Compositions and methods for inhibiting angiopoietin-like protein-3 (angpts3) expression

Abstract

To provide compositions and methods effective for lipid management in patients with low LDL-R activity or no LDL-R activity.SOLUTION: To provide an ANGPTL3dsRNA agent for reducing the expression of angiopoietin-like 3 (ANGPTL3) gene and treating ANGPTL3-related diseases and conditions, an ANGPTL3 antisense polynucleotide agent, a composition comprising the ANGPTL3dsRNA agent, and a composition comprising the ANGPTL3 antisense polynucleotide agent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compositions and methods for inhibiting the expression of angiopoietin-like 3 protein (ANGPTL3). [Background technology]

[0002] Angiopoietin-like protein 3 (ANGPTS3) is a secreted protein expressed primarily in hepatocytes (Conklin et al. Identification of a mammalian angiopoietin-related protein expressed specifically in liver. Genomics 1999, 62:477-482). It is an inhibitor of lipoprotein lipase (LPL) and endothelial lipase (EL). By inhibiting LPL and EL, ANGPTL3 reduces triglyceride (TG) hydrolysis, particularly in muscle and adipose tissue (Kersten S. Physiological regulation of lipoprotein lipase. Biochem Biophys Acta 2014;1841:919-933 Shimamura et al. Angiopoitin-like protein 3 regulates plasma HDL cholesterol through suppression of endothelial lipase. Arterioscler. Thromb. Vasc. Biol. 2007;27:366-372). Therefore, inhibition of ANGPTL3 prevents inhibition of LPL and EL activity, leading to reductions in TG and high-density lipoprotein cholesterol (HDL-C). Inhibition of ANGPTL3 may also result in a reduction of low-density lipoprotein cholesterol (LDL-C) through EL-mediated VLDL processing (Adam, et al. Angiopoitin-like protein 3 governs LDL-cholesterol levels through endothelial lipase-dependent VLDL clearance. J Lipid Res 2020;61:1271-1286).It should be noted that currently, treatments that reduce LDL-C, such as statins and PCSK9 inhibitors, are LDL-R dependent and ineffective in patients with low or no residual LDL-R activity. LDL-C reduction by inhibiting ANGPTL3 is LDL-R independent and may be an effective therapeutic approach for lipid management in patients with low or no LDL-R activity.

[0003] Hyperlipidemia is closely associated with diseases including hypertension, atherosclerosis, heart disease, diabetes, and nonalcoholic steatohepatitis (NASH). Studies have shown that loss-of-function mutations in ANGPTL3 in humans have beneficial effects. Homozygous loss of function of ANGPTL3 causes familial combined hypolipidemia, characterized by low plasma levels of triglycerides, high-density lipoprotein (HDL) cholesterol, and LDL-C, as well as a reduced risk of coronary artery disease (Romeo et al., "Rare loss-of-function mutations in ANGPTL family members contribute to plasma triglyceride levels in humans," J. Clin. Invest., 2009, 119:70-79; Musunuru et al., "Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia," N Engl J Med, 2010, 363:2220-2227). ANGPTL3 has emerged as a promising drug target for treating diseases caused by hyperlipidemia, with therapeutic modalities including antibodies, antisense oligonucleotides (ASOs), and siRNAs under development. siRNA, especially GalNAc-conjugated siRNA, has been proven to be safe, effective, and have a long duration of activity. Therefore, new ANGPTL3 siRNA agents are needed to treat multiple diseases and disorders. Summary of the Invention [Problem to be solved by the invention]

[0004] According to one aspect of the present invention, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of angiopoietin-like 3 (ANGPTS3) is provided. The dsRNA agent comprises a sense strand and an antisense strand, and the antisense strand comprises a region complementary to an ANGPTS3 RNA transcript from nucleotides 2 through 18. The complementary region comprises at least 15 contiguous nucleotides that differ by 0, 1, 1, 2, or 3 nucleotides from any one of the antisense sequences shown in any one of Tables 1 through 5, and optionally includes a targeting ligand. In some embodiments, the region complementary to the ANGPTS3 RNA transcript comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences shown in any one of Tables 1 through 5. In certain embodiments, the antisense strand of the dsRNA is at least substantially complementary to a target region of any one of SEQ ID NO:235, shown in any one of Tables 1 through 5. In some embodiments, the antisense strand of the dsRNA is fully complementary to any of the target regions of SEQ ID NO:235, provided in any of Tables 1-5. In some embodiments, the dsRNA agent comprises a sense strand sequence that is at least substantially complementary to an antisense strand sequence in a dsRNA agent shown in any one of Tables 1-5. In certain embodiments, the dsRNA agent comprises a sense strand sequence that is fully complementary to an antisense strand sequence in a dsRNA agent shown in any one of Tables 1-5. In certain embodiments, the dsRNA agent comprises an antisense strand sequence shown in any one of Tables 1-5. In some embodiments, the dsRNA agent comprises a sequence shown in a double-stranded sequence shown in any one of Tables 1-5. In some embodiments, the antisense strand of the dsRNA consists of the following nucleotide sequence II:

[0005] [ka] wherein z1 is selected from the group consisting of c, g, a, and u, and z2 is nucleotide sequence IV.

[0006] In some embodiments, z1 is u. In some embodiments, nucleotide sequence IV is 0 to 15 nucleotides in length. In some embodiments, nucleotide sequence IV is selected from the group consisting of a, au, aa, ac, ag, auu, aua, auc, aug, auug, auuu, auua, auuc, auuuu, auuuug, auucuu, auucga, auuuuga, auuuugag, auuuugaga, and auuuugagacuucca.

[0007] In some embodiments, nucleotide sequence IV is 1, 2, 3, or 4 nucleotides in length. In some embodiments, nucleotide sequence IV is selected from the group consisting of a, au, aa, ac, ag, auu, aua, auc, aug, auug, auuu, auua, or auuc.

[0008] In one embodiment, the antisense strand of the dsRNA consists of the following nucleotide sequence II':

[0009] [ka] (wherein z1 is selected from the group consisting of c, g, a, and u; z 2’ is nucleotide sequence IV'.

[0010] In some embodiments, z1 is u. In some embodiments, nucleotide sequence IV' is 0 to 15 nucleotides in length. In some embodiments, nucleotide sequence IV' is 1, 2, 3, or 4 nucleotides in length. In some embodiments, nucleotide sequence IV' is selected from the group consisting of u, a, c, g, uu, ua, uc, ug, uug, uuu, uua, and uuc.

[0011] In some embodiments, the sense strand of the dsRNA consists of the following nucleotide sequence III:

[0012] [ka] wherein z3 is a nucleotide sequence V and z4 is selected from the group consisting of c, g, a, and u.

[0013] In some embodiments, z4 is a. In some embodiments, nucleotide sequence V is 0 to 15 nucleotides in length. In some embodiments, nucleotide sequence V is selected from the group consisting of u, au, uu, gu, cu, aau, uau, gau, cau, gaau, caau, aaau, uaau, aaaau, caaaau, ucaaaau, cucaaaau, ucucaaaau, and uggaagucucaaaau.

[0014] In certain embodiments, the nucleotide sequence V is 1, 2, 3, or 4 nucleotides in length. In some embodiments, the nucleotide sequence V is selected from the group consisting of u, au, uu, gu, cu, aau, uau, gau, cau, gaau, caau, aaau, and uaau.

[0015] In certain embodiments, the sense strand of the dsRNA consists of the following nucleotide sequence III':

[0016] [ka] (In the formula, z 3’ is a nucleotide sequence V', and z4 is selected from the group consisting of c, g, a, and u.

[0017] In some embodiments, z4 is a. In some embodiments, the nucleotide sequence V' is 1, 2, 3, or 4 nucleotides in length. In some embodiments, the nucleotide sequence V' is selected from the group consisting of a, u, g, c, aa, ua, ga, ca, gaa, caa, aaa, and uaa. In some embodiments, z1 is a nucleotide sequence complementary to z4. In some embodiments, z2 is a nucleotide sequence complementary to z3. In some embodiments, z 2’ is z 3’ nucleotide sequence complementary to nucleotide sequence II or II' above. In some embodiments, the dsRNA drug comprises a sense strand and an antisense strand, wherein the antisense strand of the dsRNA consists of nucleotide sequence II or II' above, and the sense strand has a length of 30 nucleotides or less and includes a region complementary to the antisense strand comprising at least 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the dsRNA drug comprises a sense strand and an antisense strand, wherein the sense strand of the dsRNA consists of nucleotide sequence III above, and the antisense strand of the dsRNA consists of nucleotide sequence II above. In some embodiments, the dsRNA drug comprises a sense strand and an antisense strand, wherein the sense strand of the dsRNA consists of nucleotide sequence III' above, and the antisense strand of the dsRNA consists of nucleotide sequence II' above.

[0018] In some embodiments, the dsRNA comprises a sense strand and an antisense strand comprising a complementary region containing at least 15 contiguous nucleotides, wherein the contiguous nucleotides differ by 0, 1, 2, or 3 nucleotides from any one of the following nucleotide sequences:

[0019] [ka]

[0020] In certain embodiments, the sense and antisense strands comprise a nucleotide sequence selected from the following:

[0021] [ka]

[0022] In some embodiments, the dsRNA agent comprises at least one modified nucleotide, hi certain embodiments, all or substantially all of the nucleotides in the antisense strand are modified nucleotides. In some embodiments, the at least one modified nucleotide includes a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide, a 2'-deoxyribonucleotide, a 2',3'-seconucleotide mimic, a locked nucleotide, an unlocked nucleotide (UNA), a glycol nucleic acid nucleotide (GNA), a 2'-F-arabinonucleotide, a 2'-methoxyethyl nucleotide, an abasic nucleotide, ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-OMe nucleotide, an inverted 2'-deoxyribonucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, and a 3'-OMe nucleotide, a 5'-phosphorothioate group-containing nucleotide, or a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a 2'-amino-modified nucleotide, a phosphoramidite, or a non-natural base-containing nucleotide. In some embodiments, the dsRNA agent includes an E-vinylphosphonate nucleotide at the 5' end of the guide strand. In some embodiments, the dsRNA agent comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the sense strand comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the antisense strand comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In some embodiments, all or substantially all of the nucleotides in both the sense strand and the antisense strand are modified nucleotides.In some embodiments, the modified sense strand has a modified sense strand sequence shown in any one of Tables 2-5. In some embodiments, the modified antisense strand has a modified antisense strand sequence shown in any one of Tables 2-5. In some embodiments, the sense strand is complementary or substantially complementary to the antisense strand, and the complementary region is 16-23 nucleotides in length. In some embodiments, the complementary region is 19-21 nucleotides in length. In some embodiments, the complementary region is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, each strand is 30 nucleotides or less in length. In some embodiments, each strand is 25 nucleotides or less in length. In some embodiments, each strand is 23 nucleotides or less in length. In some embodiments, each strand is 21 nucleotides or less in length. In some embodiments, the dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting groups or linking groups. In some embodiments, the one or more targeting groups or linking groups are conjugated to the sense strand. In some embodiments, the targeting group or linking group comprises N-acetylgalactosamine (GalNAc). In some embodiments, the targeting group has the following structure:

[0023] [ka] JPEG2026035574000008.jpg182149 JPEG2026035574000009.jpg192149 JPEG2026035574000010.jpg181149 JPEG2026035574000011.jpg189149 JPEG2026035574000012.jpg190149 JPEG2026035574000013.jpg183149 JPEG2026035574000014.jpg172149 JPEG2026035574000015.jpg192149 JPEG2026035574000016.jpg179149 JPEG2026035574000017.jpg135149

[0024] In some embodiments, the dsRNA agent comprises a targeting group conjugated to the 5'-end of the sense strand. In some embodiments, the dsRNA agent comprises a targeting group conjugated to the 3'-end of the sense strand. In some embodiments, the antisense strand comprises an inverted abasic residue at the 3'-end. In certain embodiments, the sense strand comprises one or two inverted abasic residues at the 3' and / or 5'-ends. In some embodiments, the dsRNA agent has two blunt ends. In some embodiments, at least one strand comprises a 3' overhanging end having at least one nucleotide. In some embodiments, at least one strand comprises a 3' overhanging end having at least two nucleotides. In some embodiments, the dsRNA comprises a duplex selected from AD00108, AD00108-1, AD00112, AD00112-1, AD00112-2, AD00133, AD00134, AD00135, AD00135-2, AD00136, AD00136-1, AD00142, AD00143, AD00143-2, AD00145, and AD00146.

[0025] In some embodiments, the dsRNA comprises a duplex selected from AD00112, AD00112-1, AD00112-2, AD00135, AD00135-2, AD00136, and AD00136-1.

[0026] In some embodiments, the dsRNA comprises a duplex selected from AD00112-1, AD00112-2, AD00135-2, and AD00136-1.

[0027] In certain embodiments, the sense and antisense strands of the dsRNA comprise a nucleotide sequence and modification selected from the following:

[0028] [ka] JPEG2026035574000019.jpg153149

[0029] According to one aspect of the present invention, there is provided a composition comprising any of the dsRNA agents of the present invention in the above-described embodiments. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises one or more additional therapeutic agents. In some embodiments, the composition is packaged in a kit, container, pack, dispenser, pre-filled syringe, or vial. In some embodiments, the composition is prepared for subcutaneous or intravenous (IV) administration.

[0030] According to another aspect of the invention, there is provided a cell comprising any of the dsRNA agents of the invention in the above-described embodiments. In some embodiments, the cell is a mammalian cell, optionally a human cell.

[0031] According to another aspect of the present invention, there is provided a method for inhibiting expression of the ANGPTL3 gene in cells, comprising the step (i) of preparing cells comprising an effective amount of any of the dsRNA agents of the present invention in the above-described embodiments or any of the compositions of the present invention described above. In some embodiments, the method further comprises the step (ii) of inhibiting expression of the ANGPTL3 gene in the cells by maintaining the prepared cells for a time sufficient to achieve degradation of mRNA transcripts of the ANGPTL3 gene. In some embodiments, the cells are present in a subject, and the dsRNA agent is administered to the subject subcutaneously. In some embodiments, the cells are present in a subject, and the dsRNA agent is administered to the subject by intravenous (IV) administration. In some embodiments, the method further comprises assessing the inhibition of the ANGPTL3 gene after administering the dsRNA agent to the subject. Here, the evaluation means includes (i) identifying one or more physiological characteristics of an ANGPTL3-related disease or disorder in the subject, and (ii) comparing the identified physiological characteristics with a baseline pre-treatment physiological characteristic of the ANGPTL3-related disease or disorder and / or a control physiological characteristic of the ANGPTL3-related disease or disorder, wherein the comparison indicates one or more of the presence or absence of inhibition of ANGPTL3 gene expression in the subject. In some embodiments, the identified physiological characteristics are one or more selected from the group consisting of serum lipid levels in the subject, serum HDL levels in the subject, HDL / LDL ratio in the subject, serum triglyceride levels in the subject, and liver fat content in the subject. In some embodiments, a decrease in one or more selected from the group consisting of serum lipid levels in the subject, serum HDL levels in the subject, serum triglyceride levels in the subject, and liver fat content in the subject indicates a reduction in ANGPTL3 gene expression in the subject.

[0032] According to another aspect of the present invention, there is provided a method for inhibiting expression of the ANGPTL3 gene in a subject, comprising administering to the subject an effective amount of a dsRNA agent according to the above-described embodiments of the present invention or a composition according to the above-described embodiments of the present invention. In some embodiments, the dsRNA agent is administered subcutaneously to the subject. In some embodiments, the dsRNA agent is administered to the subject via intravenous (IV) administration. In some embodiments, the method further comprises assessing the inhibition of the ANGPTL3 gene after administering the dsRNA agent. Here, the assessment step includes (i) identifying one or more physiological characteristics of an ANGPTL3-related disease or disorder in the subject, and (ii) comparing the identified physiological characteristics with baseline physiological characteristics before treatment of the ANGPTL3-related disease or disorder and / or control physiological characteristics of the ANGPTL3-related disease or disorder. The comparison indicates one or more of the presence or absence of inhibition of ANGPTL3 gene expression in the subject. In some embodiments, the identified physiological characteristic is one or more selected from the group consisting of serum lipid levels in the subject, serum HDL levels in the subject, HDL / LDL ratio in the subject, serum triglyceride levels in the subject, and liver fat content in the subject. In some embodiments, a decrease in one or more selected from the group consisting of serum lipid levels in the subject, serum HDL levels in the subject, serum triglyceride levels in the subject, and liver fat content in the subject indicates a decrease in ANGPTL3 gene expression in the subject.

