some embodiments, the 5'-terminal phosphate group of an oligonucleotide promotes interaction with Ago2. However, oligonucleotides containing a 5'-phosphate group are susceptible to degradation by phosphatases or other enzymes, potentially limiting their in vivo bioavailability. In some embodiments, the oligonucleotide has a 5' phosphate analog that is resistant to such degradation. In some embodiments, the phosphate analog may be an oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In certain embodiments, the 1' end of the oligonucleotide chain is linked to a chemical moiety (a "phosphate mimetic") that mimics the electrostatic and steric properties of the natural 5'-phosphate group.

[0124] In some embodiments, oligonucleotides have a phosphate analog at the 4'-carbon position of the sugar (referred to as a "4'-phosphate analog"). See, for example, International Patent Application Publication No. WO2018 / 045317. In some embodiments, oligonucleotides herein include a 4'-phosphate analog at the 5'-terminal nucleotide. In some embodiments, the phosphate analog is an oxymethylphosphonate or analog thereof, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., its 4' carbon). In other embodiments, the 4'-phosphate analog is a thiomethylphosphonate or aminomethylphosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is attached to the 4'-carbon of the sugar moiety or analog thereof. In certain embodiments, the 4'-phosphate analog is an oxymethylphosphonate. In some embodiments, the oxymethylphosphonate is represented by the formula -O-CH-PO(OH) or -O-CH-PO(OR), where R is independently selected from H, CH, an alkyl group, CHCHCN, CHOCOC(CH), CHOCHCHSi(CH), or a protecting group. In certain embodiments, the alkyl group is CHCH. More typically, R is independently selected from H, CH, or CHCH.

[0125] c modified internucleoside linkage

[0126] In some embodiments, an oligonucleotide can contain a modified internucleoside linkage. In some embodiments, phosphate modification or substitution can result in an oligonucleotide containing at least one (e.g., at least one, at least two, at least three, or at least five) modified internucleotide linkages. In some embodiments, any one of the oligonucleotides disclosed herein contains about 1 to 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3, or 1 to 2) modified internucleotide linkages. In some embodiments, any one of the oligonucleotides disclosed herein contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages.

[0127] The modified internucleotide bond may be a phosphorodithioate bond, a phosphorothioate bond, a phosphotriester bond, a thionoalkylphosphonate bond, a thionealkylphosphotriester bond, a phosphoramidite bond, a phosphonate bond, or a boranophosphate bond. In some embodiments, at least one modified internucleotide bond in any one of the oligonucleotides disclosed herein is a phosphorothioate bond.

[0128] In some embodiments, the oligonucleotides described herein comprise a phosphorothioate linkage between one or more of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and / or positions 21 and 22 of the antisense strand. In some embodiments, the oligonucleotides described herein comprise a phosphorothioate linkage between positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand, respectively.

[0129] d. base modification

[0130] In some embodiments, the oligonucleotide herein has one or more modified nucleobases. In some embodiments, the modified nucleobase (also referred to herein as a base analog) is linked to the 1' position of the nucleotide sugar moiety. In certain embodiments, the modified nucleobase is a nitrogenous base. In certain embodiments, the modified nucleobase does not contain a nitrogen atom. See, for example, U.S. Patent Application Publication No. 2008 / 0274462. In some embodiments, the modified nucleotide contains a universal base. However, in certain embodiments, the modified nucleotide does not contain a nucleobase (abasic).

[0131] In some embodiments, a universal base is a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety of a modified nucleotide, or at an equivalent position of a nucleotide sugar moiety substitute, that can be paired with multiple types of bases when present in a duplex without significantly altering the structure of the duplex. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., an oligonucleotide) that is perfectly complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base has a lower T than a duplex formed with a complementary nucleic acid. m However, in some embodiments, compared to a reference single-stranded nucleic acid in which the universal base is replaced with a base resulting in one mismatch, the single-stranded nucleic acid containing the universal base exhibits a higher T than the duplex formed with the nucleic acid containing the mismatched base. m It forms a duplex with a target nucleic acid having the formula:

[0132] Non-limiting examples of universal binding nucleotides include, but are not limited to, inosine, 1-β-D-ribofuranosyl-5-nitroindole, and / or 1-β-D-ribofuranosyl-3-nitropyrrole (see U.S. Patent Application Publication No. 2007 / 0254362; Van Aerschot et al. (1995) Nucleic Acids Res. 23:4363-4370; Loakes et al. (1995) Nucleic Acids Res. 23:2361-2366; Loakes & Brown (1994) Nucleic Acids Res. 22:4039-4043).

[0133] e. Reversible modification

[0134] Although certain modifications can be made to protect oligonucleotides from the in vivo environment before reaching target cells, these modifications may reduce the efficacy or activity of oligonucleotides when they reach the cytosol of target cells.Reversible modifications can be made so that the molecule retains desired properties outside the cell, and then is removed when it enters the cytosol environment of the cell.Reversible modifications can be removed, for example, by the action of intracellular enzymes or by chemical conditions inside the cell (for example, reduction by intracellular glutathione).

[0135] In some embodiments, the reversibly modified nucleotide comprises a glutathione-sensitive moiety. Typically, nucleic acid molecules are chemically modified with a cyclic disulfide moiety to shield the negative charge generated by the internucleotide diphosphate linkage and improve cellular uptake and nuclease resistance. See U.S. Patent Application Publication No. 2011 / 0294869, International Patent Application Publication No. WO2014 / 088920, International Patent Application Publication No. WO2015 / 188197, and Meade et al. (2014) Nat. Biotechnol. 32:1256-1263. This reversible modification of the internucleotide diphosphate linkage is designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g., glutathione). An earlier example is the neutralizing phosphotriester modification, which was reported to be cleavable inside cells (see Dellinger et al. (2003) J. Am. Chem. Soc. 125:940-950).

[0136] In some embodiments, such reversible modifications allow for protection during in vivo administration (e.g., during passage through the blood and / or lysosomal / endosomal compartments of cells), where the oligonucleotide will be exposed to nucleases and other harsh environmental conditions (e.g., pH). Upon release into the cytosol of cells, where glutathione levels are higher compared to the extracellular space, the modification is reversed, resulting in cleavage of the oligonucleotide. The use of reversible glutathione-sensitive moieties allows for the introduction of sterically larger chemical groups into the oligonucleotide of interest compared to options available using irreversible chemical modifications. This is because such larger chemical groups will be removed in the cytosol and therefore will not interfere with the biological activity of the oligonucleotide within the cytosol of cells. As a result, such larger chemical groups can be engineered to confer various advantages to the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. In some embodiments, the structure of the glutathione-sensitive moiety can be engineered to modify its release kinetics.

[0137] In some embodiments, the glutathione-sensitive moiety is attached to the sugar of the nucleotide. In some embodiments, the glutathione-sensitive moiety is attached to the 2'-carbon of the sugar of the modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5'-carbon of the sugar, particularly when the modified nucleotide is the 5'-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 3'-carbon of the sugar, particularly when the modified nucleotide is the 3'-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. See, e.g., U.S. Provisional Patent Application No. 62 / 378,635, filed August 23, 2016, entitled "Compositions Comprising Reversibly Modified Oligonucleotides and Uses Thereof."

[0138] vi. Targeting Ligands

[0139] In some embodiments, it is desirable to target the oligonucleotides of the present disclosure to one or more cells or one or more organs. Through such a strategy, it is possible to avoid undesirable effects in other organs or to avoid excessive loss of the oligonucleotide to cells, tissues, or organs where the oligonucleotide is not beneficial. Thus, in some embodiments, the oligonucleotides disclosed herein are modified to facilitate targeting and / or delivery to specific tissues, cells, or organs (e.g., to facilitate delivery of the oligonucleotide to the liver). In certain embodiments, the oligonucleotides disclosed herein are modified to facilitate delivery of the oligonucleotide to hepatocytes in the liver. In some embodiments, the oligonucleotide comprises at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, or more nucleotides) bound to one or more targeting ligand(s).