[0033] According to another aspect of the present invention, there is provided a method for treating a disease or disorder associated with the presence of angiopoietin-like protein 3 (ANGPTL3), comprising suppressing ANGPTL3 gene expression by administering to a subject an effective amount of any of the dsRNA agents of the present invention in the above embodiments or any of the compositions of the present invention in the above embodiments. In some embodiments, the disease or disorder is one or more selected from the group consisting of hyperlipidemia, hypertriglyceridemia, dyslipidemia and / or cholesterol metabolism disorders, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, cardiometabolic disease, obesity, atherosclerosis, type 2 diabetes, cardiovascular disease, coronary artery disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, and pancreatitis caused by hypertriglyceridemia. In some embodiments, the method further comprises administering another treatment scheme to the subject. In some embodiments, the other treatment scheme includes treatment for an ANGPTL3-related disease or disorder. In some embodiments, another treatment scheme includes administering one or more ANGPTL3 antisense polynucleotides of the present invention to a subject, administering a non-ANGPTL3 dsRNA therapeutic agent to a subject, and modifying behavior in the subject. In some embodiments, the non-ANGPTL3 dsRNA therapeutic agent is one or more selected from the group consisting of (i) statins, (ii) one or more of PCSK9 siRNA molecules, antibodies, and antisense oligonucleotides (ASOs) capable of reducing PCSK9 expression, (iii) therapeutic agents capable of reducing lipid accumulation in a subject, and (iv) therapeutic agents capable of reducing cholesterol level and / or accumulation in a subject. In some embodiments, the dsRNA agent is administered subcutaneously to the subject. In some embodiments, the dsRNA agent is administered intravenously (IV) to the subject. In some embodiments, the method further includes determining the efficacy of the administered double-stranded ribonucleic acid (dsRNA) agent in the subject.In some embodiments, the method for determining the efficacy of a treatment in a subject includes (i) identifying one or more physiological characteristics of an ANGPTL3-related disease or disorder in the subject, and (ii) comparing the identified physiological characteristics with a baseline pre-treatment physiological characteristic of the ANGPTL3-related disease or disorder. The comparison indicates one or more of the following: the presence, absence, and level of efficacy of administering a double-stranded ribonucleic acid (dsRNA) agent to the subject. In some embodiments, the identified physiological characteristics are serum lipid levels in the subject, HDL levels in the subject, HDL / LDL ratio in the subject, serum triglyceride levels in the subject, and liver fat content in the subject. In some embodiments, a reduction in one or more of the following: serum lipid levels in the subject, serum HDL levels in the subject, serum triglyceride levels in the subject, and liver fat content in the subject indicates efficacy of administering a double-stranded ribonucleic acid (dsRNA) agent to the subject.

[0034] According to another aspect of the present invention, there is provided a method of reducing the level of angiopoietin-like protein 3 (ANGPTL3) in a subject compared to the baseline pre-treatment level of angiopoietin-like protein 3 (ANGPTL3) in the subject, comprising administering to the subject an effective amount of any dsRNA agent of the invention in any of the above-described embodiments or any composition of the invention in any of the above-described embodiments, thereby reducing the level of ANGPTL3 gene expression. In some embodiments, the dsRNA agent is administered to the subject subcutaneously or intravenously (IV).

[0035] According to another aspect of the present invention, there is provided a method for altering physiological characteristics of an ANGPTL3-related disease or disorder in a subject compared to the subject's baseline pre-treatment physiological characteristics of the ANGPTL3-related disease or disorder, comprising administering to the subject an effective amount of any of the dsRNA agents of the present invention in the above-described embodiments or any of the compositions of the present invention in the above-described embodiments, thereby altering the physiological characteristics of the ANGPTL3-related disease or disorder in the subject. In some embodiments, the dsRNA agent is administered to the subject subcutaneously or intravenously (IV). In some embodiments, the physiological characteristics are one or more selected from the group consisting of serum lipid levels in the subject, HDL levels in the subject, HDL / LDL ratio in the subject, serum triglyceride levels in the subject, and liver fat content in the subject.

[0036] According to another aspect of the present invention, the dsRNA agent is provided for use in a method for treating a disease or disorder associated with the presence of angiopoietin-like protein 3 (ANGPTL3).In some embodiments, the disease or disorder is one or more selected from the group consisting of hyperlipidemia, hypertriglyceridemia, dyslipidemia and / or cholesterol metabolism disorder, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, cardiometabolic disease, obesity, atherosclerosis, type 2 diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, and pancreatitis caused by hypertriglyceridemia.

[0037] According to another aspect of the present invention, there is provided an antisense polynucleotide agent for inhibiting expression of angiopoietin-like protein 3 (ANGPTL3), comprising 10 to 30 contiguous nucleotides, wherein at least one of the contiguous nucleotides is a modified nucleotide, and the nucleotide sequence of the agent has about 80% complementarity over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:235. In some embodiments, the equivalent region is any one of the target regions of SEQ ID NO:235, and the complementary sequence is a sequence provided in any one of Tables 1-5. In some embodiments, the antisense polynucleotide agent comprises any one of the antisense sequences shown in any one of Tables 1-5.

[0038] According to another aspect of the present invention, there is provided a composition comprising any of the antisense polynucleotide agents in the above embodiments. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises one or more additional therapeutic agents for treating an ANGPTL3-related disease or disorder. In some embodiments, the composition is packaged in a kit, container, pack, dispenser, pre-filled syringe, or vial. In some embodiments, the composition is prepared for subcutaneous or intravenous (IV) administration.

[0039] According to another aspect of the present invention, there is provided a cell comprising any of the antisense polynucleotide agents of the above embodiments. In some embodiments, the cell is a mammalian cell, optionally a human cell.

[0040] According to another aspect of the present invention, there is provided a method for inhibiting expression of the ANGPTL3 gene in a cell, comprising the step (i) of preparing a cell comprising an effective amount of any of the antisense polynucleotide agents described in the above embodiments. In some embodiments, the method further comprises the step (ii) of inhibiting expression of the ANGPTL3 gene in the cell by maintaining the cells prepared in step (i) for a time sufficient to achieve degradation of mRNA transcripts of the ANGPTL3 gene.

[0041] According to another aspect of the present invention, there is provided a method for inhibiting expression of the ANGPTL3 gene in a subject, the method comprising administering to the subject an effective amount of any of the antisense polynucleotide agents in the above embodiments.

[0042] According to another aspect of the present invention, there is provided a method for treating a disease or disorder associated with the presence of angiopoietin-like protein 3 (ANGPTL3), comprising inhibiting expression of the ANGPTL3 gene by administering to a subject an effective amount of any of the antisense polynucleotide agents of the present invention according to any of the above embodiments or any of the compositions according to the above embodiments. In some embodiments, the disease or disorder is one or more of hyperlipidemia, hypertriglyceridemia, dyslipidemia and / or dyscholesterolemia, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, cardiometabolic disease, obesity, atherosclerosis, type 2 diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, and pancreatitis due to hypertriglyceridemia.

[0043] According to another aspect of the present invention, there is provided a method of reducing the level of angiopoietin-like protein 3 (ANGPTL3) in a subject compared to the baseline pre-treatment level of angiopoietin-like protein 3 (ANGPTL3) in the subject, comprising administering to the subject an effective amount of any antisense polynucleotide agent of the present invention in any of the above embodiments or any of the compositions in the above embodiments, thereby reducing the level of ANGPTL3 gene expression. In some embodiments, the antisense polynucleotide agent is administered to the subject by subcutaneous or intravenous (IV) administration.

[0044] According to another aspect of the present invention, there is provided an antisense polynucleotide agent for inhibiting expression of the ANGPTL3 gene, comprising 10 to 30 contiguous nucleotides, wherein at least one of the contiguous nucleotides is a modified nucleotide, and the nucleotide sequence of the agent is about 80% or about 85% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:235.

[0045] According to another aspect of the present invention, there is provided a method for altering physiological characteristics of an ANGPTL3-related disease or disorder in a subject, compared to baseline pre-treatment physiological characteristics of the ANGPTL3-related disease or disorder in the subject, comprising administering to the subject an effective amount of any of the antisense polynucleotide agents of the present invention according to the above embodiments or any of the compositions according to the above embodiments, thereby altering the physiological characteristics of the ANGPTL3 disease or disorder in the subject. In some embodiments, the antisense polynucleotide agent is administered to the subject by subcutaneous or intravenous (IV) administration. In some embodiments, the physiological characteristics are one or more selected from the group consisting of serum lipid levels in the subject, HDL levels in the subject, HDL / LDL ratio in the subject, serum triglyceride levels in the subject, and liver fat content in the subject.

[0046] Sequence Description SEQ ID NOs: 1-117, 484-514 are shown in Table 1 and are sense strand sequences.

[0047] SEQ ID NOs: 118-234, 515-545 are shown in Table 1 and are antisense strand sequences.

[0048] SEQ ID NO:235 is the Homo sapiens angiopoietin-like 3 (ANGPTS3) mRNA [NCBI Reference Sequence: NM_014495.4].

[0049] [ka]

[0050] SEQ ID NO:236 is Mus musculus angiopoietin-like 3 (ANGPTS3), mRNA [NCBI Reference Sequence: NM_013913.4].

[0051] [ka]

[0052] SEQ ID NOs: 237-336, 546-605 are shown in Table 2, where the chemical modifications are: upper case 2'-fluoro, lower case 2'-OMe, * phosphorothioate.

[0053] SEQ ID NOs:337-390 are shown in Table 3. Delivery molecules are represented by "GLX-_" at the 3' end of each sense strand. Chemical modifications include uppercase 2'-fluoro, lowercase 2'-OMe, and * phosphorothioate.

[0054] SEQ ID NOs: 391-444, 606-629 are shown in Table 4. Chemical modifications include uppercase 2'-fluoro, lowercase 2'-OMe, * phosphorothioate, and Invab reverse abasic.

[0055] SEQ ID NOs:445-482 are shown in Table 5. Chemical modifications include uppercase 2'-fluoro, lowercase 2'-OMe, * phosphorothioate, and Invab inverted abasic.

[0056] SEQ ID NO:483 is the predicted cynomolgus monkey (Macaca fascicularis) angiopoietin-like 3 (ANGPTS3), mRNA [NCBI Reference Sequence: XM_005543185.2].

[0057] [ka] [Brief explanation of the drawings]

[0058] [Figure 1] FIG. 1 is a graph showing the normalized percent change in ANG3 in monkey plasma relative to day 1 (before siRNA administration). [Figure 2] FIG. 2 is a graph showing the normalized percent change in HDL in monkey plasma relative to day 1 (before siRNA administration). [Figure 3] FIG. 3 is a graph showing the normalized percent change in LDL in monkey plasma relative to day 1 (before siRNA administration). [Figure 4] FIG. 4 is a graph showing the rate of change in normalized total cholesterol (TC) in monkey plasma relative to day 1 (before siRNA administration). [Figure 5] FIG. 5 is a graph showing the rate of change in normalized triglyceride (TG) in monkey plasma relative to day 1 (before siRNA administration). [Figure 6] 1 is a graph showing the normalized percent change in ANG3 in monkey plasma relative to baseline. [Figure 7] 1 is a graph showing the normalized percent change in HDL in monkey plasma relative to baseline (before siRNA administration). [Figure 8] 1 is a graph showing the normalized percent change in LDL in monkey plasma relative to baseline (before siRNA administration). [Figure 9] 1 is a graph showing the normalized percent change in total cholesterol (TC) in monkey plasma relative to baseline (before siRNA administration). [Figure 10] 1 is a graph showing the rate of change in normalized triglycerides (TG) in monkey plasma relative to baseline (before siRNA administration). DETAILED DESCRIPTION OF THE INVENTION

[0059] Some aspects of the present invention include RNAi agents, including but not limited to double-stranded (ds) RNAi agents, that can inhibit expression of the angiopoietin-like 3 (ANGPTS3) gene. Some aspects of the present invention also include compositions containing ANGPTL3 RNAi agents and methods for using the compositions. The ANGPTL3 RNAi agents disclosed herein may be linked to a delivery compound for delivery to cells, such as hepatocytes. Pharmaceutical compositions of the present invention can include at least one ds ANGPTL3 agent and a delivery compound. In some embodiments of the compositions and methods of the present invention, the delivery compound is a GalNAc-containing delivery compound. The ANGPTL3 RNAi agents delivered to cells can inhibit expression of the ANGPTL3 gene, thereby reducing the activity of the ANGPTL3 protein product of the gene in the cell. The dsRNAi agents of the present invention can be used to treat ANGPTL3-related diseases and conditions.

[0060] In some embodiments of the present invention, diseases or disorders associated with ANGPTL3 expression are treated in a cell or a subject by reducing the expression of ANGPTL3 in the cell or the subject, respectively. Some non-limiting examples of diseases and disorders treatable by reducing the activity of ANGPTL3 include hyperlipidemia, hypertriglyceridemia, dyslipidemia and / or cholesterol metabolism disorders, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, cardiometabolic disease, obesity, atherosclerosis, type 2 diabetes, cardiovascular disease, coronary artery disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, pancreatitis due to hypertriglyceridemia, or other diseases in which reducing the level and activity of angiopoietin-like protein 3 (ANGPTL3) is medically beneficial.

[0061] The following describes how to prepare and use compositions containing ANGPTL3 single-stranded (ssRNA) and dsRNA agents to inhibit ANGPTL3 gene expression, and compositions and methods for treating diseases and conditions caused by or regulated by ANGPTL3 gene expression. The term "RNAi" is known in the art and may also be referred to as "siRNA."

[0062] As used herein, the term "RNAi" refers to an agent that contains RNA and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. As known in the art, an RNAi target region refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during gene transcription, including the messenger RNA (mRNA), an RNA processing product that is the primary transcript. The targeted portion of the sequence has a length sufficient to function as a substrate for RNAi-directed cleavage, at least at or near that portion. The target sequence may be 8-30 nucleotides in length (including the endpoints), 10-30 nucleotides in length (including the endpoints), 12-25 nucleotides in length (including the endpoints), 15-23 nucleotides in length (including the endpoints), 16-23 nucleotides in length (including the endpoints), or 18-23 nucleotides in length (including the endpoints), including all shorter lengths within each specified range. In some embodiments of the present invention, the target sequence has a length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides. In some embodiments, the target sequence is 9 to 26 nucleotides in length (inclusive), including all subranges and integers therebetween. For example, in some embodiments of the present invention, the target sequence has a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, wherein the sequence is fully or at least substantially complementary to at least a portion of an RNA transcript of the ANGPTL3 gene, but is not limited thereto. Some aspects of the present invention include pharmaceutical compositions comprising one or more ANGPTL3 dsRNA agents and a pharmaceutically acceptable carrier. In certain embodiments of the present invention, ANGPTL3 RNAi as provided herein inhibits the expression of angiopoietin-like protein 3 (ANGPTL3).

[0063] As used herein, the term "dsRNA drug" refers to a composition comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of sequence-specifically degrading or inhibiting the translation of a messenger RNA (mRNA) transcript of a target mRNA. However, without intending to be limited to a particular theory, the dsRNA drug of the present invention may act via the RNA interference mechanism (i.e., by interacting with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) in mammalian cells to induce RNA interference), or any alternative mechanism or pathway. Methods for silencing genes in plant, invertebrate, and vertebrate cells are known in the art (see, e.g., (Sharp et al., Genes Dev. 2001, 15:485; Bernstein, et al., (2001) Nature 409:363; Nykanen, et al., (2001) Cell 107:309; and Elbashir, et al., (2001) Genes Dev. 15:188), the disclosures of each of which are incorporated herein by reference in their entireties. Gene silencing programs known in the art can be used in conjunction with the disclosure provided herein to inhibit the expression of ANGPTL3.

[0064] The dsRNA agents disclosed herein consist of a sense strand and an antisense strand, and include, but are not limited to, short interfering RNA (siRNA), RNAi agents, microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates. The antisense strand of the dsRNA agent disclosed herein is complementary to at least a portion of the targeted mRNA. It is recognized in the art that dsRNA duplex structures of different lengths can be used to inhibit the expression of target genes. For example, it is known that dsRNA duplex structures with 19, 20, 21, 22, and 23 base pairs effectively induce RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). It is also known in the art that short or long RNA duplex structures can effectively induce RNA interference. In certain embodiments of the invention, ANGPTL3 dsRNAs can each comprise at least one single strand of a minimum length of 21 nt, or can have a shorter double strand that is effective when compared to a dsRNA shown in Tables 1-5 by subtracting 1, 2, 3, or 4 nucleotides from one or both ends based on one of the sequences shown in any one of Tables 1-5. In some embodiments of the invention, ANGPTL3 dsRNA agents can have a subsequence of at least 15, 16, 17, 18, 19, or 20 or more contiguous nucleotides derived from one or more sequences shown in Tables 1-5, and their ability to inhibit expression of the ANGPTL3 gene differs by no more than 5, 10, 15, 20, 25, or 30% from the level of inhibition by a dsRNA containing the complete sequence, also referred to herein as the "parent" sequence.

[0065] In some embodiments, compositions and methods of the present invention comprise single-stranded RNA in the composition and / or administered to a subject. For example, an antisense strand shown in any one of Tables 1-5 may be present in or be administered to a subject to reduce the activity of an ANGPTL3 polypeptide and / or the expression of an ANGPTL3 gene in the subject. Tables 1-5 show the core extension sequences of the antisense and sense strands of several ANGPTL3 dsRNA agents. Single-stranded antisense molecules that can be included in certain compositions of the present invention and / or administered in certain methods of the present invention are referred to herein as "single-stranded antisense agents" or "antisense polynucleotide agents." Single-stranded sense molecules that can be included in certain compositions of the present invention and / or administered in certain methods of the present invention are referred to herein as "single-stranded sense agents" or "sense polynucleotide agents." As used herein, the term "sequence" refers to a polynucleotide sequence without chemical modifications or delivery compounds. For example, the sense strand shown in Table 1 [ka] is the base sequence represented by SEQ ID NO:337 in Table 3 and SEQ ID NO:391 in Table 4. Herein, SEQ ID NO:337 and SEQ ID NO:391 are shown together with their chemical modifications and delivery compounds. Each of the sequences disclosed herein can be labeled with a label symbol. For example, a single-stranded sense sequence can be labeled with "sense strand SS#," a single-stranded antisense sequence can be labeled with "antisense strand AS#," and a duplex comprising a sense strand and an antisense strand can be labeled with "duplex AD# / AV#."