[0140] In some embodiments, the targeting ligand comprises carbohydrate, amino sugar, cholesterol, peptide, polypeptide, protein or protein part (for example, antibody or antibody fragment), or lipid.In some embodiments, the targeting ligand is an aptamer.For example, the targeting ligand can be RGD peptide used to target tumor vasculature or glioma cells, CREKA peptide for targeting tumor vasculature or stoma, transferrin, lactoferrin, or aptamer for targeting transferrin receptor expressed on CNS vasculature, or anti-EGFR antibody for targeting EGFR on glioma cells.In certain embodiments, the targeting ligand is one or more GalNAc moieties.

[0141] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, two to four nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, the targeting ligand is conjugated to two to four nucleotides at either end of the sense or antisense strand, such that the targeting ligand resembles the bristles of a toothbrush and the oligonucleotide resembles a toothbrush (e.g., the targeting ligand is conjugated to an overhang or extension of two to four nucleotides at the 5' or 3' end of the sense or antisense strand). For example, the oligonucleotide may include a stem-loop at either the 5' or 3' end of the sense strand, and one, two, three, or four nucleotides of the stem-loop may be individually conjugated to a targeting ligand. In some embodiments, the oligonucleotides (e.g., ds oligonucleotides) provided herein comprise a stem-loop at the 3' end of the sense strand, the loop of the stem-loop comprising a triloop or a tetraloop, and the three or four nucleotides comprising the triloop or tetraloop, respectively, are individually bound to a targeting ligand.

[0142] GalNAc is a high-affinity ligand for ASGPR, which is primarily expressed on the sinusoidal surface of hepatocytes and plays a major role in the binding, internalization, and subsequent excretion of circulating glycoproteins containing terminal galactose or GalNAc residues (asialoglycoproteins). Oligonucleotides of the present disclosure can be conjugated (indirectly or directly) with a GalNAc moiety to target these oligonucleotides to ASGPR expressed in cells. In some embodiments, the oligonucleotides of the present disclosure are conjugated to at least one or more GalNAc moieties, which target the oligonucleotide to ASGPR expressed in human hepatocytes (e.g., human hepatocytes). In some embodiments, the GalNAc moiety targets the oligonucleotide to the liver.

[0143] In some embodiments, the oligonucleotide of the present disclosure is directly or indirectly linked to monovalent GalNAc.In some embodiments, the oligonucleotide is directly or indirectly linked to multiple monovalent GalNAc (i.e., linked to 2, 3 or 4 monovalent GalNAc moieties, typically linked to 3 or 4 monovalent GalNAc moieties).In some embodiments, the oligonucleotide is linked to one or more divalent GalNAc, trivalent GalNAc or tetravalent GalNAc moieties.

[0144] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each linked to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of the tetraloop are each linked to a separate GalNAc. In some embodiments, 1 to 3 nucleotides of the triloop are each linked to a separate GalNAc. In some embodiments, a targeting ligand is linked to 2 to 4 nucleotides at either end of the sense or antisense strand, such that the GalNAc moieties resemble toothbrush bristles, giving the oligonucleotide a toothbrush-like appearance (e.g., the ligand is linked to an overhang or extension of 2 to 4 nucleotides at the 5' or 3' end of the sense or antisense strand). In some embodiments, the GalNAc moiety is linked to a nucleotide in the sense strand. For example, four GalNAc moieties can be linked to nucleotides within the tetraloop of the sense strand, with each GalNAc moiety linked to a single nucleotide.

[0145] In some embodiments, the oligonucleotides herein comprise a monovalent GalNAc attached to a guanine nucleotide, designated as [ademG-GalNAc] or 2'-aminodiethoxymethanol-guanine-GalNAc, as shown below. [ka]

[0146] In some embodiments, the oligonucleotides herein comprise a monovalent GalNAc attached to an adenine nucleotide, designated as [ademA-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc, as shown below. [ka]

[0147] An example of such a linkage is shown below for a loop containing the nucleotide sequence GAAA in a 5' to 3' direction (L = linker, X = heteroatom). The stem attachment points are indicated. Such a loop can be, for example, at positions 27-30 of the sense strand as described in Table 5 and shown in Figure 3. In the formula: [ka] is used to represent the point of attachment to the oligonucleotide chain. [ka]

[0148] The targeting ligand can be linked to the nucleotide using a suitable method or chemical approach (e.g., click chemistry). In some embodiments, the targeting ligand is attached to the nucleotide using a click linker. In some embodiments, an acetal-based linker is used to attach the targeting ligand to any one of the nucleotides of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO2016 / 100401. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable. An example of a loop containing the nucleotides GAAA in the 5' to 3' direction, in which a GalNAc moiety is attached to the nucleotide of the loop using an acetal linker, is shown below. Such a loop can be present, for example, at positions 27-30 of any one of the sense strands described in Table 5 and shown in Figure 3. In the chemical formula: [ka] is the point of attachment to the oligonucleotide chain. [ka]

[0149] As described above, the targeting ligand can be linked to the nucleotide using various suitable methods or chemical synthesis techniques (e.g., click chemistry). In some embodiments, the targeting ligand is linked to the nucleotide using a click linker. In some embodiments, the targeting ligand is linked to any one of the nucleotides of the oligonucleotides described herein using an acetal-based linker. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO2016 / 100401. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is a stable linker.

[0150] In some embodiments, a duplex extension (e.g., up to 3, 4, 5, or 6 bp in length) is positioned between the targeting ligand (e.g., GalNAc moiety) and the ds oligonucleotide. In some embodiments, the oligonucleotide herein does not have a GalNAc attached thereto.

[0151] III. Preparations

[0152] Various formulations have been developed to facilitate the use of oligonucleotides. For example, oligonucleotides can be delivered to a subject or cellular environment using formulations that minimize degradation, facilitate delivery and / or uptake, or impart other beneficial properties to the oligonucleotides in the formulation. In some embodiments, oligonucleotides are formulated in buffers such as phosphate-buffered saline, liposomes, micellar structures, and capsids.

[0153] Oligonucleotide formulations containing cationic lipids can be used to promote the transfection of oligonucleotides into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used. Suitable lipids include oligofectamine, lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene6 (Roche), all of which can be used according to the manufacturer's instructions.

[0154] Thus, in some embodiments, the formulation comprises a lipid nanoparticle. In some embodiments, the excipient comprises a liposome, lipid, lipid complex, microsphere, microparticle, nanosphere, or nanoparticle, or can be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof (see, e.g., Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd edition, Pharmaceutical Press, 2013).

[0155] In some embodiments, the formulations herein include an excipient. In some embodiments, the excipient confers improved stability, improved absorption, improved solubility, and / or therapeutic enhancement of the active ingredient to the composition. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, Tris base, or sodium hydroxide) or a solvent (e.g., buffer solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotide is lyophilized to extend its shelf life and then reconstituted into a solution prior to use (e.g., administration to a subject). Thus, the excipient in a composition comprising any one of the oligonucleotides described herein can be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a disintegration temperature modifier (e.g., dextran, Ficoll™, or gelatin).

[0156] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration, including parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0157] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. In many cases, it is preferable to add isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, to the composition. Sterile injectable solutions can be prepared by adding the required amount of oligonucleotide to the selected solvent, optionally with one or a combination of the ingredients listed above, followed by sterile filtration.

[0158] In some embodiments, the composition may contain at least about 0.1% or more of a therapeutic agent, although the percentage of active ingredient(s) may be from about 1% to about 80% by weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and therefore, various dosages and treatment regimens may be desirable.