[0066] Table 1 includes sense and antisense strands, and the labeled numbers of duplexes consisting of the sense and antisense strands are given in the same row in Table 1. Sense strand SEQ ID NOs: 69-117 contain random nucleobases (n) at positions 1, 2, 3, and 21. Antisense strand SEQ ID NOs: 186-234 contain random nucleobases (n) at positions 1, 19, 20, and 21. In some embodiments of the present invention, the antisense sequence contains nucleobase u or nucleobase a at the first position of the antisense sequence. In some embodiments of the present invention, the antisense sequence contains nucleobase u at the first position of the antisense sequence. In the sequences shown in Table 1, "n" may be any one of nucleobases a, u, c, g, and t, and can be selected independently for the sense and antisense strands. As used herein, the "n" in the sense strand and antisense strand refers to the nucleobase "n" at a selected position in the sense strand, which is different from the "n" in the antisense strand paired with the sense strand, but is generally complementary to the nucleobase "n" at the matching position in the antisense strand. As used herein, the term "matching position" in the sense strand and antisense strand refers to the "matching" position in each strand when the two strands are double-stranded. For example, in a 21-nucleobase sense strand and a 21-nucleobase antisense strand, the first nucleobase of the sense strand and the 21-nucleobase of the antisense strand are "matching positions". In another non-limiting example, in a 23-nucleobase sense strand and a 23-nucleobase antisense strand, the second nucleobase of the sense strand and the 22-nucleobase of the antisense strand are "matching positions". In another non-limiting example, in an 18 nucleobase sense strand and an 18 nucleobase antisense strand, the first nucleobase of the sense strand and the 18th nucleobase of the antisense strand are matched, and the fourth nucleobase of the sense strand and the 15th nucleobase of the antisense strand are matched. Those skilled in the art will understand how to identify which strands of a duplex are or may be matched in the sense and antisense strands of the paired strands.

[0067] While (n) can be any one of a, u, c, g, or t, the first "n" in the sense strand is generally complementary to the 21st (n) in the antisense strand. Two non-limiting examples include: (1) if the first in the sense strand is "g," the 21st in the antisense strand is "c"; and (2) if the first in the sense strand is "a," the 21st in the antisense strand is "u" or "t." Such complementary pairing applies to the second in the sense strand and the 20th (n) in the antisense strand, and the 21st in the sense strand and the first (n) in the antisense strand. While n can be any nucleotide at these positions, it will be understood that the nucleotides in the sense and antisense strands are generally still complementary (matching), even though there may be mismatches in some embodiments. For example, in some embodiments, "n" can be "random," meaning that it may be complementary but is not necessarily complementary, but this is not intended to be limiting. In some embodiments, "n" is complementary. As a non-limiting example, the first "n" in the antisense strand is "u" and the 21st "n" in the sense strand is "a."

[0068] The last column in Table 1 lists the duplex AD# / AV#, which is a duplex containing the sense and antisense sequences in the same row of the table. For example, Table 1 discloses a duplex designated AD#AD00007, which contains the sense strand SEQ ID NO:6 and the antisense strand SEQ ID NO:123. Thus, in Table 1, each row displays a duplex of the present invention containing the sense and antisense sequences shown in the same row, and the last column of the row displays the symbol representing each duplex.

[0069] In some embodiments of the methods of the invention, an RNAi agent comprising a polynucleotide sequence set forth in Table 1 is administered to a subject. In some embodiments of the invention, the RNAi agent administered to a subject is a duplex comprising at least one of the base sequences set forth in Table 1, comprising 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 sequence modifications. In some embodiments of the methods of the invention, the RNAi agent comprising a polynucleotide sequence set forth in Table 1 is linked to a delivery molecule, including, but not limited to, a delivery compound comprising a GalNAc compound.

[0070] [Table 1] JPEG2026035574000025.jpg219149 JPEG2026035574000026.jpg219149 JPEG2026035574000027.jpg157149

[0071] Table 2 shows the antisense and sense strand sequences of chemically modified ANGPTL3 RNAi agents of the present invention. In some embodiments of the methods of the present invention, an RNAi agent having a polynucleotide sequence shown in Table 2 is administered to a cell and / or a subject. In some embodiments of the methods of the present invention, an RNAi agent having a polynucleotide sequence shown in Table 2 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to a subject comprises the duplex shown in the first column of a row in Table 2 and includes the sequence modifications shown in the sense and antisense strand sequences, respectively, in the third and sixth columns of the same row in Table 2. In some embodiments of the methods of the present invention, the sequences shown in Table 2 can link the RNAi agent to a compound capable of delivering it to cells and / or tissues of a subject (also referred to herein as a "conjugate"). One non-limiting example of a delivery compound used in some embodiments of the present invention is a GalNAc-containing compound. Column 1 of Table 2 shows the AD# / AV# duplexes of the base sequences shown in Table 1. For double-stranded AD# / AV#, the base sequence is shown, and the indicated sense and antisense strands contain the base sequence and have the indicated chemical modifications shown in the same row of Table 2. For example, Table 1 shows the base sequences of single strands represented by SEQ ID NO:1 (sense) and SEQ ID NO:118 (antisense), which together form a duplex represented by double-stranded AD#AD00001. Table 2 shows double-stranded AD#AD00001, which is composed of SEQ ID NO:237 and SEQ ID NO:287, each containing the base sequences of SEQ ID NO:1 and SEQ ID NO:118, respectively, and having the sense and antisense sequences shown in columns 3 and 6, respectively. The "sense strand SS#" in column 2 of Table 2 is a symbol representing the sense sequence (including modifications) shown in column 3 of the same row. "Antisense strand AS#" in the fifth column of Table 2 is a symbol representing the antisense sequence (including modifications) shown in the sixth column.

[0072] [Table 2] JPEG2026035574000029.jpg225149 JPEG2026035574000030.jpg231149

[0073] Table 3 shows antisense and sense strand sequences of chemically modified ANGPTL3 RNAi agents of the present invention. In some embodiments of the methods of the present invention, an RNAi agent shown in Table 3 is administered to a cell and / or a subject. In some embodiments of the methods of the present invention, an RNAi agent having a polynucleotide sequence shown in Table 3 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to a subject comprises the duplex shown in the first column of a row in Table 3, and includes the sequence modifications and / or delivery compounds shown in the respective sense and antisense strand sequences in columns 3 and 6 of the same row in Table 3. These sequences are used in several in vivo testing studies described later in this specification. In some embodiments of the methods of the present invention, the sequences shown in Table 3 can be linked to a delivery compound (also referred to herein as a "conjugate"). The delivery compound is, by way of non-limiting example, a GalNAc-containing compound, represented in column 3 of Table 3 as "GLX-n" on the sense strand. As used herein and as shown in Table 3, "GLX-n" is used to indicate that the linked GalNAc-containing compound is any one selected from the group consisting of GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, the structures of which are described later in this specification. The first column of Table 3 lists the sense and antisense duplexes in that row, designated as duplex AD#. For example, duplex AD#AD00102 is a duplex of sense strand SEQ ID NO: 337 and antisense strand SEQ ID NO: 364. Each row in Table 3 provides the sense and antisense strands and discloses the duplex between the indicated sense and antisense strands.In the second column of Table 3, "Sense chain SS#" is a symbol representing the sense sequence (including modifications) shown in the third column of the same row. In the fifth column of Table 3, "Antisense chain AS#" is a symbol representing the antisense sequence (including modifications) shown in the sixth column. While the symbol for one GLO compound containing linked GalNAc is shown as GLO-0, it should be understood that other GLO-n or GLS-n compounds may be substituted for the compound represented by GLO-0, and the resulting compounds are included within the embodiments of the methods and / or compositions of the present invention.

[0074] [Table 3]

[0075] Table 4 shows the antisense and sense strand sequences of several chemically modified ANGPTL3 RNAi agents of the present invention. In some embodiments of the methods of the present invention, an RNAi agent having a polynucleotide sequence shown in Table 4 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to a subject includes the duplex shown in column 1 of a row in Table 4 and contains the sequence modifications and / or delivery compounds shown in the respective sense and antisense strand sequences in columns 3 and 6 of the same row in Table 4. In some embodiments of the methods of the present invention, the sequences shown in Table 4 can link the RNAi agent to a compound capable of delivering it to cells and / or tissues of a subject. A delivery compound used in some embodiments of the present invention is, by way of non-limiting example, a GalNAc-containing compound. As shown in Table 4, the term "GLX-n" refers to a GalNAc-containing compound in the sense strand. In Table 4, GLX-n is used to indicate that the linked GalNAc-containing compound is any one selected from the group consisting of GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16.

[0076] Their structures are described later in this specification. The first column of Table 4 lists the duplex AD# corresponding to the duplex shown in Table 3. Duplex AD# indicates the duplex sequence in Table 3, and indicates that the sense sequence, antisense sequence, and duplex sequence in Table 4 also have the same base sequence as the duplex AD# shown in Table 3. However, the sequences and duplexes in Table 4 have different chemical modifications and / or delivery compounds compared to the corresponding sequences and duplexes shown in Table 3. For example, SEQ ID NO: 337 (sense), SEQ ID NO: 364 (antisense), and duplex AD#AD00102 shown in Table 3 have the same base sequence as SEQ ID NO: 391, SEQ ID NO: 418, and AD#AD00102-1, respectively, shown in Table 4. Their chemical modifications and / or delivery compounds are indicated in each table.

[0077] [Table 4] JPEG2026035574000033.jpg225149 JPEG2026035574000034.jpg225149

[0078] Table 5 shows the antisense and sense strand sequences of several chemically modified ANGPTL3 RNAi agents of the invention. In some embodiments of the methods of the invention, an RNAi agent having a polynucleotide sequence shown in Table 5 is administered to a subject. In some embodiments of the invention, the RNAi agent administered to a subject includes the duplex shown in the first column of a row in Table 5 and contains the sequence modifications and / or delivery compounds shown in the sense and antisense strand sequences, respectively, in the third and sixth columns of the same row in Table 5. In some embodiments of the methods of the invention, the sequences shown in Table 5 can be linked to a compound capable of delivering the RNAi agent to cells and / or tissues of a subject. The delivery compound used in some embodiments of the invention is, by way of non-limiting example, a GalNAc-containing compound. Note that the terms "GLO-0" and "GLS-5" in Table 5 refer to various GalNAc-containing compounds linked to the exemplified sense strand. It is understood that the compound represented by GLO-0 can be replaced with another GLO-n or GLS-n compound, and the resulting compound is included in embodiments of the methods and / or compositions of the invention. Similarly, a compound represented by GLS-5 may be replaced with another GLS-n or GLO-n compound, and the resulting compound is included in embodiments of the methods and / or compositions of the present invention. It will be understood that some embodiments of the invention include an RNAi agent of the invention having a sequence as shown in Table 5, but linked to any one of GalNAc-containing compounds selected from the group consisting of GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16.

[0079] Their structures are described later in this specification. Column 1 of Table 5 lists the duplex AD# numbers AD00178 to AD#AD00187, and the numbers in each row designate duplexes containing the sense and antisense strands shown in columns 3 and 6 of the same row, respectively, and having a 3' GLO- or GLS-delivery compound and modification linked to the sense strand. Duplexes AD#AD00178 to AD00187 are perfectly complementary to the mouse ANGPTL3 mRNA sequence but have zero or one mismatch with the human ANGPTL3 mRNA sequence.

[0080] The first column of Table 5 lists the duplex AD# numbers AD00179-1, AD00180-1, AD00181-1, AD00103-1, AD00183-1, AD00184-1, AD 00185-1, AD 00186-1, and AD 00187-1, and the numbers in each row designate the duplex containing the sense and antisense strands, respectively, and having a 5' or 3' GLS-n or GLO-n delivery compound and modification linked to the sense strand, as shown in columns 3 and 6 of the same row. The modifications include uppercase 2'-fluoro, lowercase 2'-OMe, * phosphorothioate, and Invab inverted abasic. The duplexes AD00179-1, AD00180-1, AD00181-1, AD00103-1, AD00183-1, AD00184-1, AD 00185-1, AD 00186-1 and AD 00187-1 are perfectly complementary to the human ANGPTL3 mRNA sequence but have zero or one mismatch with the mouse ANGPTL3 mRNA sequence.

[0081] [Table 5] JPEG2026035574000036.jpg226149

[0082] mismatch As known to those skilled in the art, efficacy of dsRNA can tolerate mismatches, particularly mismatches within the terminal regions of the dsRNA. Some mismatches are more tolerant, for example, mismatches with wobble base pairs G:U and A:C are more tolerant of efficacy (Du et al., A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismached target sites. Nucleic Acids Res. 2005 Mar 21;33(5):1671-7. Doi:10.1093 / nar / gki312. Nucleic Acids Res. 2005;33(11):3698). In some embodiments of the methods and compounds of the present invention, the ANGPTL3 dsRNA agent can contain one or more mismatches with respect to the ANGPTL3 target sequence. In some embodiments, the ANGPTL3 dsRNA agent of the present invention contains no mismatches. In certain embodiments, the ANGPTL3 dsRNA agent of the present invention contains one or fewer mismatches. In some embodiments, the ANGPTL3 dsRNA agent of the present invention contains two or fewer mismatches. In certain embodiments, the ANGPTL3 dsRNA agent of the present invention contains three or fewer mismatches. In some embodiments of the present invention, the antisense strand of the ANGPTL3 dsRNA agent contains a mismatch to the ANGPTL3 target sequence that is not located in the center of the complementary region. In some embodiments, the antisense strand of the ANGPTL3 dsRNA agent contains one, two, three, four, or more mismatches within the last 5, 4, 3, 2, or 1 nucleotide from either or both of the 5' or 3' ends of the complementary region. To determine whether an ANGPTL3 dsRNA agent containing a mismatch to the ANGPTL3 target sequence effectively inhibits the expression of the ANGPTL3 gene, methods described herein and / or known in the art can be used.

[0083] Complementarity Unless otherwise specified, the term "complementary" as used herein, when used to describe a first nucleotide sequence (e.g., the sense strand of an ANGPTL3 dsRNA agent or a targeting ANGPTL3 mRNA) related to a second nucleotide sequence (e.g., the antisense strand of an ANGPTL3 dsRNA agent or a single-stranded antisense polynucleotide), refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to an oligonucleotide or polynucleotide comprising the second nucleotide sequence to form a double-stranded or double-helical structure under specific conditions (by forming hydrogen bonds between base pairs under physiological conditions in mammals (or similar in vitro conditions)). Other conditions, such as physiologically relevant conditions present in vivo, may also be applicable. The optimal set of conditions for testing the complementarity of two sequences can be determined by one skilled in the art depending on the ultimate application of the hybridized nucleotides. Complementary sequences that meet, at least to some extent, the above hybridization requirements include Watson-Crick or non-Watson-Crick base pairs, and also include natural or modified nucleotides or nucleotide mimetics. Sequence identity or complementarity is independent of modification.

[0084] Complementary sequences include, for example, those found in the ANGPTL3 dsRNA described herein, including base pairs between an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or two nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. In some embodiments, if two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, it is understood that such overhangs are not considered mismatches with respect to the specified complementarity herein. For example, if an ANGPTL3 dsRNA agent includes an oligonucleotide having a length of 19 nucleotides and an oligonucleotide having a length of 20 nucleotides, and the longer oligonucleotide contains a 19-nucleotide sequence that is completely complementary to the shorter oligonucleotide, then for purposes described herein, this would be considered "fully complementary." Thus, as used herein, "perfectly complementary" means that all (100%) of the bases in a contiguous sequence of a first polynucleotide hybridize to the same number of bases in a contiguous sequence of a second polynucleotide. The contiguous sequence can include all or part of the first or second nucleotide sequence.

[0085] As used herein, the term "substantially complementary" means that in a hybridized nucleic acid sequence pair, at least about 85%, but not all, of the bases in a contiguous sequence of a first polynucleotide hybridize to the same number of bases in a contiguous sequence of a second polynucleotide. The term "substantially complementary" refers to a first sequence relative to a second sequence, where the two sequences, when hybridized to form a duplex of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs (bp), contain one or more (e.g., at least one, two, three, four, or five) mismatched base pairs, while simultaneously maintaining the ability to hybridize under conditions most relevant to their end application (e.g., inhibiting ANGPTL3 gene expression via the RISC pathway). As used herein, the term "partially complementary" can be used to refer to nucleobase sequence hybridization in which at least 75% but not all of the bases in a contiguous sequence of a first polynucleotide hybridize to the same number of bases in a contiguous sequence of a second polynucleotide. In some embodiments, "partially complementary" means that at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bases in a contiguous sequence of a first polynucleotide hybridize to the same number of bases in a contiguous sequence of a second polynucleotide.

[0086] The terms "complementary," "fully complementary," "substantially complementary," and "partially complementary" are used herein to refer to base matches between the sense strand and antisense strand of an ANGPTL3 dsRNA agent, between the antisense strand of an ANGPTL3 dsRNA agent and the sequence of a target ANGPTL3 mRNA, or between a single-stranded antisense oligonucleotide and the sequence of a target ANGPTL3 mRNA. It will be understood that the term "antisense strand of an ANGPTL3 dsRNA agent" can refer to the same sequence of an "ANGPTL3 antisense polynucleotide agent."

[0087] As used herein, the term "substantially the same" or "substantially identical" when referring to a nucleic acid sequence refers to a nucleic acid sequence that contains a sequence having at least about 85% or more sequence identity, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, relative to a reference sequence. The percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions where identical nucleic acid bases appear in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The inventions disclosed herein encompass nucleic acid sequences that are substantially the same as those disclosed herein (e.g., Tables 1-5). In some embodiments, the sequences disclosed herein are completely identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, a sequence disclosed herein (e.g., in Tables 1-5).

[0088] As used herein, the term "strand containing a sequence" refers to an oligonucleotide containing a chain of nucleotides described by a sequence referenced in standard nucleotide nomenclature. As used herein, the term "double-stranded RNA" or "dsRNA" refers to an RNAi molecule or molecular conjugate having a hybridized double-stranded region containing two antiparallel, substantially or completely complementary nucleic acid strands, said to have "sense" and "antisense" orientations relative to the target ANGPTL3 RNA. The double-stranded region may have any length that allows specific degradation of the desired target ANGPTL3 RNA by the RISC pathway, but typically has a length of 9 to 30 base pairs, e.g., 15 to 30 base pairs. When a double strand of 9 to 30 base pairs is used, the double strand may be any length within the range of, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or any length within any subrange therebetween, specifically 15 to 30 base pairs, 15 to 26 base pairs, 15 to 22 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 19 base pairs, 15 to 17 base pairs, 18 to 30 base pairs, 18 to 26 base pairs, 18 to 23 base pairs, 18 to 30 base pairs, 18 to 40 base pairs, 18 to 42 base pairs, 18 to 50 base pairs, 18 to 52 base pairs, 18 to 60 base pairs, 18 to 62 base pairs, 18 to 70 base pairs, 18 to 80 base pairs, 18 to 90 base pairs, 18 to 100 base pairs, 18 to 120 base pairs, 18 to 130 base pairs, 18 to 210 base pairs, 18 to 220 base pairs, 18 to 230 base pairs, 18 to 300 base pairs, 18 to 26 base pairs, 18 to 23 base pairs, 18 to 30 ...400 base pairs, 18 to 52 The length of the dsRNA may be, but is not limited to, 19 to 22 base pairs, 18 to 21 base pairs, 18 to 20 base pairs, 19 to 30 base pairs, 19 to 26 base pairs, 19 to 23 base pairs, 19 to 22 base pairs, 19 to 21 base pairs, 19 to 20 base pairs, 20 to 30 base pairs, 20 to 26 base pairs, 20 to 25 base pairs, 20 to 24 base pairs, 20 to 23 base pairs, 20 to 22 base pairs, 20 to 21 base pairs, 21 to 30 base pairs, 21 to 26 base pairs, 21 to 25 base pairs, 21 to 24 base pairs, 21 to 23 base pairs, or 21 to 22 base pairs. ANGPTL3 dsRNA agents produced in cells by Dicer and similar enzyme processing generally have a length of 19 to 22 base pairs. One strand of the double-stranded region of the ANGPTL3 dsDNA agent comprises a sequence that is essentially complementary to a region of the target ANGPTL3 RNA.The two strands forming the double-stranded structure may be derived from a single RNA molecule having at least one self-complementary region, or may be composed of two or more independent RNA molecules. When the double-stranded region is formed from the two strands of a single molecule, the molecule may have a double-stranded region separated by a single nucleotide chain (referred to herein as a "hairpin loop") between the 3'-end of one strand forming the double-stranded structure and the 5'-end of the corresponding other strand. In some embodiments of the present invention, the hairpin loop contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more unpaired nucleotides. When the substantially complementary two strands of an ANGPTL3 dsRNA agent are composed of a single RNA molecule, the molecules may be linked by a covalent bond, but such a bond is not required. When the two strands are covalently linked in a manner other than a hairpin loop, the connecting structure is referred to as a "linker." The term "siRNA" is also used herein to refer to the dsRNA agents described herein.

[0089] In some embodiments of the present invention, an ANGPTL3 dsRNA agent can comprise a sense sequence and an antisense sequence with unpaired nucleotides or nucleotide analogs at one or two ends of the dsRNA agent.Ends without unpaired nucleotides are called "blunt ends" and do not have overhanging nucleotides.If both ends of a dsRNA agent are blunt, the dsRNA is called "blunt-ended".In some embodiments of the present invention, the dsRNA agent has a blunt end at the first end.In some embodiments of the present invention, the dsRNA agent has a blunt end at the second end.In some embodiments of the present invention, the ANGPTL3 dsRNA agent has both ends blunt.

[0090] In some embodiments of the dsRNA agent of the present invention, the dsRNA does not have one or two blunt ends. In this case, there is at least one unpaired nucleotide at the end of the strand of the dsRNA agent. For example, a nucleotide overhang exists when the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa. The dsRNA can include an overhang having at least 1, 2, 3, 4, 5, 6, or more nucleotides. The nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. In some embodiments, the nucleotide overhang is present in the sense strand of the dsRNA agent, the antisense strand of the dsRNA agent, or both ends of the dsRNA agent, and it will be understood that the nucleotides at the overhang may be present at the 5' end, 3' end, or both ends of the antisense or sense strand of the dsRNA. In some embodiments of the present invention, one or more nucleotides at the overhang are replaced by nucleoside phosphorothioates.

[0091] As used herein, the term "antisense strand" or "guide strand" refers to the strand of an ANGPTL3 dsRNA agent that comprises a region that is substantially complementary to an ANGPTL3 target sequence.As used herein, the term "sense strand" or "lagging strand" refers to the strand of an ANGPTL3 dsRNA agent that comprises a region that is substantially complementary to a region of the antisense strand of the ANGPTL3 dsRNA agent.

[0092] qualification In some embodiments of the present invention, the RNA of the ANGPTL3 RNAi agent is chemically modified to enhance stability and / or one or more other beneficial characteristics. In some aspects of the present invention, nucleic acids can be synthesized and / or modified by methods well established in the art, for example, as described in "Current protocols in Nucleic Acid Chemistry," Beaucage, SLet et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. In certain embodiments, modifications that may be present in ANGPTL3 dsRNA agents of the present invention include, for example, (a) end modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted ligation, etc.), 3'-end modifications (conjugation, DNA nucleotides, inverted ligation, etc.), (b) base modifications, such as stabilized bases, destabilized bases, or base substitutions that base pair with an expanded repertoire of partners, abasic (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) backbone modifications, including phosphodiester bond modifications or substitutions. Some specific examples of RNA compounds that may be used in certain embodiments of ANGPTL3 dsRNA agents, ANGPTL3 antisense polynucleotides, and ANGPTL3 sense polynucleotides of the present invention include, but are not limited to, RNAs containing modified backbones or linked by non-natural internucleotide linkages. As a non-limiting example, RNAs with modified backbones may lack a phosphorus atom in the backbone. RNA that does not have a phosphorus atom in its internucleotide backbone is sometimes called an oligonucleotide. In certain embodiments of the invention, the modified RNA has a phosphorus atom in its internucleotide backbone.

[0093] The terms "RNA molecule," "RNA," or "ribonucleic acid molecule" should be understood to encompass not only RNA molecules expressed or discovered in nature, but also RNA analogs and derivatives containing one or more ribonucleotide / ribonucleoside analogs or derivatives, as described herein or known in the art. The terms "ribonucleoside" and "ribonucleotide" may be used interchangeably herein. RNA molecules may be modified in the nucleobase structure or sugar-phosphate backbone structure (e.g., as described herein below), provided that molecules containing nucleoside analogs or derivatives retain the ability to form double strands. As some non-limiting examples, the RNA molecule may further comprise at least one modified nucleoside, including, but not limited to, a 2'-O-methyl group modified nucleoside, a nucleoside containing a 5'-phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or a lauric acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluorine modified nucleoside, a 2'-amino group modified nucleoside, a 2'-alkyl group modified nucleoside, a morpholino nucleoside, a phosphoramidate, or a nucleoside containing a non-natural base, or any combination thereof. In some embodiments of the present invention, the RNA molecule comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to its full length, ribonucleosides of the ANGPTL3 dsRNA drug molecule, wherein the ribonucleosides are modified ribonucleosides.The multiple modified ribonucleosides in such an RNA molecule do not need to have the same modifications.

[0094] In some embodiments, a dsRNA agent, ANGPTL3 antisense polynucleotide, and / or ANGPTL3 sense polynucleotide of the invention can comprise one or more independently selected modified nucleotides and / or one or more independently selected non-phosphodiester linkages. As used herein, the term "independently selected" when referring to selected elements (e.g., modified nucleotides, non-phosphodiester linkages, etc.) means that two or more selected elements can be, but are not necessarily, identical to one another.

[0095] As used herein, "nucleotide base," "nucleotide," or "nucleobase" refers to a heterocyclic pyrimidine or purine compound that is a standard component of all nucleic acids and forms nucleotides. The term "nucleotide base" includes adenine (a), guanine (g), cytosine (c), thymine (t), and uracil (u). Nucleobases may be further modified, including, but not limited to, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. The term "ribonucleotide" or "nucleotide" as used herein can refer to unmodified nucleotides, modified nucleotides, or moieties substituted with alternatives. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil may be substituted with other moieties without substantially altering the complementary base-pairing properties of oligonucleotides containing nucleotides with such substitutions.

[0096] In one embodiment, the modified RNA used in the methods and compositions herein is a peptide nucleic acid (PNA) that has the ability to form a desired double-stranded structure and enables or mediates the specific degradation of the target RNA by the RISC pathway. In one embodiment of the present invention, the ANGPTL3 RNA interference agent includes a single-stranded RNA that interacts with a target ANGPTL3 RNA sequence to guide the cleavage of the target ANGPTL3 RNA.

[0097] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkylphosphonates, including 3'-alkylenephosphonates, as well as chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thiophosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'→5' linked analogs, and those with reverse polarity, where the internucleoside linkage is 3'→5' to 5'→3' or 2'→5' to 5'→2'. Salts, mixed salts, and free acid forms are also included. Methods for preparing phosphorus-containing linkages are well known in the art, and such methods can be used to prepare certain modified ANGPTL3 dsRNA agents, certain modified ANGPTL3 antisense polynucleotides, and / or certain modified ANGPTL3 sense polynucleotides of the invention.

[0098] Modified RNA backbones that do not contain phosphorus atoms include backbones consisting of short alkyl or cycloalkyl internucleoside linkages, heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages, morpholine linkages (some of which are formed in the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and backbones containing mixed N, O, S, and CH moieties. Methods for preparing modified RNA backbones that do not contain phosphorus atoms are well known in the art, and such methods can be used to prepare certain modified ANGPTL3 dsRNA agents, certain modified ANGPTL3 antisense polynucleotides, and / or certain modified ANGPTL3 sense polynucleotides of the present invention.

[0099] In certain embodiments of the present invention, RNA mimics are included in ANGPTL3 dsRNA, ANGPTL3 antisense polynucleotides, and / or ANGPTL3 sense polynucleotides, for example, but not limited to, by replacing the backbone, the linkage between the sugar and nucleoside of a nucleotide unit, with a novel group. In such embodiments, the base units are maintained for hybridization with an appropriate ANGPTL3 nucleic acid targeting compound. Such oligomeric compounds, which are RNA mimics with excellent hybridization properties, are called peptide nucleic acids (PNAs). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are linked directly or indirectly to the aza-nitrogen atoms of the backbone amide moiety. Methods for preparing RNA mimics are well known in the art, and such methods can be used to prepare several modified ANGPTL3 dsRNA agents of the present invention.

[0100] Some embodiments of the present invention include, among others, RNAs with phosphorothioate backbones and oligonucleotides with heteroatom backbones, such as -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- (referred to as a methylene(methylimino) or MMI backbone), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2- (wherein a natural phosphodiester backbone is represented as -OPO-CH2-). Methods for producing RNAs with phosphorothioate backbones and oligonucleotides with heteroatom backbones are well known in the art, and such methods can be used to produce certain modified ANGPTL3 dsRNA agents, certain ANGPTL3 antisense polynucleotides, and / or certain ANGPTL3 sense polynucleotides of the present invention.

[0101] The modified RNA may contain one or more substituted sugar moieties. The ANGPTL3 dsRNA, ANGPTL3 antisense polynucleotide, and / or ANGPTL3 sense polynucleotide of the present invention may contain, at the 2' position, one selected from the group consisting of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 and alkenyl and alkynyl. Some exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are 1 to 10. In some other embodiments, the dsRNA has at the 2'-position a C1 to C 10The substituent includes one selected from the group consisting of lower alkyl, substituted lower alkyl, alkylaryl, aralkyl, O-alkylaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporting group, intercalator, group for improving the pharmacokinetic properties of an ANGPTL3 dsRNA agent, or group for improving the pharmacological properties of an ANGPTL3 dsRNA agent, ANGPTL3 antisense polynucleotide and / or ANGPTL3 sense polynucleotide, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy. Another exemplary modification is 2'-dimethylaminooxyethoxy (i.e., O(CH2)2ON(CH3)2-, also known as 2'-DMAOE), and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE, i.e., 2'-O-CH2-O-CH2-N(CH2)2), as described in the Examples below. Methods for preparing modified RNAs (e.g., as described above) are well known in the art, and such methods can be used to prepare some modified ANGPTL3 dsRNA agents of the invention.

[0102] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can be made at other positions on the RNA of an ANGPTL3 dsRNA agent, ANGPTL3 antisense polynucleotide, and / or ANGPTL3 sense polynucleotide of the invention, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked ANGPTL3 dsRNA, ANGPTL3 antisense polynucleotide, or ANGPTL3 sense polynucleotide, and the 5' position of the 5'-terminal nucleotide. ANGPTL3 dsRNA agents, ANGPTL3 antisense polynucleotides, and / or ANGPTL3 sense polynucleotides can also have sugar mimetics, such as, for example, a cyclobutyl moiety replacing the pentofuranosyl sugar. Methods for preparing modified RNAs (e.g., as described above) are well known in the art, and such methods can be used to prepare certain modified ANGPTL3 dsRNA agents, ANGPTL3 antisense polynucleotides, and / or ANGPTL3 sense polynucleotides of the invention.

[0103] In some embodiments, an ANGPTL3 dsRNA agent, an ANGPTL3 antisense polynucleotide, and / or an ANGPTL3 sense polynucleotide may contain a modified or substituted nucleobase (commonly abbreviated in the art as "base"). As used herein, "unmodified" or "natural" nucleobase includes the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives and other alkyl derivatives of adenine and guanine, 2-propyl derivatives and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5- These include uracil (pseudouridine), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogenated, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Nucleobases that may be included in certain embodiments of the ANGPTL3 dsRNA agents of the present invention are well known in the art, see, e.g., Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. Ed. Wiley-VCH, 2008; The Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, Kroschwitz, JL, Ed. John Wiley & Sons, 1990; English et al., Angewandte, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, S. and Lebleu, B., Ed., CRC Press, 1993. Methods for producing dsRNAs, ANGPTL3 antisense strand polynucleotides, and / or ANGPTL3 sense strand polynucleotides comprising nucleobase modifications and / or substitutions (e.g., as described herein) are well known in the art, and such methods can be used to produce modified ANGPTL3 dsRNA agents, ANGPTL3 sense polynucleotides, and / or ANGPTL3 antisense polynucleotides of the present invention.

[0104] In some embodiments, the ANGPTL3 dsRNA agents, ANGPTL3 antisense polynucleotides, and / or ANGPTL3 sense polynucleotides of the present invention comprise RNA modified to contain one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with a modified ribose moiety that contains an additional bridge connecting the 2' and 4' carbons. Such structures are effective in "locking" the ribose in a 3'-endo structural conformation. Addition of a locked nucleic acid to the ANGPTL3 dsRNA drug, ANGPTL3 antisense polynucleotide, and / or ANGPTL3 sense polynucleotide of the present invention can improve stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O.R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Methods for preparing dsRNA drugs, ANGPTL3 antisense polynucleotides, and / or ANGPTL3 sense polynucleotides containing locked nucleic acids are well known in the art, and such methods can be used to prepare certain modified ANGPTL3 dsRNA drugs of the present invention.

[0105] In some embodiments, the ANGPTL3 dsRNA compound, sense polynucleotide, and / or antisense polynucleotide of the present invention comprises at least one modified nucleotide, such as, for example, 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxyribonucleotide, 2'-3'-seconucleotide mimic, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, and 3'-OMe nucleotide, 5'-phosphorothioate group-containing nucleotide, or terminal nucleotide linked to cholesterol derivative or dodecanoic acid bisdecylamide group, 2'-amino-modified nucleotide, phosphoramidate, or unnatural base-containing nucleotide. In some embodiments, the ANGPTL3 dsRNA compound comprises an E-vinylphosphonate nucleotide at the 5'-end of the antisense strand, also referred to herein as the guide strand.

[0106] In some embodiments, the ANGPTL3 dsRNA compounds of the invention contain at least one modified nucleotide, such as an abasic nucleotide, ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-omega nucleotide, or an inverted 2'-deoxyribonucleotide, at the 3' and 5' ends of the sense polynucleotide and / or the 3' end of the antisense polynucleotide. As known to those skilled in the art, the inclusion of an abasic or inverted abasic nucleotide at the end of an oligonucleotide enhances stability (Czauderna et al. Structural variations and stabilizing modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res. 2003;31(11):2705-2716. doi:10.1093 / nar / gkg393).

[0107] In some embodiments, the ANGPTL3 dsRNA compounds and antisense polynucleotides of the present invention contain at least one modified nucleotide, such as an unlocked nucleic acid nucleotide (UNA) and / or a glycol nucleic acid nucleotide (GNA). UNA and GNA are thermally unstable chemical modifications known to those skilled in the art to significantly improve the off-target profile of siRNA compounds (Janas et al., Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun. 2018;9(1):723. doi:10.1038 / s41467-018-02989-4; Laursen et al., Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo. Mol BioSyst. 2010;6:862-70).