[0159] In some embodiments, liver-targeted delivery of any of the oligonucleotides herein is directed, although targeting of other tissues is also contemplated.

[0160] IV.How to use

[0161] i. Reducing ANGPTL3 expression in cells

[0162] The present disclosure provides methods for contacting or delivering an effective amount of any one of the oligonucleotides herein to a cell or cell population to reduce ANGPTL3 expression. These methods can include the steps described herein, which may, but need not, be performed in the order described. However, other orders are possible. Furthermore, individual or multiple steps may be performed in parallel and / or overlapping in time, and / or may be repeated individually or multiple times. Furthermore, these methods may include additional, unspecified steps.

[0163] The methods herein are useful with any suitable cell type. In some embodiments, the cells are any cells that express mRNA (e.g., hepatocytes, macrophages, monocyte-derived cells, prostate cancer cells, brain cells, endocrine tissue, bone marrow, lymph nodes, lung, gallbladder, liver, duodenum, small intestine, pancreas, kidney, gastrointestinal tract, urinary bladder, adipose and soft tissue, and skin). In some embodiments, the cells are primary cells obtained from a subject. In some embodiments, primary cells have undergone a limited number of passages, thereby allowing the cells to substantially maintain their native phenotypic characteristics. In some embodiments, the cells to which the oligonucleotides are delivered are ex vivo or in vitro (i.e., cells in culture or can be delivered to the organism in which the cells reside).

[0164] In some embodiments, the oligonucleotides herein are delivered using a suitable nucleic acid delivery method, including, but not limited to, injection of a solution containing the oligonucleotide, bombardment with particles coated with the oligonucleotide, exposure of a cell or cell population to a solution containing the oligonucleotide, or electroporation of the cell membrane in the presence of the oligonucleotide. Other suitable methods for delivering oligonucleotides to cells can also be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection, such as calcium phosphate.

[0165] In some embodiments, the reduction of ANGPTL3 expression can be determined by a suitable assay or technique for evaluating one or more characteristics or features of a cell or cell population associated with ANGPTL3 expression (e.g., using an ANGPTL3 expression biomarker), or by an assay or technique for evaluating a molecule that directly indicates ANGPTL3 expression (e.g., ANGPTL3 mRNA or ANGPTL3 protein). In some embodiments, the extent to which the oligonucleotide herein reduces ANGPTL3 expression is assessed by comparing the expression of ANGPTL3 in a cell or cell population contacted with the oligonucleotide with an appropriate control (e.g., a suitable cell or cell population not contacted with the oligonucleotide, or a suitable cell or cell population contacted with a control oligonucleotide). In some embodiments, after delivery of the RNAi molecule, a suitable control level of mRNA expression in protein is set as a predetermined level or value, thereby eliminating the need to measure the control level every time. The predetermined level or value can take various forms. In some embodiments, the predetermined level or value can be a single cutoff value, such as a median or mean.

[0166] In some embodiments, administration of the oligonucleotides herein reduces ANGPTL3 expression in a cell or cell population. In some embodiments, the reduction in ANGPTL3 expression is about 1% or less, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less, compared to an appropriate control level of mRNA. An appropriate control level can be the level of mRNA expression and / or protein translation in a cell or cell population not contacted with the oligonucleotides herein. In some embodiments, the effect of delivery of the oligonucleotides to cells by the methods herein is evaluated after a period of time. For example, the level of mRNA in the cells can be analyzed at least about 8 hours, about 12 hours, about 18 hours, or about 24 hours after introduction of the oligonucleotide into the cells, or at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or up to 14 days.

[0167] In some embodiments, the oligonucleotide is delivered in the form of a transgene engineered to express the oligonucleotide or strands comprising the oligonucleotide (e.g., its sense and antisense strands) in a cell. In some embodiments, the oligonucleotide is delivered using a transgene engineered to express any of the oligonucleotides disclosed herein. The transgene can be delivered using a viral vector (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or a non-viral vector (e.g., a plasmid or synthetic mRNA). In some embodiments, the transgene can be directly injected into the subject.

[0168] ii. Medical Use

[0169] The present disclosure also provides oligonucleotides for use or adaptable for use in treating a subject who would benefit from reducing the expression of ANGPTL3 (e.g., a human having a disease, disorder, or condition associated with the expression of ANGPTL3). In some respects, the present disclosure provides oligonucleotides for use or adaptable for use in treating a subject having a disease, disorder, or condition associated with the expression of ANGPTL3. The present disclosure provides oligonucleotides for use or adaptable for use in the manufacture of a medicament or pharmaceutical composition for treating a disease, disorder, or condition associated with the expression of ANGPTL3. In some embodiments, the oligonucleotides for use or adaptable for use target ANGPTL3 mRNA (e.g., via the RNAi pathway) and reduce the expression of ANGPTL3. In some embodiments, the oligonucleotides for use or adaptable for use target ANGPTL3 mRNA and are used or adaptable for use to reduce the amount or level of ANGPTL3 mRNA, ANGPTL3 protein, and / or ANGPTL3 activity.

[0170] Additionally, the following methods can include selecting a subject having or predisposed to a disease, disorder, or condition associated with ANGPTL3 expression. In some cases, the methods can include selecting an individual having or predisposed to a marker of ANGPTL3 expression, such as elevated TG or cholesterol (or altered LPL and / or EL activity).

[0171] Also, as described in more detail below, the method may thus include steps such as measuring or obtaining a baseline value of a marker of ANGPTL3 expression, and then comparing the value so obtained with one or more other baseline values ​​or values ​​obtained after administering the oligonucleotide to assess the effectiveness of the treatment.

[0172] iii.Treatment method

[0173] The present disclosure also provides methods of treating a subject having, suspected of having, or at risk of developing a disease, disorder, or condition using the oligonucleotides herein. In some embodiments, the present disclosure provides methods of treating or ameliorating the onset or progression of a disease, disorder, or condition associated with expression of ANGPTL3 using the oligonucleotides herein. In other embodiments, the present disclosure provides methods of achieving one or more therapeutic effects in a subject having a disease, disorder, or condition associated with expression of ANGPTL3 using the oligonucleotides herein. In some embodiments of the methods herein, the subject is treated by administering a therapeutically effective amount of any one or more of the oligonucleotides herein. In some embodiments, the treatment includes reducing expression of ANGPTL3. In some embodiments, the subject is treated therapeutically. In some embodiments, the subject is treated prophylactically.

[0174] In some embodiments of this method, an oligonucleotide described herein, or a pharmaceutical composition comprising the oligonucleotide, is administered to a subject having a disease, disorder, or condition associated with ANGPTL3 expression, thereby treating the subject by reducing ANGPTL3 expression in the subject. In some embodiments, the amount or level of ANGPTL3 mRNA is reduced in the subject. In some embodiments, the amount or level of ANGPTL3 protein is reduced in the subject. In some embodiments, the amount or level of ANGPTL3 activity is reduced in the subject. In some embodiments, the amount or level of triglycerides (TG) (e.g., one or more TGs or total TG) is reduced in the subject. In some embodiments, the amount or level of cholesterol (e.g., total cholesterol, LDL cholesterol, and / or HDL cholesterol) is reduced in the subject. In some embodiments, the amount or level of low-density lipoprotein (LDL) cholesterol is reduced in the subject. In some embodiments, the amount or activity of LPL is altered in the subject. In some embodiments, the amount or activity of EL is altered in the subject. In some embodiments, any combination of the following is reduced or altered in a subject: ANGPTL3 expression, amount or level of ANGPTL3 mRNA, amount or level of ANGPTL3 protein, amount or level of ANGPTL3 activity, amount or level of TG, amount or level of cholesterol, and / or amount or activity of LPL and / or EL.