[0108] In some embodiments, the RNA of the ANGPTL3 dsRNA agent, ANGPTL3 antisense polynucleotide, and / or ANGPTL3 sense polynucleotide of the present invention may include other modifications. Such modifications include chemically attaching one or more ligands, moieties, or conjugates to the RNA that enhance one or more characteristics of the ANGPTL3 dsRNA agent, ANGPTL3 antisense polynucleotide, and / or ANGPTL3 sense polynucleotide, respectively. Some non-limiting examples of characteristics that can be enhanced include the activity of the ANGPTL3 dsRNA agent, ANGPTL3 antisense polynucleotide, and / or ANGPTL3 sense polynucleotide; cellular distribution; delivery of the ANGPTL3 dsRNA agent; pharmacokinetic properties of the ANGPTL3 dsRNA agent; and cellular uptake of the ANGPTL3 dsRNA agent. In some embodiments of the present invention, the ANGPTL3 dsRNA agent includes one or more targeting groups or linking groups conjugated to the sense strand of the ANGPTL3 dsRNA agent of the present invention. A non-limiting example of a targeting group is a compound containing N-acetylgalactosamine (GalNAc). The terms "targeting group," "targeting agent," "linking agent," "targeting compound," and "targeting ligand" may be used interchangeably herein. In some embodiments of the invention, an ANGPTL3 dsRNA agent comprises a targeting compound conjugated to the 5'-end of the sense strand. In some embodiments of the invention, an ANGPTL3 dsRNA agent comprises a targeting compound conjugated to the 3'-end of the sense strand. In some embodiments of the invention, an ANGPTL3 dsRNA agent comprises a targeting group comprising GalNAc. In some embodiments of the invention, an ANGPTL3 dsRNA agent does not comprise a targeting compound conjugated to one or both of the 3'-end and 5'-end of the sense strand.In some embodiments of the invention, an ANGPTL3 dsRNA agent does not include a targeting compound that includes GalNAc conjugated to one or both of the 5' and 3' ends of the sense strand.

[0109] Although other targeting agents and linkers are known herein, for example, targeting agents and linkers that may be used in some embodiments of the present invention include, for example, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), thioethers such as cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), beryl-S-tritylthiol (Manoharan et al., Ann. N. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992,20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10:1111-1118; Kabanov et al., FEBS Lett., 1990,259:327-330; Svinarchuk et al., Biochimie, 1993,75:49-54), di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moieties (Mishra et al., Biochim. Biophys.Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0110] In some embodiments, compositions comprising an ANGPTL3 dsRNA agent, an ANGPTL3 antisense polynucleotide, and / or an ANGPTL3 sense polynucleotide can include a ligand that alters the distribution, targeting, etc. of the ANGPTL3 dsRNA agent. In some embodiments, compositions comprising an ANGPTL3 dsRNA agent of the present invention include a ligand that enhances affinity for a selected target (e.g., a molecule, a cell or cell type, a cell or organ compartment, a tissue, an organ, or a region of the body) compared to, for example, the absence of such a ligand. Ligands that can be used in the compositions and / or methods of the present invention can be naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), or lipids. Ligands can also be recombinant molecules or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids or polyamines. Some examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, lactic acid-glycolic acid copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, and polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, polyamine dendrimers, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary ammonium salts of polyamines, and α-helical peptides.

[0111] The ligand included in the compositions and / or methods of the present invention may include a targeting group, some non-limiting examples of which include cell or tissue targeting agents such as lectins, glycoproteins, lipids, or proteins, such as antibodies that bind to specific cell types (e.g., kidney cells or liver cells). The targeting group may be thyroid-stimulating hormone, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine, N-acetylglucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptidomimetic.

[0112] Other examples of ligands include dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC 4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenazine), and peptidases. Examples of suitable nucleotides include nucleotides (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bizimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ conjugates of macrocyclic tetraaza compounds), dinitrophenyl, HRP, or AP.

[0113] The ligand included in the compositions and / or methods of the invention may be a protein (e.g., a glycoprotein), a peptide (e.g., a molecule with a specific affinity for a co-ligand), or an antibody (e.g., an antibody that binds to a specific cell type (e.g., cancer cells, endothelial cells, cardiomyocytes, or bone cells)). In one embodiment, the ligand used in the compositions and / or methods of the invention may be a hormone or hormone receptor. In one embodiment, the ligand used in the compositions and / or methods of the invention may be a lipid, a lectin, a carbohydrate, a vitamin, a cofactor, multivalent lactose, multivalent galactose, N-acetylgalactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. In one embodiment, the ligand used in the compositions and / or methods of the invention may be a substance that can improve cellular uptake of an ANGPTL3 dsRNA agent, for example, by disrupting the cytoskeleton in the cell, e.g., by disrupting microtubules, microfilaments, and / or intermediate filaments in the cell. Some non-limiting examples of such substances include cytochalasin, vincristine, vinblastine, cytolysin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, and myoservin.

[0114] In some embodiments, the ligand linked to the ANGPTL3 dsRNA agent of the present invention functions as a pharmacokinetic (PK) modulator. Examples of PK modulators that can be used in the compositions and methods of the present invention include, but are not limited to, lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein-linking agents, PEG, vitamins, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and serum protein-binding aptamers. Furthermore, because oligonucleotides containing many phosphorothioate linkages are known to bind to serum proteins, short oligonucleotides containing multiple phosphorothioate linkages in the backbone, such as oligonucleotides of about 5, 10, 15, or 20 bases, may also be used as ligands in the compositions and / or methods of the present invention.

[0115] ANGPTL3 dsRNA drug compositions In some embodiments of the present invention, the ANGPTL3 dsRNA agent is present in a composition. The composition of the present invention may include one or more ANGPTL3 dsRNA agents and one or more selected from the group consisting of pharmaceutically acceptable carriers, drug delivery agents, targeting agents, detectable markers, etc. Non-limiting examples of targeting agents that can be used in some embodiments of the methods of the present invention include agents that target and deliver the ANGPTL3 dsRNA agents of the present invention to cells and / or cells to be treated. Targeting agents are selected based on factors such as the nature of the ANGPTL3-related disease or condition and the target cell type. In some non-limiting examples of some embodiments of the present invention, the ANGPTL3 dsRNA agent is desirably targeted to hepatocytes and / or hepatocytes. It should be understood that in some embodiments of the methods of the present invention, the therapeutic agent includes an ANGPTL3 dsRNA agent that contains only a delivery agent (e.g., a delivery agent containing N-acetylgalactosamine (GalNAc)) without an additional linking element. For example, in some embodiments of the present invention, an ANGPTL3 dsRNA agent is linked to a delivery compound comprising GalNAc, included in a composition comprising a pharmaceutically acceptable carrier, and administered to a cell or subject without the use of any detectable label, targeting agent, or the like, linked to the ANGPTL3 dsRNA agent.

[0116] When the ANGPTL3 dsRNA agent of the present invention is administered and / or linked with one or more agents selected from the group consisting of a delivery agent, a targeting agent, a labeling agent, etc., those skilled in the art will understand how to appropriately select and use the agent applied to the method of the present invention. In some methods of the present invention, a labeling agent can be used to determine the location of the ANGPTL3 dsRNA agent in cells and tissues, and can also be used to determine the location of a therapeutic composition containing an ANGPTL3 dsRNA agent administered in a method of the present invention in cells, tissues, or organs. Methods for linking and using labeling agents, such as enzyme labels, dyes, and radiolabels, are known in the art. It should be understood that in some embodiments of the compositions and methods of the present invention, a labeling agent is linked to one or both of the sense polynucleotide and the antisense polynucleotide contained in the ANGPTL3 dsRNA agent.

[0117] Delivery of ANGPTL3 dsRNA and ANGPTL3 antisense polynucleotide drugs Some embodiments of the methods of the present invention include delivering an ANGPTL3 dsRNA agent to a cell. As used herein, the term "delivery" means promoting or affecting cellular uptake or absorption. Absorption or uptake of the ANGPTL3 dsRNA agent can occur through independent diffusion or active cellular processes, or by using a delivery agent, targeting agent, or the like that can bind to the ANGPTL3 dsRNA agent of the present invention. Delivery means suitable for the methods of the present invention include, but are not limited to, in vivo delivery, in which the ANGPTL3 dsRNA agent is administered by injection at a tissue site or systemically. In some embodiments of the present invention, the ANGPTL3 dsRNA agent is linked to a delivery agent.

[0118] Some non-limiting examples of methods for delivering ANGPTL3 dsRNA agents to cells, tissues, and / or subjects include ANGPTL3 dsRNA-GalNAc conjugates, SAMiRNA technology, LNP-based delivery methods, and naked RNA delivery. These and other delivery methods have been successfully used in the art to deliver therapeutic RNAi agents to treat various diseases and conditions, including, but not limited to, liver disease, acute intermittent porphyria (AIP), hemophilia, and pulmonary fibrosis. For details of several delivery means, see, e.g., Nikam, RRKR Gore (2018) Nucleic Acid Ther, 28(4), 209-224 Aug 2018; Springer ADSFDowdy (2018) Nucleic Acid Ther. Jun 1, 28(3):109-118; Lee, K. et al., (2018) Arch Pharm, 41(9), 867-874; and Nair, JK et al., (2014) J. Am. Chem. Soc. 136:16958-16961, the contents of each of which are incorporated herein by reference.

[0119] In some embodiments of the present invention, ANGPTL3 dsRNA agents of the present invention may be delivered to cells, tissues, and / or subjects using lipid nanoparticles (LNPs). LNPs are typically used to deliver ANGPTL3 dsRNA agents, including therapeutic ANGPTL3 dsRNA agents, in vivo. One advantage of using LNPs or other delivery agents is that the stability of the ANGPTL3 RNA agent is improved when it is delivered to a subject using LNPs or other delivery agents. In some embodiments of the present invention, the LNPs comprise cationic LNPs carrying one or more ANGPTL3 RNAi molecules of the present invention. When the LNPs containing the ANGPTL3 RNAi molecules are administered to a subject, the LNPs and the ANGPTL3 RNAi molecules linked thereto are taken up into the cells by endocytosis and, once present, release an RNAi trigger molecule that mediates RNAi.

[0120] In embodiments of the present invention, another non-limiting example of a delivery agent that can be used to deliver the ANGPTL3 dsRNA agent of the present invention to cells, tissues, and / or subjects is an agent that contains GalNAc linked to the ANGPTL3 dsRNA agent of the present invention and delivers the ANGPTL3 dsRNA agent to cells, tissues, and / or subjects. Some examples of other GalNAc-containing delivery agents that can be used in certain embodiments of the methods and compositions of the present invention are disclosed in PCT WO2020191183A1. Non-limiting examples of GalNAc targeting ligands for delivering ANGPTL3 dsRNA agents to cells that can be applied to the compositions and methods of the present invention include targeting ligand clusters. Some examples of targeting ligand clusters described herein are referred to as GalNAc ligands with phosphodiester linkages (GLO) and GalNAc ligands with phosphorothioate linkages (GLS). As used herein, the term "GLX-n" may be used to refer to a linked GalNAc-containing compound that is any one selected from the group consisting of GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16. Each of the above structures is represented below by the rightmost position (""). [ka] "). It is understood that any RNAi and dsRNA molecule of the present invention can be linked to the structures GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, i.e., GLO-1 through GLO-16, and GLS-1 through GLS-16.

[0121] [ka] JPEG2026035574000039.jpg174149 JPEG2026035574000040.jpg182149 JPEG2026035574000041.jpg176149 JPEG2026035574000042.jpg182149 JPEG2026035574000043.jpg187149 JPEG2026035574000044.jpg186149 JPEG2026035574000045.jpg174149 JPEG2026035574000046.jpg192149 JPEG2026035574000047.jpg176149 JPEG2026035574000048.jpg189149

[0122] In some embodiments of the present invention, in vivo delivery can be achieved using a β-glucan delivery system, as described, for example, in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005 / 0281781, which are incorporated herein by reference in their entireties. ANGPTL3 RNAi agents can also be introduced into cells in vitro using methods known in the art, such as electroporation or lipid transfection. In some embodiments of the methods of the present invention, ANGPTL3 dsRNAs are delivered without a targeting agent. These RNAs can be delivered as naked RNA molecules. As a non-limiting example, the ANGPTL3 dsRNAs of the present invention can be administered to a subject in a pharmaceutical composition containing an RNAi agent but not a targeting agent (e.g., a GalNAc-targeting compound) to treat an ANGPTL3-related disease or disorder, such as liver disease, in the subject.

[0123] It is understood that the examples of RNAi delivery means described herein are not limited to those described herein, and that delivery means used in the art, in addition to some of the delivery means described herein, can also be used in combination with the ANGPTL3 RNAi agents and treatment methods described in the embodiments herein.

[0124] The ANGPTL3 dsRNA agents of the present invention can be administered to a subject in an amount and manner that effectively reduces the level and activity of ANGPTL3 polypeptide in a cell and / or a subject. In some embodiments of the methods of the present invention, one or more ANGPTL3 dsRNA agents are administered to a cell and / or a subject to treat a disease or disorder associated with ANGPTL3 expression and activity. In some embodiments, the methods of the present invention include administering one or more ANGPTL3 dsRNA agents to a subject in need of such treatment to reduce a disease or disorder associated with ANGPTL3 expression in the subject. Additionally, the ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents of the present invention are administered to reduce ANGPTL3 expression and / or activity in one or more of in vitro, ex vivo, and in vivo cells.

[0125] In some embodiments of the present invention, an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent is delivered (e.g., incorporated) into a cell, thereby reducing the level of ANGPTL3 polypeptide in the cell and thus reducing its activity. Targeting agents and methods can be used to assist in delivering an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent to a specific cell type, cell subtype, organ, spatial region in a subject, and / or subcellular region in a cell. In some methods of the present invention, an ANGPTL3 dsRNA agent can be administered alone or in combination with one or more additional ANGPTL3 dsRNA agents. In some embodiments, two, three, or four or more independent ANGPTL3 dsRNA agents are administered to a subject.

[0126] In some embodiments of the present invention, an ANGPTL3 dsRNA agent is administered to a subject in combination with one or more other treatment schemes for treating an ANGPTL3-related disease or disorder to treat the disease or disorder. Some non-limiting examples of other treatment schemes include administering one or more ANGPTL3 antisense polynucleotides according to the present invention, administering a non-ANGPTL3 dsRNA therapeutic agent, and behavioral changes. The other treatment schemes can be administered at one or more times selected from the group consisting of before, simultaneously, and after the administration of the ANGPTL3 dsRNA agent of the present invention. As used herein, simultaneous administration refers to within 5 minutes from time zero, within 10 minutes from time zero, within 30 minutes from time zero, within 45 minutes from time zero, and within 60 minutes from time zero. It should be understood that "time zero" refers to the time when the ANGPTL3 dsRNA agent of the present invention is administered to the subject. Some non-limiting examples of non-ANGPTL3 dsRNA therapeutics include one or more statins; one or more statins capable of reducing the expression of proprotein convertase subtilisin / kexin type 9 (PCSK9) (German CA, Shapiro MD. Small Interfering RNA Therapeutic Inclisiran: A New Approach to Targeting PCSK9. BioDrugs. 2020 Feb;34(1):1-9. Doi:10.1007 / s40259-019-6. PMID:31782112.); one or more siRNA molecules or antibodies or antisense oligonucleotides (ASOs); a therapeutic agent capable of reducing lipid accumulation in a subject; and a therapeutic agent capable of reducing cholesterol levels and / or accumulation in a subject. Some non-limiting examples of behavioral changes include dietary plans, counseling, and exercise plans.These and other therapeutic agents and behavioral changes are known in the art and can be used to treat an ANGPTL3 disease or disorder in a subject, and can be administered to a subject in combination with the administration of one or more ANGPTL3 dsRNA agents of the invention to treat an ANGPTL3 disease or disorder. ANGPTL3 dsRNA agents of the invention administered to a cell or a subject to treat an ANGPTL3-associated disease or disorder can act synergistically with one or more other therapeutic agents or active agents to improve the effectiveness of the one or more therapeutic agents or active agents and / or improve the effectiveness of the ANGPTL3 dsRNA agent in treating an ANGPTL3-associated disease or disorder.

[0127] The therapeutic methods of the present invention, which include administering ANGPTL3 dsRNA, can be used before the onset of an ANGPTL3-related disease or disorder and / or while an ANGPTL3-related disease or disorder is present, as well as at any time during the early, middle, or late stages of the disease or disorder. The methods of the present invention can also be used to treat subjects who have previously been treated for an ANGPTL3-related disease or condition with one or more other therapeutic agents and / or therapeutic activities, but who have experienced minimal success and / or failure in treating the ANGPTL3-related disease or condition in the subject.

[0128] Vector encoding dsRNA In some embodiments of the present invention, ANGPTL3 dsRNA agents can be delivered to cells using vectors. The ANGPTL3 dsRNA agent transcription unit can be contained in a DNA or RNA vector. The construction and use of vectors encoding transgenes to deliver sequences to cells and / or subjects is known in the art. In the methods of the present invention, the use of vectors can result in transient expression of ANGPTL3 dsRNA for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more hours, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weeks. The length of transient expression can be determined using conventional methods based on factors such as, but not limited to, the specific vector construct selected and the target cell and / or tissue. Such transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, and can be integrating or non-integrating vectors. Transgenes can also be constructed to be inherited as extrachromosomal plasmid genes (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0129] Single or multiple strands of an ANGPTL3 dsRNA agent can be transcribed from a promoter on an expression vector. When expressing two independent strands to produce, for example, dsRNA, two independent expression vectors can be co-introduced into cells using means such as transfection or infection. In some embodiments, each independent strand of an ANGPTL3 dsRNA agent of the present invention can be transcribed by two promoters contained in the same expression vector. In some embodiments, an ANGPTL3 dsRNA agent of the present invention is expressed as an inverted repeat polynucleotide linked by a linker polynucleotide sequence, thereby resulting in an ANGPTL3 dsRNA agent with a stem and loop structure.