[0175] In some embodiments of the methods herein, an oligonucleotide herein, or a pharmaceutical composition comprising the oligonucleotide, is administered to a subject having a disease, disorder, or condition associated with ANGPTL3 such that the expression of ANGPTL3 in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the expression of ANGPTL3 before administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the expression of ANGPTL3 is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% in a subject compared to the expression of ANGPTL3 in a subject not receiving the oligonucleotide or pharmaceutical composition, or in a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject).

[0176] In some embodiments of the methods herein, an oligonucleotide herein, or a pharmaceutical composition comprising the oligonucleotide, is administered to a subject having a disease, disorder, or condition associated with ANGPTL3 expression such that the amount or level of ANGPTL3 mRNA in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the amount or level of ANGPTL3 mRNA before administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the amount or level of ANGPTL3 mRNA is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% in a subject compared to the amount or level of ANGPTL3 mRNA in a subject not receiving the oligonucleotide or pharmaceutical composition, or in a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference subject or control subject).

[0177] In some embodiments of the methods herein, an oligonucleotide herein, or a pharmaceutical composition comprising the oligonucleotide, is administered to a subject having a disease, disorder, or condition associated with ANGPTL3 expression such that the amount or level of ANGPTL3 protein in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the amount or level of ANGPTL3 protein before administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the amount or level of ANGPTL3 protein is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% in a subject compared to the amount or level of ANGPTL3 protein in a subject not receiving the oligonucleotide or pharmaceutical composition, or in a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference subject or control subject).

[0178] In some embodiments of the methods herein, an oligonucleotide herein, or a pharmaceutical composition comprising the oligonucleotide, is administered to a subject having a disease, disorder, or condition associated with ANGPTL3 such that the activity or expression of ANGPTL3 in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the amount or level of ANGPTL3 activity before administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the amount or level of ANGPTL3 activity is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% in a subject compared to the amount or level of ANGPTL3 activity in a subject not receiving the oligonucleotide or pharmaceutical composition, or in a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference subject or control subject).

[0179] In some embodiments of the methods herein, an oligonucleotide herein, or a pharmaceutical composition comprising the oligonucleotide, is administered to a subject having a disease, disorder, or condition associated with ANGPTL3 expression such that the amount or level of TG (e.g., one or more TGs or total TG) in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the amount or level of TG before administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the amount or level of TG is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% in a subject compared to the amount or level of TG in a subject not receiving the oligonucleotide or pharmaceutical composition, or in a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject).

[0180] Generally, the normal or desirable TG range for a human subject is <150 mg / dL of blood, with <100 mg / dL being ideal. In some embodiments, a subject selected for treatment or treated is identified or determined to have a TG amount or level of ≥150 mg / dL. In some embodiments, a subject selected for treatment or treated is identified or determined to have a TG amount or level in the range of 150 mg / dL to 199 mg / dL, which is considered borderline high TG levels. In some embodiments, a subject selected for treatment or treated is identified or determined to have a TG amount or level in the range of 200 mg / dL to 499 mg / dL, which is considered high TG levels. In some embodiments, a subject selected for treatment or treated is identified or determined to have a TG amount or level in the range of 500 mg / dL or greater (i.e., ≥500 mg / dL), which is considered a very high TG level. In some embodiments, a subject selected for treatment or treated is identified or determined to have a TG amount or level of ≧150 mg / dL, ≧200 mg / dL, or ≧500 mg / dL. In some embodiments, a subject selected for treatment or treated is identified or determined to have a TG amount or level of 200 mg / dL to 499 mg / dL, or ≧500 mg / dL. In some embodiments, a patient selected for treatment or treated is identified or determined to have a TG amount or level of ≧200 mg / dL.

[0181] In some embodiments of the methods herein, an oligonucleotide herein, or a pharmaceutical composition comprising the oligonucleotide, is administered to a subject having a disease, disorder, or condition associated with ANGPTL3 expression such that the amount or level of cholesterol (e.g., total cholesterol, LDL cholesterol, and / or HDL cholesterol) in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the amount or level of cholesterol before administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the amount or level of cholesterol is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% in a subject compared to the amount or level of cholesterol in a subject not receiving the oligonucleotide or pharmaceutical composition, or in a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject).

[0182] Generally, the normal or desirable cholesterol range (total cholesterol) for an adult human patient is <200 mg / dL in the blood. In some embodiments, the patient selected for treatment or the patient to be treated is identified or determined to have a cholesterol amount or level of ≥200 mg / dL. In some embodiments, the patient selected for treatment or the patient to be treated is identified or determined to have a cholesterol amount or level in the range of 200 mg / dL to 239 mg / dL, which is considered to be borderline high cholesterol levels. In some embodiments, the patient selected for treatment or the patient to be treated is identified or determined to have a cholesterol amount or level in the range of 240 mg / dL or higher (i.e., ≥240 mg / dL), which is considered to be a high cholesterol level. In some embodiments, the patient selected for treatment or the patient to be treated is identified or determined to have a cholesterol amount or level of 200 mg / dL to 239 mg / dL, or ≥240 mg / dL. In some embodiments, patients selected for treatment or treated are identified or determined to have a cholesterol amount or level of ≧200 mg / dL or ≧240 mg / dL or greater.

[0183] In some embodiments of the methods herein, an oligonucleotide herein, or a pharmaceutical composition comprising the oligonucleotide, is administered to a subject having a disease, disorder, or condition associated with ANGPTL3 such that the amount or level of LDL cholesterol in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the amount or level of LDL cholesterol before administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the amount or level of LDL cholesterol is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% in a subject compared to the amount or level of LDL cholesterol in a subject not receiving the oligonucleotide or pharmaceutical composition (e.g., a reference or control subject), or a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment.

[0184] Generally, the normal or desirable range for LDL cholesterol in an adult human subject is <100 mg / dL in the blood. In some embodiments, a subject selected for treatment or a subject to be treated is identified or determined to have an amount or level of LDL cholesterol of ≥100 mg / dL. In some embodiments, a subject selected for treatment or a subject to be treated is identified or determined to have an amount or level of LDL cholesterol in the range of 100 mg / dL to 129 mg / dL, which is considered above optimal. In some embodiments, a subject selected for treatment or a subject to be treated is identified or determined to have an amount or level of LDL cholesterol in the range of 130 mg / dL to 159 mg / dL, which is considered borderline high. In some embodiments, a subject selected for treatment or a subject to be treated is identified or determined to have an amount or level of LDL cholesterol in the range of 160 mg / dL to 189 mg / dL, which is considered to have a high LDL cholesterol level. In some embodiments, a subject selected for treatment or treated is identified or determined to have an amount or level of LDL cholesterol in the range of 190 mg / dL or greater (i.e., ≧190 mg / dL), which is considered an extremely high LDL cholesterol level. In some embodiments, a subject selected for treatment or treated is identified or determined to have an amount or level of LDL cholesterol of ≧100 mg / dL, ≧130 mg / dL, ≧160 mg / dL, or ≧190 mg / dL or greater, preferably ≧160 mg / dL or ≧190 mg / dL or greater. In some embodiments, a subject selected for treatment or treated is identified or determined to have an amount or level of LDL cholesterol of 100 mg / dL to 129 mg / dL, 130 mg / dL to 159 mg / dL, 160 mg / dL to 189 mg / dL, or ≧190 mg / dL.

[0185] Suitable methods for determining ANGPTL3 expression, ANGPTL3 mRNA, ANGPTL3 protein, ANGPTL3 activity, the amount or level of TG and / or LDL cholesterol, and the amount or activity of LPL and / or EL in a subject or a sample obtained from a subject are known in the art. Further, the Examples described herein exemplify methods for determining ANGPTL3 expression.