[0130] Some non-limiting examples of RNA expression vector are DNA plasmid or virus vector.The expression vector used in the embodiment of the present invention can be adapted to eukaryotic cells.Eukaryotic cell expression vectors are commonly used in the art and can be obtained from many commercial sources.The delivery of ANGPTL3 dsRNA expression vector can be systemic, for example, by introducing it into the subject by intravenous or intramuscular administration, or by administering it to the target cell explanted from the subject, or by any other means that can be incorporated into desired target cell.

[0131] Viral vector systems that can be included in the methods of embodiments of the present invention include, but are not limited to, (a) adenoviral vectors, (b) retroviral vectors, including but not limited to lentiviral vectors and Moloney murine leukemia virus, (c) adeno-associated virus vectors, (d) herpes simplex virus vectors, (e) SV40 vectors, (f) polyomavirus vectors, (g) papillomavirus vectors, (h) picornavirus vectors, (i) poxvirus vectors, such as orthopox vectors, e.g., vaccinia virus vectors, or avipox vectors, e.g., canarypox or fowlpox vectors, and (j) auxiliary-dependent or enteric-less adenoviruses. Constructs for recombinantly expressing ANGPTL3 dsRNA agents can include regulatory elements, such as promoters, enhancers, etc., selected to provide constitutive expression or regulated / inducible expression. The use of promoters, enhancers, etc., as well as viral vector systems, are well known in the art and can be used in conjunction with the methods and compositions described herein.

[0132] Some embodiments of the present invention include the use of viral vector to deliver ANGPTL3 dsRNA agent to cells.Many adenovirus-based delivery systems are commonly used in the art, for example, to deliver to lung, liver, central nervous system, endothelial cell and muscle.Some non-limiting examples of viral vectors that can be used in the method of the present invention include AAV vector, vaccinia virus such as vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC, and bird pox such as fowlpox or canarypox.

[0133] Some embodiments of the present invention comprise the method of using vector to deliver ANGPTL3 dsRNA agent to cell.Such vector can be in a pharmaceutically acceptable vector, and can comprise the sustained release matrix that gene delivery vector is incorporated into, but does not necessarily comprise.In some embodiments, the vector for delivering ANGPTL3 dsRNA can be produced from recombinant cell, and the pharmaceutical composition of the present invention can comprise one or more ANGPTL3 dsRNA delivery system producing cells.

[0134] Pharmaceutical Compositions Comprising ANGPTL3 dsRNA or ssRNA Agents Some embodiments of the present invention include the use of a pharmaceutical composition comprising an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent and a pharmaceutically acceptable vector. Pharmaceutical compositions comprising an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent can be used in methods of the present invention to reduce ANGPTL3 gene expression and ANGPTL3 activity in cells and treat ANGPTL3-related diseases or conditions. Such pharmaceutical compositions can be prepared based on the delivery method. Non-limiting examples of formulations depending on the delivery mode include compositions for subcutaneous delivery, compositions for systemic administration via parenteral delivery, compositions for intravenous (IV) delivery, compositions for intrathecal delivery, and compositions for direct delivery to the brain. To deliver ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents into cells, the pharmaceutical compositions of the present invention can be administered using one or more of the following means: topical (e.g., transdermal patch); pulmonary, nebulizer spray, or aerosol inhalation or insufflation; intratracheal; intranasal; epidermal and transdermal; oral, or parenteral. Parenteral administration includes, for example, intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous administration using an implantable device, or intracranial administration, such as intraparenchymal, intrathecal, or intraventricular administration. ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents can also be delivered directly to target tissues, such as the liver or kidney. It should be understood that "delivery of an ANGPTL3 dsRNA agent" or "delivery of an ANGPTL3 antisense polynucleotide agent" to a cell encompasses directly delivering the ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent, respectively, causing the cell to express the ANGPTL3 dsRNA agent from an encoding vector delivered to the cell, or any suitable means of causing the ANGPTL3 dsRNA or ANGPTL3 antisense polynucleotide agent to appear in the cell. The preparation and use of formulations and means for delivering inhibitory RNA are known and commonly used in the art.

[0135] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of an ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent of the present invention and a pharmaceutically acceptable vector. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerin, ethanol, and combinations thereof. However, the term specifically excludes cell culture media. Pharmaceutically acceptable carriers for orally administered drugs include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, adhesives, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives. Suitable inert diluents include sodium carbonate, calcium carbonate, sodium phosphate, calcium phosphate, and lactose, although cornstarch and alginic acid are suitable disintegrants. Adhesives may also include starch and gelatin. When a lubricant is required, magnesium stearate, stearic acid, or talc can typically be used as the lubricant. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. The drugs contained in the pharmaceutical formulations are further described below.

[0136] As used herein, terms such as "pharmacologically effective amount," "therapeutically effective amount," and "effective amount" refer to the amount of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent of the present invention that produces a desired pharmacological, therapeutic, or preventive result. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or disorder is reduced by at least 10%, then the therapeutically effective amount of a drug for treating the disease or disorder is the amount required to reduce the parameter by at least 10%. For example, a therapeutically effective amount of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent can reduce ANGPTL3 polypeptide levels by at least 10%.

[0137] Effective dose In some embodiments, the methods of the present invention include contacting a cell with an effective amount of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent to reduce expression of the ANGPTL3 gene in the contacted cell. Some embodiments of the methods of the present invention include administering an effective amount of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent to a subject to reduce expression of the ANGPTL3 gene in the subject and treat an ANGPTL3-related disease or disorder. An "effective amount" used to reduce ANGPTL3 expression and / or treat an ANGPTL3-related disease or disorder is an amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent for treating an ANGPTL3-related disease or disorder may be the amount necessary (i) to reduce or stop the progression of the disease or disorder, or (ii) to reverse, alleviate, or eliminate one or more symptoms of the disease or disorder. In some embodiments of the present invention, an effective amount is the amount of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent that, when administered to a subject in need of treatment for an ANGPTL3-related disease or disorder, results in a therapeutic response that prevents and / or treats the disease or disorder. In some embodiments of the present invention, an effective amount is the amount of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent of the present invention that, when combined with or co-administered with another therapy for an ANGPTL3-related disease or disorder, results in a therapeutic response that prevents and / or treats the disease or disorder. In some embodiments of the present invention, the biological effect of treating a subject with an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent of the present invention may be the improvement and / or complete elimination of symptoms caused by an ANGPTL3-related disease or disorder. In some embodiments of the present invention, the biological effect is the complete elimination of the ANGPTL3-related disease or disorder, as evidenced, for example, by a diagnostic test showing that the subject is free of an ANGPTL3-related disease or disorder.Non-limiting examples of detectable physiological symptoms include reduced lipid accumulation in the liver of a subject after administration of an agent of the present invention. To determine the effect of the agent and / or method of the present invention on an ANGPTL3-related disease or disorder, other means known in the art for assessing the status of an ANGPTL3-related disease or disorder can be used.

[0138] Typically, the effective amount of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent for reducing ANGPTL3 polypeptide activity to a level that treats an ANGPTL3-related disease or disorder is determined in a clinical trial, thereby establishing the effective dose for a test population relative to a control population in a blinded study. In some embodiments, the effective amount is an amount that produces a desired response, for example, an amount that alleviates an ANGPTL3-related disease or disorder in cells, tissues, and / or subjects with the disease or disorder. Thus, the effective amount of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent for treating an ANGPTL3-related disease or disorder that can be treated by reducing ANGPTL3 polypeptide activity can be an amount that, when administered, reduces the amount of ANGPTL3 polypeptide activity in a subject to be lower than the amount present in cells, tissues, and / or subjects without administering the ANGPTL3 dsRNA agent or the ANGPTL3 antisense polynucleotide agent. In certain embodiments of the present invention, the level of ANGPTL3 polypeptide activity and / or ANGPTL3 gene expression present in cells, tissues, and / or subjects that have been uncontacted or administered an ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent of the present invention is referred to as a "control" amount. In some embodiments of the methods of the present invention, the control amount for a subject is the amount before treatment of the subject. In other words, the level in a subject before administration of an ANGPTL3 agent may be the control level for the subject, and is compared to the level of ANGPTL3 polypeptide activity and / or ANGPTL3 gene expression in the subject after administration of an ANGPTL3 agent to the subject. When treating an ANGPTL3-related disease or disorder, the desired response may be a reduction or elimination of one or more symptoms of the disease or disorder in cells, tissues, and / or subjects. The reduction or elimination may be temporary or permanent. It should be understood that the status of an ANGPTL3-related disease or condition can be monitored using methods to determine ANGPTL3 polypeptide activity, ANGPTL3 gene expression, symptom assessment, clinical trials, etc.In some embodiments of the invention, the expected response to treating an ANGPTL3-associated disease or condition is to delay or prevent the onset of the disease or condition.

[0139] The effective amount of a compound that reduces the activity of ANGPTL3 polypeptide can also be determined by evaluating the physiological effects on a cell or subject of administering an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent (e.g., alleviation of an ANGPTL3-related disease or condition after administration). Subject measurement and / or symptom monitoring can be used to determine the efficacy of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent of the present invention, which can be administered to a pharmaceutical compound of the present invention, and can also be used to determine whether or not the subject responds to treatment. Non-limiting examples include one or more serum lipid mass spectrometry tests known in the art. Another non-limiting example includes one or more liver function tests known in the art that can be used to determine the status of an ANGPTL3-related liver disease or condition in a subject before and after treatment with an ANGPTL3 dsRNA agent of the present invention. In another non-limiting example, one or more liver cholesterol accumulation tests known in the art are used to determine the status of an ANGPTL3-related disease in a subject. In this example, the disease involves cholesterol accumulation, and the test is performed to determine cholesterol levels in the subject before and after treating the subject with an ANGPTL3 dsRNA agent of the invention.

[0140] Some embodiments of the present invention include methods for determining the efficacy of a dsRNA agent or an ANGPTL3 antisense polynucleotide agent of the present invention administered to a subject to treat an ANGPTL3-related disease or disorder by assessing and / or monitoring one or more "physiological characteristics" of the ANGPTL3-related disease or disorder in the subject. Some non-limiting examples of physiological characteristics of an ANGPTL3-related disease or disorder include serum lipid levels in the subject, LDL levels in the subject, HDL levels in the subject, the LDL / HDL ratio in the subject, triglyceride levels in the subject, liver fat content in the subject, physical symptoms, etc. Standard means for determining such physiological characteristics are known in the art and include, but are not limited to, blood tests, imaging studies, physical examinations, etc.

[0141] It should be understood that the amount of an ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent administered to a subject can be modified at least in part based on the determined disease and / or disorder status and / or physiological characteristics of the subject. The amount of treatment can be varied, for example, by increasing or decreasing the amount of the ANGPTL3-dsRNA agent or ANGPTL3 antisense polynucleotide agent, changing the composition of the separately administered ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent, changing the route of administration, or changing the time of administration. The effective amount of an ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent will vary depending on the specific condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent treatment (if any), the specific route of administration, and other factors within the knowledge and professional health care of the physician. For example, the effective amount will depend on the expected level of ANGPTL3 polypeptide activity and / or ANGPTL3 gene expression that will effectively treat the ANGPTL3-related disease or condition. The effective amount of a particular ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent of the present invention used in the method of the present invention can be determined empirically by those skilled in the art without undue experimentation. By selecting from multiple ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents of the present invention and weighting factors (e.g., efficacy, relative bioavailability, patient weight, severity of adverse side effects, and preferred administration method) based on the teachings provided herein, an effective prophylactic or therapeutic treatment regimen can be designed to effectively treat a particular subject. As used in some embodiments of the present invention, an effective amount of an ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent of the present invention may be an amount that produces a desired biological effect in cells when contacted with cells.

[0142] It should be understood that ANGPTL3 gene silencing can be determined by any appropriate measurement in any cells that express ANGPTL3 constitutively or modified by genome engineering. In some embodiments of the present invention, administration of an ANGPTL3 dsRNA agent of the present invention reduces ANGPTL3 gene expression by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments of the present invention, administration of an ANGPTL3 dsRNA agent of the present invention reduces ANGPTL3 gene expression by 5% to 10%, 5% to 25%, 10% to 50%, 10% to 75%, 25% to 75%, 25% to 100%, or 50% to 100%.

[0143] Administration The ANGPTL3 dsRNA agent and the ANGPTL3 antisense polynucleotide agent are delivered in a pharmaceutical composition at a dose sufficient to inhibit expression of the ANGPTL3 gene. In some embodiments of the present invention, the dose of the ANGPTL3 dsRNA agent or the ANGPTL3 antisense polynucleotide agent is 0.01 to 200.0 mg / kg of subject body weight per day, typically 1 to 50 mg / kg body weight, 5 to 40 mg / kg body weight, 10 to 30 mg / kg body weight, 1 to 20 mg / kg body weight, 1 to 10 mg / kg body weight, or 4 to 15 mg / kg body weight per day, including the endpoints. For example, The dsRNA agent or ANGPTL3 antisense polynucleotide agent may be administered at a dose of about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, or 1.9 mg / kg. g / kg, 1.9mg / kg, 2mg / kg, 2.1mg / kg, 2.2mg / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg, 3.9mg / kg, 4mg / kg, 4.1mg / kg, 4.2mg / kg, 4.3mg / kg, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / kg, 4.8mg / kg, 4.9mg / kg, 5mg / kg, 5.1mg / kg, 5.2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg , 5.9mg / kg, 6mg / kg, 6.1mg / kg, 6.2mg / kg, 6.3mg / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7mg / kg, 7.1mg / kg, 7.2mg / kg, 7.3mg / kg, 7.4mg / kg, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg, 7.8mg / kg, 7.9mg / kg, 8mg / kg, 8.1mg / kg, 8.2mg / kg, 8.3mg / kg, 8.4mg / kg, 8.5mg / kg, 8.6mg / kg, 8.7mg / kg, 8.8mg / kg, 8.9mg / kg, 9mg / kg, 9.1mg / kg, 9.2mg / kg, 9.3mg / kg, 9.4mg / kg, 9.5mg / kg, 9.6mg / kg, 9.7mg / kg, 9.8mg / kg, 9.9mg / kg, 10mg / kg, 11mg / kg, 12mg / kg, 13mg / kg, 14mg / kg, 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 21mg / kg, 22mg / kg, 23mg / kg, 24mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30mg / kg, 31mg / kg, 32mg / kg, 33mg / kg, 34mg / kg, 35mg / body weight. .

[0144] When determining the dose and delivery time of the ANGPTL3 dsRNA agent of the present invention, various factors are taken into consideration.The absolute amount of the delivered ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent depends on various factors, including concurrent treatment, number of doses, and individual parameters, including age, physical condition, body type, and weight.These are factors known to those skilled in the art and can be obtained by routine experimentation.In some embodiments, the maximum dose can be used, which is the maximum safe dose based on sound medical judgment.

[0145] In some embodiments, the methods of the present invention may include administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent to a subject. In some cases, the subject may be administered a pharmaceutical compound (e.g., a drug compound comprising an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent) at least daily, every other day, weekly, every other week, monthly, etc. The drug may be administered once daily or more than once daily, for example, two, three, four, five, or more doses within a 24-hour period. The pharmaceutical composition of the present invention may be administered once daily, or the ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent may be administered as two, three, or more divided doses at appropriate intervals throughout the day, or continuous infusion or oral delivery may be used. In some embodiments of the methods of the invention, a pharmaceutical composition of the invention is administered to a subject one or more times per day, one or more times per week, one or more times per month, or one or more times per year.

[0146] In some embodiments of the methods of the present invention, the drug compound may be administered alone or in combination with one or more other ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents, and / or in combination with other drug treatments, therapies, or regimens administered to a subject with an ANGPTL3-related disease or disorder. The drug compound may be administered as a pharmaceutical composition. The pharmaceutical composition used in the methods of the present invention is sterile and may contain a certain amount of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent that reduces the activity of an ANGPTL3 polypeptide to a level sufficient to produce the desired response, in a weight or volume unit suitable for administration to a subject. The dose of a pharmaceutical composition containing an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent that reduces angiopoietin-like protein 3 (ANGPTL3) activity administered to a subject can be selected depending on various parameters, particularly the administration method used and the condition of the subject. Other factors include the desired treatment duration. If the subject responds inadequately to the initial dose administered, higher doses (or substantially higher doses through a different, more localized delivery route) can be employed, to the extent tolerated by the patient.

[0147] treatment ANGPTL3-related diseases and conditions, in which a decrease in the level and / or activity of ANGPTL3 polypeptide effectively treats the disease or condition, can be treated by inhibiting ANGPTL3 expression using the methods and ANGPTL3 dsRNA agents of the present invention. Some examples of diseases and conditions that can be treated by the ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents of the present invention and the treatment methods of the present invention include, but are not limited to, hyperlipidemia, hypertriglyceridemia, abnormal lipid and / or cholesterol metabolism, homo- and heterofamilial hypercholesterolemia, statin-resistant hypercholesterolemia, cardiometabolic disease, obesity, atherosclerosis, type 2 diabetes, cardiovascular disease, coronary artery disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, and hypertriglyceridemia-related pancreatitis. Such diseases and disorders are sometimes referred to herein as "ANGPTL3-related diseases and disorders" and "diseases and disorders caused and / or regulated by ANGPTL3."

[0148] In some embodiments of the present invention, the ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent of the present invention can be administered to a subject at one or more time points before or after diagnosing an ANGPTL3-related disease or disorder. In some embodiments of the present invention, the subject is at risk of suffering from or developing an ANGPTL3-related disease or disorder. A subject at risk of developing an ANGPTL3-related disease or disorder is a subject who is more likely to develop the ANGPTL3-related disease or disorder than a control subject who is at higher risk of developing the ANGPTL3-related disease or disorder. In some embodiments of the present invention, the risk level can be statistically significant compared to the control risk level. At-risk subjects include, for example, subjects who have a pre-existing disease and / or genetic abnormality and are therefore more susceptible to an ANGPTL3-related disease or disorder than control subjects without a pre-existing disease or genetic abnormality, subjects with a family and / or personal history of an ANGPTL3-related disease or disorder, and subjects who have been treated for an ANGPTL3-related disease or disorder. It is understood that a pre-existing disease and / or genetic abnormality that renders a subject more susceptible to an ANGPTL3-associated disease or condition may also be a disease and / or genetic abnormality that has been identified as pre-existing and that makes the subject more likely to develop an ANGPTL3-associated disease or condition.

[0149] It should be understood that an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent can be administered to a subject depending on the subject's medical condition. For example, healthcare provided to a subject can include assessing lipid levels in a sample obtained from the subject and determining whether there is a need to lower the subject's lipid levels by administering an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent of the present invention. In this example, even if the subject has not been diagnosed with an ANGPTL3-related disease (e.g., one of those disclosed herein), lipid levels are considered to be a physiological characteristic of an ANGPTL3-related disease. A healthcare provider can monitor changes in the subject's lipid levels as the efficacy of the administered ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent of the present invention increases. In a non-limiting example, a biological sample, such as a blood or serum sample, can be obtained from the subject, and the subject's lipid levels can be determined in the sample. An ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent is administered to a subject, and blood or serum samples are collected from the subject after administration to measure lipid levels, and the results are compared with results determined from the subject's sample before (previous) administration. A reduction in the subject's lipid levels in the post-administration sample compared to pre-administration levels indicates the efficacy of the administered ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent in reducing lipid levels in the subject.

[0150] Some embodiments of the methods of the present invention include adjusting a treatment comprising administering a dsRNA agent or ANGPTL3 antisense polynucleotide agent of the present invention to a subject based at least in part on an assessment of one or more changes in the physiological characteristics of the subject's ANGPTL3-related disease or disorder caused by the treatment. For example, in some embodiments of the present invention, determining the effect of an administered dsRNA agent or ANGPTL3 antisense polynucleotide agent of the present invention on the subject is used to help adjust the amount of a dsRNA agent or ANGPTL3 antisense polynucleotide agent of the present invention subsequently administered to the subject. In a non-limiting example, the subject's lipid levels are determined after administration of a dsRNA agent or ANGPTL3 antisense polynucleotide agent of the present invention to the subject, and based at least in part on the determined levels, it is determined that a larger amount of the dsRNA agent or ANGPTL3 antisense polynucleotide agent is desirable, thereby improving the physiological effect of the administered agent, e.g., lowering or further lowering the subject's lipid levels. In another non-limiting example, it is desirable to determine the lipid levels of a subject after administering a dsRNA agent or an ANGPTL3 antisense polynucleotide agent of the present invention to the subject, and to administer a lower amount of the dsRNA agent or ANGPTL3 antisense polynucleotide agent to the subject based at least in part on the determined levels.

[0151] Some embodiments of the present invention also include assessing changes in one or more physiological characteristics of a subject due to previous treatment in order to adjust the amount of a dsRNA agent or ANGPTL3 antisense polynucleotide agent of the present invention administered to the subject. Some embodiments of the methods of the present invention also include determining physiological characteristics of an ANGPTL3-related disease or disorder one, two, three, four, five, six, or more times to evaluate and / or monitor the efficacy of an administered ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent of the present invention, and optionally using the results of the determinations to adjust the dose, administration scheme, and / or administration frequency of the dsRNA agent or ANGPTL3 antisense polynucleotide agent of the present invention to treat an ANGPTL3-related disease or disorder in the subject. In some embodiments of the methods of the invention, administering an effective amount of a dsRNA agent or ANGPTL3 antisense polynucleotide agent of the invention to a subject is expected to result in a reduction in the subject's lipid levels, serum lipid levels, LDL levels, LDL / HDL ratio, triglyceride levels, liver fat in the subject, etc., compared to previous levels or control levels determined for the subject.

[0152] As used herein, the terms "treatment," "treated," or "under treatment," when used in reference to an ANGPTL3-related disease or disorder, may refer to prophylactic treatment that reduces the likelihood of an ANGPTL3-related disease or disorder occurring in a subject, or may refer to treatment after an ANGPTL3-related disease or disorder has occurred in a subject, such that the level of the ANGPTL3-related disease or disorder is eliminated or reduced, the ANGPTL3-related disease or disorder is prevented from developing at a later stage (e.g., more severe), and / or the progression of the ANGPTL3-related disease or disorder in the subject is alleviated compared to a subject in the absence of treatment to reduce the activity of an ANGPTL3 polypeptide in the subject.

[0153] In some embodiments, the agents, compositions, and methods of the present invention can be used to inhibit the expression of the ANGPTL3 gene. As used herein, the terms "inhibition," "silencing," "reducing," "downregulating," and "knockdown" of the expression of the ANGPTL3 gene refer to a decrease in the expression of the ANGPTL3 gene, as measured, for example, by one or more of the level of RNA transcribed from the gene, the activity level of expressed ANGPTL3, and the level of ANGPTL3 polypeptide, protein, or protein subunit translated from mRNA, in a cell, cell group, tissue, organ, or subject that transcribes the ANGPTL3 gene, when the cell, cell group, tissue, organ, or subject is contacted with (e.g., treated with) an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent of the present invention, compared to a control level of RNA transcribed from the ANGPTL3 gene, the activity level of expressed ANGPTL3, or the level of ANGPTL3 translated from mRNA. In some embodiments, the control level is the level in a cell, tissue, organ, or subject not contacted with (eg, treated with) an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent.

[0154] Administration method Various routes of administration of ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents can be used in the methods of the present invention. The specific delivery mode selected will depend, at least in part, on the particular disorder being treated and the dosage required for therapeutic efficacy. Generally, the methods of the present invention can be practiced using any medically acceptable administration method that produces levels effective for treating ANGPTL3-related diseases or disorders while not causing clinically unacceptable adverse effects. In some embodiments of the present invention, ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents can be administered via oral, enteral, mucosal, transdermal, and / or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal, and intrasternal injection or infusion. Other routes include, but are not limited to, nasal (e.g., gastric nasogastric tube), transdermal, vaginal, rectal, sublingual, and inhalation. Delivery routes of the present invention may include intrathecal, intracardiac, or intracranial routes. In some embodiments of the present invention, ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents can be administered by placing them in a slow-release matrix and placing the matrix in a subject. In some aspects of the present invention, ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents can be delivered to target cells using nanoparticles coated with delivery agents that target specific cells or organelles. Various delivery means, methods, and agents are known in the art. Some non-limiting examples of delivery methods and delivery agents are provided elsewhere herein. In some aspects of the present invention, the term "delivery" when referring to ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents refers to administering one or more "naked" ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agent sequences to cells or subjects.In certain embodiments of the present invention, "delivery" refers to administering to a cell or a subject by means of transfection, delivering to a subject a cell containing an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent, delivering to a cell and / or subject a vector encoding the ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent, etc. Delivering an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent by transfection can include administering a vector to a cell and / or subject.

[0155] In some methods of the present invention, one or more ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents can be used in formulations that may be administered as pharmaceutically acceptable solutions, typically containing pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible vectors, auxiliary agents, and any other therapeutic ingredients. In some embodiments of the present invention, ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents can be formulated together with other therapeutic agents for simultaneous administration. According to the methods of the present invention, ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents can be used in pharmaceutical compositions. Typically, pharmaceutical compositions include an ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent and any pharmaceutically acceptable vector. Pharmaceutically acceptable vectors are well known to those skilled in the art. As used herein, a pharmaceutically acceptable vector refers to a non-toxic material that does not inhibit the biological effectiveness of the active ingredient (e.g., the ability of an ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent to inhibit ANGPTL3 gene expression in a cell or subject). Many methods for administering and delivering dsRNA agents or ANGPTL3 antisense polynucleotide agents used in therapeutic applications are known in the art and can be used in the methods of the present invention.

[0156] Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Pat. No. 5,211,657 and are known to those skilled in the art. Such formulations may typically contain salts, buffers, preservatives, compatible carriers, and any other therapeutic agents. When used in medicine, these salts are pharmaceutically acceptable; however, pharmaceutically unacceptable salts may be conveniently used in the preparation of pharmaceutically acceptable salts and are not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, and succinic acid. Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth metal salts, e.g., sodium, potassium, or calcium salts.

[0157] Some embodiments of the methods of the present invention include directly administering one or more ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents to a tissue. In some embodiments, the tissue to which the compound is administered is a tissue in which an ANGPTL3-related disease or disorder exists or can occur, such as, but not limited to, the liver or kidney. Direct tissue administration can be achieved by direct injection or other methods. Because many orally administered compounds naturally reach and pass through the liver and kidney, some embodiments of the therapeutic methods of the present invention include orally administering one or more ANGPTL3 dsRNA agents to a subject. ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents, alone or in combination with other therapeutic agents, may be administered once or multiple times. When multiple doses of ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents are administered, they can be administered by different routes. For example, but not limited to, the first (or previous multiple) doses can be administered subcutaneously, and one or more additional doses can be administered orally and / or systemically.

[0158] In some embodiments of the present invention where systemic administration of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent is desired, the ANGPTL3 dsRNA agent or the ANGPTL3 antisense polynucleotide agent may be prepared for parenteral administration by injection (e.g., push injection or continuous infusion). Injectable preparations are present in unit dosage forms, such as ampoules or multi-dose containers, with or without the addition of preservatives. ANGPTL3 dsRNA agent formulations (also called pharmaceutical compositions) can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain compounding agents such as suspending agents, stabilizers, and / or dispersing agents.

[0159] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Some examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, as well as saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's glucose), and the like. Other additives, such as preservatives, antibacterial agents, antioxidants, chelating agents, and inert gases, may also be added. Other forms of administration, such as intravenous administration, require relatively low dosages. If the subject does not respond adequately to the initial dose administered, a higher dose (or a substantially higher dose through a different, more localized delivery route) can be used, as long as the patient is able to tolerate it. If necessary, multiple doses per day can be used to achieve adequate systemic or local levels of one or more ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents and to achieve an appropriate reduction in angiopoietin-like protein 3 (ANGPTL3) activity.

[0160] In some other embodiments, the methods of the present invention include using a delivery vehicle suitable for implantation into a subject (e.g., a subject), such as a biocompatible microparticle, nanoparticle, or implant. An exemplary bioerodible implant for use in accordance with this method is described in PCT Publication No. WO 95 / 24929 (herein incorporated by reference), which describes a biocompatible, biodegradable polymer matrix comprising a biopolymer.

[0161] In the methods of the present invention, both non-biodegradable and biodegradable polymer matrices can be used to deliver one or more ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents to a subject. In some embodiments, the matrix may be biodegradable. The matrix polymer may be a natural or synthetic polymer. The polymer may be selected based on the desired release time, which typically ranges from about several hours to a year or more. Typically, release occurs within a period of several hours to 3-12 months. The polymer may be in the form of a hydrogel capable of absorbing up to about 90% of its weight in water, and may optionally be crosslinked with multivalent ions or other polymers.

[0162] Generally, in some embodiments of the present invention, ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent can be delivered by using biodegradable implant, by diffusion or degradation of polymer matrix.The exemplary synthetic polymer used for such purpose is known in the art.Biodegradable polymer and non-biodegradable polymer can be used to deliver ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent by using the method known in the art. Biodegradable adhesive polymers (e.g., biodegradable hydrogels) (see H.S. Sawhney, C.P. Pathak, and J.A. Hubbell in Macromolecules, 1993, 26, 581-587). Other suitable delivery systems include timed-, delayed-, or sustained-release delivery systems. Such systems can avoid duplicate administration of ANGPTL3 dsRNA or ANGPTL3 antisense polynucleotide agents, providing increased convenience for patients and healthcare professionals. A variety of release delivery systems are available and known to those skilled in the art (e.g., U.S. Patent Nos. 5,075,109; 4,452,775; 4,675,189; 5,736,152; 3,854,480; 5,133,974; and 5,407,686, the teachings of each of which are incorporated herein by reference). In addition, pump-based hardware delivery systems may be used, some of which are suitable for implantation.

[0163] The use of long-term sustained release implants can be applied to the prophylactic treatment of subjects and subjects at risk of developing recurrent ANGPTL3-related diseases or conditions.As used herein, long-term sustained release means that the implant is constructed and arranged to deliver therapeutic levels of ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents within at least 10 days, 20 days, 30 days, 60 days, 90 days, 6 months, 1 year or more.Long-term sustained release implants are well known to those skilled in the art and include some of the release systems mentioned above.

[0164] The molecule or compound having the desired purity can be mixed with any pharmaceutically acceptable carrier, excipient, or stabilizer [Remington's Pharmaceutical Sciences, 21st Edition (2006)] to prepare and store the ANGPTL3 dsRNA agent or ANGPTL3 antisense polynucleotide agent as a lyophilized formulation or aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to subjects at the dosages and concentrations used, and include, for example, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethylamine chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); and low-molecular-weight (less than about 10 residues) polypeptides. proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, and dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN, PLURONICS®, or polyethylene glycol (PEG).

[0165] Cells, subjects and controls The methods of the present invention can be used in conjunction with cells, tissues, organs, and / or subjects. In some embodiments of the present invention, the subject is a human or a vertebrate mammal, including, but not limited to, a dog, cat, horse, cow, sheep, mouse, rat, and primate such as a monkey. Thus, the present invention can be used to treat ANGPTL3-related diseases or conditions in human and non-human subjects. In some embodiments of the present invention, the subject may be a farm animal, a zoo animal, a domestic animal, or a non-domestic animal, and the methods of the present invention can be used for veterinary prophylaxis and treatment. In some embodiments of the present invention, the subject is a human, and the methods of the present invention can be used in human prophylactic and therapeutic regimens.

[0166] Some non-limiting examples of subjects to which the present invention is applicable include subjects diagnosed as suffering from, suffering from, or at risk of suffering from a disease or disorder associated with higher than desired ANGPTL3 expression and / or activity (also referred to as "elevated ANGPTL3 expression levels"). Some non-limiting examples of diseases and conditions associated with higher than desired ANGPTL3 expression and / or activity are described elsewhere herein. The methods of the present invention can be applied to subjects diagnosed at the time of treatment as suffering from a disease or condition associated with higher than desired ANGPTL3 expression and / or activity, or subjects believed to be suffering from or at risk of developing a disease or condition associated with higher than desired ANGPTL3 expression and / or activity. In some embodiments of the present invention, the disease or disorder associated with higher than desired ANGPTL3 expression and / or activity levels is an acute disease or disorder, and in some embodiments of the present invention, the disease or disorder associated with higher than desired ANGPTL3 expression and / or activity levels is a chronic disease or condition.

[0167] In a non-limiting example, an ANGPTL3 dsRNA agent of the present invention is administered to a subject diagnosed with, suspected of having, or at risk for statin-resistant hypercholesterolemia, a disease in which reduced expression of ANGPTL3 is expected. The method of the present invention can be applied to subjects diagnosed with the disease or disorder at the time of treatment, or subjects thought to be at risk for having or developing the disease or disorder.

[0168] In another non-limiting example, the ANGPTL3 dsRNA agent of the present invention is administered to a subject diagnosed with, suspected of having, or at risk for hyperlipidemia, in which ANGPTL3 expression is expected to be reduced. The method of the present invention can be applied to subjects diagnosed with the disease or disorder at the time of treatment, or subjects thought to be at risk for having or developing the disease or disorder.

[0169] Cells to which the methods of the present invention can be applied include in vitro, in vivo, and ex vivo cells. Cells can be in a subject, in culture and / or in suspension, or in any other suitable state or condition. Cells to which the methods of the present invention can be applied may be liver cells, hepatocytes, cardiomyocytes, pancreatic cells, cardiovascular cells, kidney cells, or other types of vertebrate cells, including human and non-human mammalian cells. In certain aspects of the present invention, cells to which the methods of the present invention can be applied are healthy cells known to be free of disease. In certain embodiments of the present invention, the cells to which the methods and compositions of the present invention can be applied are liver cells, hepatocytes, cardiomyocytes, pancreatic cells, cardiovascular cells, and / or kidney cells. While in some aspects of the present invention, the control cells are normal cells, it should be understood that in certain cases, cells with a disease or condition may be used as control cells, for example, to compare results between treated cells with a disease or condition and untreated cells with a disease or condition.