[0186] In some embodiments, the amount or level of ANGPTL3 expression, ANGPTL3 mRNA, ANGPTL3 protein, ANGPTL3 activity, TG, LDL cholesterol, LPL protein, LPL activity, EL protein, EL activity, or any combination thereof is reduced in a cell (e.g., a hepatocyte), reduced in a cell population or group (e.g., an organoid), reduced in an organ (e.g., a liver), reduced in blood or a fraction thereof (e.g., plasma), reduced in a tissue (e.g., liver tissue), reduced in a sample (e.g., a liver biopsy sample), or other suitable biological material obtained or isolated from a subject. In some embodiments, the amount or level of ANGPTL3 expression, ANGPTL3 mRNA, ANGPTL3 protein, ANGPTL3 activity, TG, LDL cholesterol, LPL protein, LPL activity, EL protein, EL activity, or any combination thereof, is reduced in two or more types of cells (e.g., hepatocytes and one or more other types of cells), reduced in multiple cell groups, reduced in multiple organs (e.g., liver and one or more other organs), reduced in multiple fractions of blood (e.g., plasma and one or more other blood fractions), reduced in multiple types of tissues (e.g., liver tissue and one or more other types of tissue), reduced in multiple types of separated samples (e.g., liver biopsy sample and one or more other types of biopsy sample), etc.

[0187] Examples of diseases, disorders, or conditions associated with ANGPTL3 expression include, but are not limited to, hypertriglyceridemia, obesity, hyperlipidemia, dyslipidemia and / or cholesterol metabolism disorders, atherosclerosis, type II diabetes (T2D), cardiovascular disease, chronic kidney disease, coronary artery disease, NASH, NAFLD, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other ANGPTL3-associated metabolic disorders and diseases. Of particular interest herein are cardiovascular disease, T2D, hypertriglyceridemia, NASH, obesity, or a combination thereof.

[0188] Due to their high specificity, the oligonucleotides herein specifically target the mRNA of target genes in diseased cells and tissues. In disease prevention, the target gene may be one that is necessary for the onset or maintenance of the disease or one that has been identified as being associated with a higher risk of developing the disease. In disease treatment, the oligonucleotide may be contacted with diseased cells or tissues. For example, an oligonucleotide substantially identical to all or part of a wild-type (i.e., native) or mutant gene associated with a disorder or condition associated with ANGPTL3 expression may be contacted with or introduced into a cell or tissue type of interest, such as hepatocytes or other liver cells.

[0189] In some embodiments, the target gene can be a target gene from any mammal, such as a human. Any gene can be silenced by the methods described herein.

[0190] The methods described herein typically involve administering an oligonucleotide to a subject in an effective amount, i.e., an amount that can produce a desired therapeutic result. A therapeutically acceptable amount can be an amount that can treat a disease or disorder. The appropriate dosage for any one subject will depend on certain factors, including the subject's size, body surface area, age, the specific composition administered, the active ingredient(s) in the composition, the time and route of administration, general health, and other drugs that are administered at the same time.

[0191] In some embodiments, any one of the compositions herein is administered to a subject enterally (for example, orally, by gastric feeding tube, by duodenal feeding tube, through gastrostomy tube or rectally), parenterally (for example, subcutaneous injection, intravenous injection or infusion, intraarterial injection or infusion, intraosseous injection, intramuscular injection, intracerebral injection, intraventricular injection, intrathecal injection), topically (for example, transdermally, inhalation, eye drop or via mucosa), or by direct injection into target organ (for example, the liver of a subject).Typically, the oligonucleotide herein is administered intravenously or subcutaneously.

[0192] As a non-limiting example, the oligonucleotide of the present disclosure is usually administered quarterly (once every 3 months), bimonthly (once every 2 months), monthly, or weekly.For example, the oligonucleotide can be administered every week, or once every 2 or 3 weeks.Alternatively, the oligonucleotide can be administered daily.In some embodiments, the subject is administered one or more loading doses of the oligonucleotide, followed by one or more maintenance doses of the oligonucleotide.

[0193] In some embodiments, the subject to be treated is a human or non-human primate or other mammalian subject. Other exemplary subjects include companion animals such as dogs and cats, farm animals such as horses, cows, pigs, sheep, goats, chickens, and animals such as mice, rats, guinea pigs, and hamsters.

[0194] V. Kit

[0195] In some embodiments, the present disclosure provides kits containing the oligonucleotides described herein and instructions for use. In some embodiments, the kits include the oligonucleotides described herein, a package insert containing instructions for use of the kit, and / or any of its components. In some embodiments, the kits contain the oligonucleotides described herein, one or more controls, and various buffers, reagents, enzymes, and other standard components well known in the art in a suitable container. In some embodiments, the container includes at least one vial, well, test tube, flask, bottle, syringe, or other container means for receiving and, optionally, appropriately dispensing the oligonucleotide. In some embodiments providing additional components, the kits include additional containers into which the components are placed. The kits also include means for tightly containing the oligonucleotides and any other reagents for commercial sale. Such containers may include injection-molded or blow-molded plastic containers into which the desired vials are retained. The containers and / or kits may include labels bearing instructions and / or warnings.

[0196] In some embodiments, the kit comprises an oligonucleotide described herein and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotide, and instructions for treating or delaying the progression of a disease, disorder, or condition associated with ANGPTL3 expression in a subject in need thereof. [Example]

[0197] While the present disclosure has been described with reference to specific embodiments illustrated in the following examples, those skilled in the art will recognize that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the present disclosure. Furthermore, the following examples are presented for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Furthermore, particular conditions, materials, compositions of matter, processes, and process steps may be modified to adapt them to the objective, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the present disclosure. Standard techniques well known in the art or those specifically described below are utilized.

[0198] Example 1: Preparation of double-stranded RNAi oligonucleotides

[0199] Oligonucleotide synthesis and purification

[0200] The ds RNAi oligonucleotides described in the preceding examples are chemically synthesized using methods described herein. Generally, ds RNAi oligonucleotides are synthesized using solid-phase oligonucleotide synthesis methods described for 19-mer to 23-mer siRNAs (see, e.g., Scaringe et al. (1990) Nucleic Acids Res. 18:5433-5441, and Usman et al. (1987) J. Am. Chem. Soc. 109:7845-7845; see also U.S. Patent Nos. 5,804,683, 5,831,071, 5,998,203, 6,008,400, 6,111,086, 6,117,657, 6,353,098, 6,362,323, 6,437,117, and 6,469,158).

[0201] Individual RNA strands are synthesized and HPLC-purified according to standard methods (Integrated DNA Technologies, Coralville, IA). For example, RNA oligonucleotides are synthesized using solid-phase phosphoramidite chemistry, deprotected, and desalted on a NAP-5 column (Amersham Pharmacia Biotech, Piscataway, NJ) using standard techniques (Damha & Olgivie (1993) Methods Mol. Biol. 20:81-114; Wincott et al. (1995) Nucleic Acids Res. 23:2677-2684). Oligomers are purified using ion-exchange high-performance liquid chromatography (IE-HPLC) on an Amersham Source 15Q column (1.0 cm x 25 cm, Amersham Pharmacia Biotech) using a 15-minute linear step gradient. The gradient changes from 90:10 buffer A:B to 52:48 buffer A:B. where Buffer A is 100 mM Tris, pH 8.5, and Buffer B is 100 mM Tris, pH 8.5, 1 M NaCl. Samples are monitored at 260 nm, and peaks corresponding to full-length oligonucleotide species are collected, pooled, desalted on a NAP-5 column, and lyophilized.

[0202] The purity of each oligomer is determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc.; Fullerton, CA). The CE capillary has an inner diameter of 100 μm and contains ssDNA 100R Gel (Beckman-Coulter). Typically, approximately 0.6 nmoles of oligonucleotide is injected into the capillary, run at an electric field of 444 V / cm, and detected by UV absorbance at 260 nm. Denaturing Tris-borate-7M-urea flow buffer is purchased from Beckman-Coulter. Oligoribonucleotides that are at least 90% pure, as assessed by CE, are obtained for use in the experiments described below. Compound identity is verified by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry on a Voyager DE™ Biospectometry Work Station (Applied Biosystems; Foster City, CA) following the manufacturer's recommended protocol. Relative molecular weights of all oligomers are obtained, often within 0.2% of the expected molecular weight.

[0203] Preparation of duplexes

[0204] For example, resuspend ssRNA oligomers (e.g., at a concentration of 100 μM) in a double buffer consisting of 100 mM potassium acetate, 30 mM HEPES, pH 7.5. Complementary sense and antisense strands are mixed in equimolar amounts to obtain a final solution of, for example, 50 μM duplex. Heat the sample to 100°C for 5 minutes in RNA buffer (IDT) and allow to cool to room temperature before use. Store dsRNA oligonucleotides at -20°C. Store ssRNA oligomers at -80°C, either lyophilized or in nuclease-free water.

[0205] Example 2: Inhibition of ANGPTL3 Expression In Vitro by RNAi Oligonucleotides

[0206] Identification of ANGPTL3 target sequences

[0207] To identify RNAi oligonucleotide inhibitors of ANGPTL3 expression, a computer-based algorithm is used to generate ANGPTL3 target sequences suitable for assaying inhibition of ANGPTL3 expression via the RNAi pathway. The algorithm provides RNAi oligonucleotide guide strand sequences complementary to suitable ANGPTL3 target sequences of human ANGPTL3 mRNA (e.g., SEQ ID NO: 128, Table 1). Exemplary target sequences of human ANGPTL3 mRNA are shown in Table 2. Some of the guide strand sequences identified by the algorithm are also complementary to the corresponding monkey ANGPTL3 target sequences and / or mouse ANGPTL3 mRNA (SEQ ID NOs: 129 and 130, respectively, Table 1). 384 ds RNAi oligonucleotides (formatted as DsiRNA oligonucleotides) are generated, each with a unique guide strand having a region complementary to the ANGPTL3 target sequence identified by the algorithm.

[0208] [Table 1]

[0209] [Table 2]

[0210] In vitro cell-based assays

[0211] The ability of each of the 384 DsiRNAs to inhibit ANGPTL3 expression was determined using an in vitro cell-based assay. Briefly, HuH-7 human hepatocytes stably expressing ANGPTL3 were transfected with each DsiRNA (0.5 nM) in separate wells of a multi-well cell culture plate. The cells were maintained for 24 hours after transfection, and the remaining ANGPTL3 mRNA levels from the transfected cells were then determined using a TAQMAN®-based qPCR assay. Two qPCR assays, a 3' assay and a 5' assay, were used to determine mRNA levels measured by HEX and FAM probes, respectively.

[0212] The results of a HuH-7 cell-based assay using 384 DsiRNAs are shown in Figures 1 and 2. Figure 1 shows the results of a HuH-7 cell-based assay using 109 DsiRNAs with guide strands complementary to human, monkey, and mouse ANGPTL3 mRNA ("Triple Common"). Transfections of the triple common DsiRNAs that result in 35% or less of ANGPTL3 mRNA remaining in cells compared to the negative control are considered candidate inhibitors of ANGPTL3 expression (referred to herein as "hits"). Figure 2 shows the results of a HuH-7 cell-based assay using 275 DsiRNAs with guide strands complementary to human and monkey ANGPTL3 mRNA ("human-monkey"). Human-monkey DsiRNAs that result in 30% or less of ANGPTL3 mRNA remaining compared to the negative control are also considered hits. Figures 1 and 2 show the percentage of remaining mRNA in the 3' assay (circles) and 5' assay (diamonds), respectively.

[0213] These results demonstrate that DsiRNAs designed to target human ANGPTL3 mRNA inhibit ANGPTL3 expression in cells (as determined by a decrease in the amount of ANGPTL3 mRNA in DsiRNA-transfected cells), and that nucleotide sequences containing DsiRNA hits are useful for generating RNAi oligonucleotides that inhibit ANGPTL3 expression. Furthermore, these results demonstrate that multiple ANGPTL3 target sequences are suitable for RNAi-mediated inhibition of ANGPTL3 expression.

[0214] Example 3: Inhibition of ANGPTL3 expression in vivo by RNAi oligonucleotides

[0215] Of the 384 DsiRNAs screened in the HuH-7 cell-based assay described in Example 2, the nucleotide sequences of 55 DsiRNA hits (Table 3) were selected for further in vivo evaluation. Briefly, the nucleotide sequences of the 55 selected DsiRNAs were used to generate 55 corresponding double-stranded RNAi oligonucleotides containing a nicked tetraloop-GalNAc-linked structure with a 36-mer passenger strand and a 22-mer guide strand (referred to herein as "GalNAc-linked ANGPTL3 oligonucleotides"). Furthermore, the nucleotide sequences containing the passenger and guide strands of the GalNAc-linked ANGPTL3 oligonucleotides have distinct patterns of modified nucleotides and phosphorothioate linkages (see, e.g., Figure 3, which is a schematic diagram of the general structure and chemical modification pattern of GalNAc-linked ANGPTL3 oligonucleotides). Three adenosine nucleotides comprising the tetraloop are each linked to a GalNAc moiety. (CAS Number: 14131-60-3)

[0216] [Table 3-1] [Table 3-2]

[0217] Mouse studies

[0218] The GalNAc-conjugated ANGPTL3 oligonucleotides shown in Table 3 are evaluated in mice engineered to transiently express human ANGPTL3 mRNA in hepatic parenchymal cells. Three GalNAc-conjugated ANGPTL3 oligonucleotides (ANGPTL3-0204-M2, ANGPTL3-0327-M2, and ANGPTL3-1327-M2) are used as benchmark controls. Briefly, 6- to 8-week-old female CD-1 mice are subcutaneously treated with GalNAc-conjugated ANGPTL3 oligonucleotides at a dose level of 1 mg / kg. Three days later (72 hours), the mice are hydrodynamically injected with a DNA plasmid encoding the complete human ANGPTL3 gene under the control of the ubiquitous cytomegalovirus (CMV) promoter sequence. One day after plasmid transfer, liver samples are collected. Total RNA from these mice is compared with mice treated with an equal volume of PBS alone for qRT-PCR analysis of ANGPTL3 mRNA. Values ​​are normalized to transfection efficiency using the NeoR gene contained in the plasmid.

[0219] As shown in Figure 4, all GalNAc-linked ANGPTL3 oligonucleotides tested inhibit ANGPTL3 expression, as determined by a decrease in the amount of ANGPTL3 mRNA in liver samples from oligonucleotide-treated mice compared with PBS-treated mice. The average percent remaining ANGPTL3 mRNA in liver samples from mice treated with the benchmark GalNAc-linked ANGPTL3 oligonucleotide ANGPTL3-0327 relative to PBS-treated mice is shown as a black bar. Figure 4 shows that 26 of the 55 GalNAc-linked ANGPTL3 oligonucleotides tested inhibit ANGPTL3 expression to a greater extent than the benchmark GalNAc-linked ANGPTL3 oligonucleotide ANGPTL3-0327. Based on these results, 10 of the 55 GalNAc-linked ANGPTL3 oligonucleotides, indicated by arrows in Figure 4 and listed in Table 4, were selected for evaluation of their ability to inhibit ANGPTL3 expression in NHPs. The 10 GalNAc-linked ANGPTL3 oligonucleotides listed in Table 4 contain chemically modified nucleotides with either pattern M1 or M2, as described in Figure 3.

[0220] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13] [Table 4-14] [Table 4-15] [Table 4-16] [Table 4-17] [Table 4-18] [Table 4-19] [Table 4-20] [Table 4-21] In the modification patterns of Table A, "M" refers to a 2'-OMe modified nucleotide. "F" refers to a 2'-F modified nucleotide. "S" refers to a nucleotide with a 3'-phosphorothioate linkage. "{MS}" refers to a 2'-OMe modified nucleotide with a 3'-phosphorothioate linkage. "{FS}" refers to a 2'-F modified nucleotide with a 3'-phosphorothioate linkage. "[adem-GalNAc]" refers to a nucleotide having a 2'-GalNAc linkage. [ka] "{Px-MS}" refers to a 2'-OMe modified nucleotide with a 3'-phosphorothioate linkage and a 5' phosphonate. In the modified sequences of Table A: "mN" refers to a 2'-OMe modified nucleotide. "[fN]" refers to a 2'-F modified nucleotide. "[mNs]" refers to 2'-OMe modified nucleotides with 3'-phosphorothioate linkages. "[fNs]" refers to 2'-F modified nucleotides with 3'-phosphorothioate linkages. "[ademG-GalNAc]" refers to a G nucleotide with a 2'-GalNAc linkage. [ka] "[ademA-GalNAc]" refers to an A nucleotide with a 2'-GalNAc linkage. [ka] "[Mephosphonate-4O-mUs]" refers to 5'-phosphonate-4'-oxy-2'-OMe uridine with a 3'-phosphorothioate linkage. [ka]

[0221] Non-human primate (NHP) research

[0222] The GalNAc-conjugated ANGPTL3 oligonucleotides listed in Table 4 were evaluated in cynomolgus monkeys (Macaca fascicularis). In this study, NHPs were grouped so that the mean body weight (approximately 5.4 kg) was comparable between the control and experimental groups. Each cohort included two male and three female subjects. GalNAc-conjugated ANGPTL3 oligonucleotides were administered subcutaneously on study day 0. Blood samples were collected on study days -8, -5, and 0, as well as weekly after administration. Ultrasound-guided core needle liver biopsies were collected on study days 28, 56, and 84. At each time point, total RNA from the liver biopsy samples was analyzed by qRT-PCR to measure ANGPTL3 mRNA in oligonucleotide-treated NHPs compared to NHPs treated with an equivalent volume of PBS. To normalize the data, measurements were performed relative to the geometric mean of two reference genes, PPIB and 18S rRNA. As shown in Figure 5A (day 28), Figure 5B (day 56), and Figure 5C (day 84), treatment of NHPs with the GalNAc-conjugated ANGPTL3 oligonucleotides listed in Table 4 inhibits ANGPTL3 expression in the liver, as determined by a reduction in the amount of ANGPTL3 mRNA in liver samples from oligonucleotide-treated NHPs compared with PBS-treated NHPs. The mean percent reduction in ANGPTL3 mRNA in liver samples from treated NHPs is shown above the set of data points for each treatment group, and a plot of the mean values ​​over time is shown in Figure 6. At all time points evaluated, ANGPTL3-1412 inhibits ANGPTL3 expression to a greater extent than the benchmark GalNAc-conjugated ANGPTL3 oligonucleotide, ANGPTL3-0327. Inhibition of ANGPTL3 expression was also determined from the same NHP study by measuring ANGPTL3 protein in serum prepared from predose and weekly blood samples by ELISA. A significant decrease in serum ANGPTL3 protein is observed in NHPs treated with GalNAc-conjugated ANGPTL3 oligonucleotides compared to NHPs treated with PBS, as shown in Figure 7. The values ​​of the three pre-treatment samples were averaged and set to 100%, and data are reported as relative values ​​compared to the pre-treatment mean.Taken together, these results demonstrate that treatment of NHPs with GalNAc-conjugated ANGPTL3 oligonucleotides reduces the amount of ANGPTL3 mRNA in the liver and concomitantly reduces the amount of ANGPTL3 protein in the serum.

[0223] Taken together, these results demonstrate that GalNAc-linked ANGPTL3 oligonucleotides designed to target human ANGPTL3 mRNA inhibit ANGPTL3 expression in vivo (as determined by reduced levels of ANGPTL3 mRNA and ANGPTL3 protein in treated animals).

[0224] Sequence Listing The following nucleic acid and / or amino acid sequences are set forth in the above disclosure and are provided below by reference.

[0225] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

Claims

1. An oligonucleotide that reduces the expression of ANGPTL3, comprising an antisense strand comprising a sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, and 116.

2. 2. The oligonucleotide of claim 1, comprising a sense strand comprising a sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115.

3. The oligonucleotide of claim 1 or 2, wherein the antisense strand comprises a sequence set forth in any one of SEQ ID NOs: 100, 102, 104, 20, 26, 50, 72, 74, 76, 80, and 114.

4. The oligonucleotide of claim 2 or 3, wherein the sense strand comprises a sequence set forth in any one of SEQ ID NOs: 99, 101, 103, 19, 25, 49, 71, 73, 75, 79, and 113.

5. 1. An oligonucleotide that reduces expression of ANGPTL3, comprising an antisense strand 15 to 30 nucleotides in length and a sense strand 15 to 40 nucleotides in length, wherein the antisense strand has a region complementary to a target sequence of ANGPTL3 set forth in any one of SEQ ID NOs: 125, 126, 127, 118, 119, 120, 121, 122, 123, 124, and 117, and the complementary region is at least 15 contiguous nucleotides in length.

6. 6. The oligonucleotide of claim 5, wherein the complementary region is perfectly complementary to the target sequence of ANGPTL3.

7. The oligonucleotide of any one of claims 1 to 6, wherein the antisense strand is 19 to 27 nucleotides in length.

8. 8. The oligonucleotide of any one of claims 1 to 7, wherein the antisense strand is 21 to 27 nucleotides in length, and optionally the antisense strand is 22 nucleotides in length.

9. The oligonucleotide according to any one of claims 2 to 8, wherein the sense strand forms a duplex region with the antisense strand.

10. 10. The oligonucleotide of claim 9, wherein the sense strand is 19 to 40 nucleotides in length, and optionally the sense strand is 36 nucleotides in length.

11. 11. The oligonucleotide of claim 9 or 10, wherein the duplex region is at least 19 nucleotides in length.

12. 12. The oligonucleotide of any one of claims 9 to 11, wherein the duplex region is at least 21 nucleotides in length, and optionally the duplex region is 20 nucleotides in length.

13. The oligonucleotide of any one of claims 5 to 12, wherein the region complementary to ANGPTL3 is at least 19 contiguous nucleotides in length.

14. The oligonucleotide of any one of claims 5 to 13, wherein the region complementary to ANGPTL3 is at least 21 contiguous nucleotides in length.

15. 15. The oligonucleotide of any one of claims 5 to 14, wherein the antisense strand comprises a sequence set forth in any one of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, and 116.

16. The oligonucleotide of any one of claims 5 to 15, wherein the sense strand comprises a sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115.

17. The oligonucleotide of any one of claims 5 to 16, wherein the antisense strand comprises a sequence set forth in any one of SEQ ID NOs: 100, 102, 104, 20, 26, 50, 72, 74, 76, 80, and 114.

18. The oligonucleotide of any one of claims 5 to 17, wherein the sense strand comprises a sequence set forth in any one of SEQ ID NOs: 99, 101, 103, 19, 25, 49, 71, 73, 75, 79, and 113.

19. The oligonucleotide of any one of claims 2 to 18, wherein the sense strand comprises a stem-loop at its 3' end shown as S1-L-S2, where S1 is complementary to S2, and L forms a loop of 3 to 5 nucleotides in length between S1 and S2.

20. An oligonucleotide that reduces the expression of ANGPTL3, comprising an antisense strand and a sense strand; the antisense strand is 21 to 27 nucleotides in length and has a region complementary to ANGPTL3; the sense strand comprises a stem-loop at its 3' end shown as S1-L-S2, where S1 is complementary to S2, and L forms a loop 3 to 5 nucleotides in length between S1 and S2; The oligonucleotide, wherein the antisense strand and the sense strand form a duplex structure having a length of at least 19 nucleotides, but they are not covalently linked.

21. 21. The oligonucleotide of claim 20, wherein the complementary region is perfectly complementary to at least 19 contiguous nucleotides of ANGPTL3 mRNA.

22. The oligonucleotide of any one of claims 19 to 21, wherein L is a tetraloop.

23. 23. The oligonucleotide of any one of claims 19 to 22, wherein L is 4 nucleotides in length.

24. The oligonucleotide of any one of claims 19 to 23, wherein L comprises the sequence written as GAAA.

25. 25. The oligonucleotide of any one of claims 5 to 24, wherein the antisense strand is 27 nucleotides in length and the sense strand is 25 nucleotides in length, or optionally, the antisense strand is 22 nucleotides in length and the sense strand is 36 nucleotides in length.

26. 26. The oligonucleotide of claim 25, wherein the antisense strand and the sense strand form a duplex region that is 25 nucleotides in length, optionally the duplex is 20 nucleotides in length.

27. The oligonucleotide of any one of claims 20 to 24, comprising a 3' overhang sequence on the antisense strand that is 2 nucleotides in length.

28. The oligonucleotide of any one of claims 9 to 18, wherein the oligonucleotide comprises an antisense strand and a sense strand, each ranging from 21 to 23 nucleotides in length.

29. 29. The oligonucleotide of claim 28, wherein the oligonucleotide has a double-stranded structure ranging from 19 to 21 nucleotides in length.

30. 30. The oligonucleotide of claim 28 or 29, wherein the oligonucleotide comprises a 3' overhang sequence of one or more nucleotides in length, the 3' overhang sequence being present in the antisense strand, the sense strand, or the antisense strand and the sense strand.

31. 30. The oligonucleotide of claim 28 or 29, wherein the oligonucleotide comprises a 3' overhang sequence 2 nucleotides in length, the 3' overhang sequence being present in the antisense strand, the sense strand being 21 nucleotides in length, and the antisense strand being 23 nucleotides in length, such that the sense strand and the antisense strand form a duplex 21 nucleotides in length.

32. 10. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified nucleotide.

33. 33. The oligonucleotide of claim 32, wherein the modified nucleotide comprises a 2' modification.

34. 34. The oligonucleotide of claim 33, wherein the 2'-modification is a modification selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid.

35. The oligonucleotide according to any one of claims 32 to 34, wherein all of the nucleotides of the oligonucleotide are modified.

36. 10. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one modified internucleotide bond.

37. 37. The oligonucleotide of claim 36, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

38. 10. The oligonucleotide of any one of the preceding claims, wherein the 4'-carbon of the sugar of the 5' nucleotide of the antisense strand comprises a phosphate analogue.

39. 39. The oligonucleotide of claim 38, wherein the phosphate analog is an oxymethylphosphonate, a vinylphosphonate, or a malonylphosphonate.

40. 10. The oligonucleotide of claim 1, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands.

41. 41. The oligonucleotide of claim 40, wherein each targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a lipid.

42. 41. The oligonucleotide of claim 40, wherein each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

43. 43. The oligonucleotide of claim 42, wherein the GalNAc moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

44. 25. The oligonucleotide of any one of claims 19 to 24, wherein up to four nucleotides of L of the stem loop are each linked to a monovalent GalNAc moiety.

45. 10. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide is an RNAi oligonucleotide.

46. 10. A pharmaceutical composition comprising an oligonucleotide according to any one of the preceding claims and a pharmaceutically acceptable carrier, delivery agent or excipient.

47. 47. A method of delivering an oligonucleotide to a subject, comprising administering to the subject the pharmaceutical composition of claim 46.

48. 1. A method of reducing expression of ANGPTL3 in a cell, a cell population, or a subject, comprising: i. contacting said cell or said cell population with the oligonucleotide of any one of claims 1 to 45 or the pharmaceutical composition of claim 46; ii. administering to the subject the oligonucleotide of any one of claims 1 to 45 or the pharmaceutical composition of claim 46.

49. 49. The method of claim 48, wherein reducing the expression of ANGPTL3 comprises reducing the amount or level of ANGPTL3 mRNA, the amount or level of ANGPTL3 protein, or both.

50. 47. A method for reducing the amount or level of triglycerides (TG) in a subject, the method comprising administering to the subject an oligonucleotide according to any one of claims 1 to 45 or a pharmaceutical composition according to claim 46.

51. 47. A method for reducing the amount or level of cholesterol in a subject, the method comprising administering to the subject an oligonucleotide according to any one of claims 1 to 45 or a pharmaceutical composition according to claim 46.

52. 52. The method of any one of claims 48 to 51, wherein the subject has a disease, disorder, or condition associated with expression of ANGPTL3.

53. A method for treating a subject having a disease, disorder, or condition associated with expression of ANGPTL3, comprising administering to the subject a therapeutically effective amount of the oligonucleotide of any one of claims 1 to 45 or the pharmaceutical composition of claim 46, thereby treating the subject.

54. 1. A method for treating a subject having a disease, disorder, or condition associated with expression of ANGPTL3, comprising administering to the subject a therapeutically effective amount of an oligonucleotide or a pharmaceutical composition thereof, the oligonucleotide comprising a sense strand of 15 to 50 nucleotides in length and an antisense strand of 15 to 30 nucleotides in length, wherein the sense strand forms a duplex region with the antisense strand, and the sense strand is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115, and the antisense strand is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, and 116, thereby treating the subject.

55. A method for treating a subject having a disease, disorder, or condition associated with expression of ANGPTL3, comprising administering to the subject a therapeutically effective amount of an oligonucleotide comprising a pair of sense and antisense strands selected from the rows shown in Table 5, or a pharmaceutical composition thereof, thereby treating the subject.

56. 56. The method of any one of claims 52 to 55, wherein the disease, disorder, or condition associated with ANGPTL3 expression is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, dyslipidemia and / or cholesterol metabolism disorders, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, and statin-resistant hypercholesterolemia.

57. 57. The method of claim 56, wherein the disease, disorder, or condition associated with expression of ANGPTL3 is cardiovascular disease, type II diabetes, hypertriglyceridemia, NASH, obesity, or a combination thereof.

58. 58. The method of any one of claims 53 to 57, wherein the oligonucleotide or pharmaceutical composition is administered in combination with a second composition or therapeutic agent.

59. Use of the oligonucleotide of any one of claims 1 to 45 or the pharmaceutical composition of claim 46 in the manufacture of a medicament for the treatment of a disease, disorder or condition associated with expression of ANGPTL3.

60. An oligonucleotide according to any one of claims 1 to 45 or a pharmaceutical composition according to claim 46 for use or adapted for use in the treatment of a disease, disorder or condition associated with expression of ANGPTL3.

61. 46. ​​A kit comprising the oligonucleotide of any one of claims 1 to 45 and an optional pharmaceutically acceptable carrier, and a package insert containing instructions for administration to a subject having a disease, disorder, or condition associated with ANGPTL3 expression.

62. 62. The use of claim 59, the oligonucleotide or pharmaceutical composition for use of claim 60, or the kit of claim 61, wherein the disease, disorder, or condition associated with ANGPTL3 expression is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, dyslipidemia and / or cholesterol metabolism disorders, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, and statin-resistant hypercholesterolemia.

63. 62. The use of claim 59, the oligonucleotide or pharmaceutical composition for use of claim 60, or the kit of claim 61, wherein the disease, disorder, or condition associated with expression of ANGPTL3 is cardiovascular disease, type II diabetes, hypertriglyceridemia, NASH, obesity, or a combination thereof.

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