[0170] According to the methods of the present invention, the level of ANGPTL3 polypeptide activity can be determined and compared with a control level of ANGPTL3 polypeptide activity. The control may be a predetermined value and may take various forms. It may be a single cutoff value, such as a median or mean value. It may be constructed based on a comparison group, such as a group with normal levels of ANGPTL3 polypeptide and / or ANGPTL3 polypeptide activity or a group with elevated levels of ANGPTL3 polypeptide and / or ANGPTL3 polypeptide activity. Other non-limiting examples of comparison groups include a group with one or more symptoms of an ANGPTL3-associated disease or disorder or diagnosed with an ANGPTL3-associated disease or disorder; a group without one or more symptoms of the disease or disorder or not diagnosed with the disease or disorder; a group administered the subject siRNA treatment of the present invention; or a group not administered the subject siRNA treatment of the present invention. Typically, the control can be based on apparently healthy normal individuals or apparently healthy cells in an appropriate age group. It should be understood that the control according to the present invention may be a sample of material tested in parallel with the experimental material, in addition to a predetermined value. For example, it may include a sample from a control group or a control sample prepared in parallel with the experimental sample. In some embodiments of the present invention, the control may be a cell or subject that has not been contacted or treated with an ANGPTL3 dsRNA agent of the present invention, and in such cases, the control level of ANGPTL3 polypeptide and / or ANGPTL3 polypeptide activity can be compared with the level of ANGPTL3 polypeptide and / or ANGPTL3 polypeptide activity in a cell or subject that has been contacted with an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent of the present invention.

[0171] In some embodiments of the present invention, the ANGPTL3 polypeptide level determined for a subject may be a control level relative to an ANGPTL3 polypeptide level determined for the same subject at a different time. In a non-limiting example, the ANGPTL3 level is determined from a biological sample obtained from a subject who has not received the ANGPTL3 treatment of the present invention. In some embodiments, the biological sample is a serum sample. The ANGPTL3 polypeptide level determined from a sample obtained from the subject can be used as the subject's baseline or control value. In the treatment methods of the present invention, after one or more administrations of an ANGPTL3 dsRNA agent to the subject, one or more additional serum samples can be obtained from the subject, and the ANGPTL3 polypeptide level in the subsequent sample or samples can be compared with the subject's control / baseline level. Such comparisons can be used to assess the onset, progression, or resolution of an ANGPTL3-related disease or disorder in a subject. For example, a higher level of ANGPTL3 polypeptide in a baseline sample obtained from a subject than the level obtained from the same subject after administering an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent of the invention to the subject indicates regression of an ANGPTL3-related disease or condition and indicates that the administered ANGPTL3 dsRNA agent of the invention is effective in treating an ANGPTL3-related disease or disorder.

[0172] In some embodiments of the present invention, one or more of the levels of ANGPTL3 polypeptide and / or ANGPTL3 polypeptide activity determined for a subject can be used as a control value to compare later levels of ANGPTL3 polypeptide and / or ANGPTL3 activity in the same subject, thereby enabling assessment of changes in "baseline" ANGPTL3 polypeptide activity in the subject. Thus, an initial level of ANGPTL3 polypeptide and / or initial level of ANGPTL3 polypeptide activity can be present in and / or determined in a subject, and methods and compounds of the present invention can be used to reduce the level of ANGPTL3 polypeptide and / or ANGPTL3 polypeptide activity in a subject using the initial level as the control level for the subject.

[0173] Using the methods of the present invention, an ANGPTL3 dsRNA agent and / or an ANGPTL3 antisense polynucleotide agent of the present invention can be administered to a subject. The efficacy of the administration and treatment of the present invention can be evaluated if the ANGPTL3 polypeptide level in a serum sample obtained from the subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the pre-administration level of ANGPTL3 polypeptide in a serum sample obtained from the subject at a previous time point, or compared to an untreated control level (e.g., the ANGPTL3 polypeptide level in a control serum sample). Note that both the ANGPTL3 polypeptide level and the activity level of the ANGPTL3 polypeptide are related to the expression level of the ANGPTL3 gene. Some embodiments of the methods of the present invention include administering an ANGPTL3 dsRNA and / or an ANGPTL3 antisense agent of the present invention to a subject, thereby reducing the ANGPTL3 polypeptide level and the activity level of the ANGPTL3 polypeptide in the subject.

[0174] Some embodiments of the present invention involve determining the presence, absence, and / or amount (also referred to herein as level) of ANGPTL3 polypeptide in one or more biological samples obtained from one or more subjects. This measurement can be used to assess the efficacy of a treatment method of the invention. For example, the methods and compositions of the present invention can be used to determine the level of ANGPTL3 polypeptide in a biological sample obtained from a subject previously treated with an ANGPTL3 dsRNA agent and / or an ANGPTL3 antisense agent of the present invention. An ANGPTL3 polypeptide level determined from a serum sample obtained from the treated subject that is at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more lower than the pre-treatment level of ANGPTL3 polypeptide determined for the subject or the level in an uncontacted control biological sample indicates an effective level of the treatment administered to the subject.

[0175] In some embodiments of the present invention, physiological characteristics of an ANGPTL3-related disease or disorder determined for a subject may be determined as a control for comparison with physiological characteristics determined for the same subject at a different time. As a non-limiting example, physiological characteristics, such as lipid levels and / or HDL / LDL ratios, are determined from a biological sample (e.g., a serum sample) obtained from a subject who has not been administered an ANGPTL3 treatment of the present invention. The lipid levels and / or HDL / LDL ratios (and / or other physiological characteristics of an ANGPTL3 disease or condition) determined from a sample obtained from the subject can be used as the subject's baseline or control values. In the treatment methods of the present invention, after one or more administrations of an ANGPTL3 dsRNA agent to the subject, one or more additional serum samples can be obtained from the subject, and the lipid levels and / or HDL / LDL ratios in the subsequent one or more samples can be compared with the subject's control / baseline levels and / or ratios, respectively. Such comparisons can be used to assess the onset, progression, or resolution of an ANGPTL3-related disease or disorder in a subject. For example, lipid levels in a baseline sample obtained from a subject that are higher than lipid levels determined in a sample obtained from the same subject after administration of an ANGPTL3 dsRNA agent or an ANGPTL3 antisense polynucleotide agent of the present invention to the subject indicate regression of an ANGPTL3-related disease or condition and that the administered ANGPTL3 dsRNA agent of the present invention is effective in treating an ANGPTL3-related disease or disorder.

[0176] In some embodiments of the present invention, one or more values ​​of a physiological characteristic of an ANGPTL3-related disease or disorder determined for a subject may be used to compare with a control value of the physiological characteristic at a later time in the same subject, thus enabling assessment of changes in the "baseline" physiological characteristic in the subject. Thus, an initial physiological characteristic can be present in and / or determined in a subject, and the methods and compounds of the present invention can be used to reduce the level of ANGPTL3 polypeptide and / or ANGPTL3 polypeptide activity in the subject, using the initial physiological characteristic determination as a control for the subject.

[0177] Using the methods of the present invention, an effective amount of an ANGPTL3 dsRNA agent and / or an ANGPTL3 antisense polynucleotide agent of the present invention can be administered to a subject to treat an ANGPTL3 disease or disorder. The efficacy of the administration and treatment of the present invention can be evaluated by determining changes in one or more physiological characteristics of an ANGPTL3 disease or disorder. In a non-limiting example, lipid levels in a serum sample obtained from the subject are reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the lipid levels before administration in a serum sample obtained from the subject at a previous time point, or compared to untreated control levels (e.g., lipid levels in a control serum level). It should be understood that lipid levels, HDL levels, HDL / LDL ratios, triglyceride levels, and liver fat content of a subject are each related to ANGPTL3 gene expression levels. Some embodiments of the methods of the invention include administering to a subject an effective amount of an ANGPTL3 dsRNA and / or ANGPTL3 antisense agent of the invention to inhibit ANGPTL3 gene expression, thereby reducing lipid levels, HDL levels, HDL / LDL ratio, triglyceride levels, the amount of liver fat in the subject, or positively affecting physiological characteristics of an ANGPTL3-related disease or condition in the subject.

[0178] In some embodiments of the present invention, methods of determining the presence or absence and / or changes in physiological characteristics of an ANGPTL3-related disease or disorder can be used to assess the efficacy of the treatment methods of the present invention, including, but not limited to, determining the presence or absence and / or changes in physiological characteristics of an ANGPTL3-related disease or disorder by, for example, (1) assessing physiological characteristics of one or more biological samples obtained from one or more subjects; (2) imaging the subject (e.g., but not limited to, obtaining liver images); and (3) physical examination of the subject. Such determinations can be used to assess the efficacy of the treatment methods of the present invention.

[0179] kit Kits are also within the scope of the present invention and include one or more ANGPTL3 dsRNA agents and / or ANGPTL3 antisense polynucleotide agents, as well as instructions for use in the methods of the present invention. Kits of the present invention may include one or more agents selected from the group consisting of ANGPTL3 dsRNA agents, ANGPTL3 sense polynucleotides, and ANGPTL3 antisense polynucleotide agents that can be used to treat ANGPTL3-related diseases or conditions. Kits containing one or more ANGPTL3 dsRNA agents, ANGPTL3 sense polynucleotides, and ANGPTL3 antisense polynucleotides can be prepared for use in the therapeutic methods of the present invention. The components of the kits of the present invention may be packaged in aqueous media or in lyophilized form. Kits of the present invention may include a compartmentalized carrier for sealingly receiving one or more container means or a series of container means, such as test tubes, vials, flasks, bottles, syringes, etc., therein. A first container device or a series of container devices may contain one or more compounds, such as an ANGPTL3 dsRNA agent and / or an ANGPTL3 sense or antisense polynucleotide agent. The second container device or series of container devices can contain a targeting agent, labeling agent, delivery agent, etc., which may be included as part of the ANGPTL3 dsRNA agent and / or ANGPTL3 antisense polynucleotide to be administered in the treatment methods of the invention in one embodiment.

[0180] The kits of the invention may include instructions, typically in written form, that provide guidance for administering treatment with the kit and for making decisions based thereon.

[0181] The following examples are provided to illustrate specific embodiments of the present invention, but are not intended to limit the scope of the present invention. As will be apparent to those skilled in the art, the present invention can be applied in a variety of compositions and methods. [Example]

[0182] Example 1 Synthesis of ANGPTL3 RNAi Agents The ANGPTL3 RNAi drug duplexes shown in Tables 2 to 5 above were synthesized according to the following general procedure.

[0183] Sense and antisense siRNA sequences were synthesized on an oligonucleotide synthesizer using a well-established solid-phase synthesis method based on phosphoramidite chemistry. Oligonucleotide chain elongation was achieved through a four-step cycle of deprotection, condensation, capping, and oxidation or sulfurization to add each nucleotide. Synthesis was performed on a solid-phase support made of controlled pore glass (CPG, 1000 Å). The phosphoramidites used were commercially available. Phosphoramidites bearing GalNAc ligand clusters (non-limiting examples: GLPA1 and GLPA2) were synthesized according to the procedures described in Examples 2 and 3 herein. siRNAs used for in vitro screening (Table 2) were synthesized at a 2 μmol scale, while siRNAs used for in vivo testing (Tables 3, 4, and 5) were synthesized at a 5 μmol or larger scale. When a GalNAc ligand (non-limiting example: GLO-0) was linked to the 3' end of the sense strand, a CPG solid support with the GalNAc ligand linked to it was used. When a GalNAc ligand (GLS-1 or GLS-2, for example) was linked to the 5' end of the sense strand, a GalNAc phosphoramidite (GLPA1 or GLPA2, for example) was used in the final coupling reaction. 3% trichloroacetic acid (TCA) in dichloromethane was used to deprotect the 4,4'-dimethoxytrityl protecting group (DMT). 5-Ethylthio-1H-tetrazole was used as an activating agent. I2 in THF / Py / HO and phenylacetodisulfide (PADS) in pyridine / MeCN were used for the oxidation and sulfurization reactions, respectively. After the final solid-phase synthesis step, the solid-support-bound oligomer was cleaved and the protecting groups were removed by treatment with a 1:1 volume mixture of 40% (wt%) aqueous methylamine and 28% ammonium hydroxide. The crude mixture was concentrated to synthesize siRNA for in vitro screening. The remaining solid was dissolved in 1.0 M NaOAc, and ice-cold EtOH was added to precipitate the single-stranded product as the sodium salt, which was used for annealing without further purification.To synthesize siRNA for in vivo testing, the crude single-stranded product was further purified by ion-pairing reversed-phase HPLC (IP-RP-HPLC). The purified single-stranded oligonucleotide product from IP-RP-HPLC was converted to the sodium salt by dissolving in 1.0 M NaOAc and precipitating with ice-cold EtOH. Equimolar complementary sense and antisense oligonucleotides were annealed in water to form the double-stranded siRNA product, which was then lyophilized to yield a fluffy white solid.

[0184] [Table 6] JPEG2026035574000050.jpg176149

[0185] Example 2 Preparation of Intermediate A and Intermediate B As shown in Scheme 1 below, intermediate A was synthesized by treating commercially available galactosamine pentaacetate with trimethylsilyl trifluoromethanesulfonate (TMSOTf) in dichloromethane (DCM). Subsequent glycosylation with Cbz-protected 2-(2-aminoethoxy)ethan-1-ol afforded compound II. The Cbz protecting group was removed by hydrogenation to afford intermediate A as the trifluoroacetic acid (TFA) salt. Intermediate B was synthesized according to the same scheme, except that Cbz-protected 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol was used as the starting material.

[0186] [ka]

[0187] To a solution of compound I (20.0 g, 51.4 mmol) in 100 mL of 1,2-dichloroethane (DCE), TMSOTf (17.1 g, 77.2 mmol) was added. The resulting reaction solution was stirred at 60 °C for 2 hours and then at 25 °C for 1 hour. Cbz-protected 2-(2-aminoethoxy)ethan-1-ol (13.5 g, 56.5 mmol) in DCE (100 mL) dried over 4 Å molecular sieve powder (10 g) was added dropwise to the above reaction solution at 0 °C under a N atmosphere. The resulting reaction mixture was stirred at 25 °C for 16 hours under a N atmosphere. The reaction mixture was filtered and washed with saturated aqueous sodium bicarbonate (200 mL), water (200 mL), and saturated brine (200 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give the crude product, which was then triturated with 2-methyltetrahydrofuran / heptane (5 / 3, v / v, 1.80 L) for 2 h. The resulting mixture was filtered and dried to give compound II (15.0 g, 50.3% yield) as a white solid.

[0188] To a dry, argon-purged hydrogenation bottle, 10% Pd / C (1.50 g) was carefully added, followed by 10 mL of tetrahydrofuran (THF), followed by a solution of compound II (15.0 g, 26.4 mmol) in THF (300 mL) and TFA (trifluoroacetic acid, 3.00 g, 26.4 mmol). The resulting mixture was degassed and purged with H2 three times and stirred under H2 (45 psi) at 25 °C for 3 h. Thin layer chromatography (TLC, DCM:MeOH = 10:1) indicated that compound II was completely consumed. The reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in anhydrous DCM (500 mL) and concentrated. This process was repeated three times to give intermediate A (14.0 g, 96.5% yield) as a foamy white solid. 1H NMR (400MHz DMSO-d6): δppm 7.90(d,J=9.29Hz,1H),7.78(br s,3H),5.23(d,J=3.26Hz,1H),4.98(dd,J=11.29,3.26Hz,1H),4.56(d,J=8. 53Hz,1H),3.98-4.07(m,3H),3.79-3.93(m,2H),3.55-3.66(m,5H),2.98(br d,J=4.77Hz,2H),2.11(s,3H),2.00(s,3H),1.90(s,3H),1.76(s,3H).

[0189] Intermediate B was synthesized in a similar manner to intermediate A. 1 H NMR(400MHz DMSO-d6):δppm 7.90(br d,J=9.03Hz,4 H),5.21(d,J=3.51Hz,1H),4.97(dd,J=11.1Hz,1H),4.54(d,J=8.53Hz,1H),3.98-4 .06(m,3H),3.88(dt,J=10.9Hz,1H),3.76-3.83(m,1H),3.49-3.61(m,9H),2.97(br s,2H),2.10(s,3H),1.99(s,3H),1.88(s,3H),1.78(s,3H).C 20 H 34 N2O 11 Calculated mass: 478.22; Measured mass: 479.3 (M+H + ).

[0190] Example 3 Synthesis of GalNAc Ligand Cluster Phosphoramidites GLPA1, GLPA2, and GLPA15. GLPA1 and GLPA2 were prepared according to Scheme 2 below. Starting from benzyl-protected propane-1,3-diamine, tert-butyl 2-bromoacetate was alkylated to give triester compound I. The benzyl protecting group was removed by hydrogenation to give secondary amine compound II. The amide was coupled with 6-hydroxyhexanoic acid to give compound III. Subsequent treatment with HCl in dioxane resulted in removal of the tert-butyl protecting group, generating tricarboxylic acid compound IV. Amide coupling of tricarboxylic acid compound IV with intermediate A or intermediate B gave compound Va or Vb. Phosphoramidite GLPA1 or GLPA2 was synthesized by phosphorylating compound Va or Vb using 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole.

[0191] [ka]

[0192] To a solution of N-benzyl-1,3-propanediamine (5.00 g, 30.4 mmol) in dimethylformamide (DMF, 100 mL) was added tert-butyl 2-bromoacetate (23.7 g, 121 mmol), followed by dropwise addition of diisopropylethylamine (DIEA, 23.61 g, 182 mmol). The resulting reaction mixture was stirred at 25–30°C for 16 h. LCMS showed complete consumption of N-benzyl-1,3-propanediamine. The reaction mixture was diluted with HO (500 mL) and extracted with EtOAc (500 mL × 2). The combined organic layer was washed with saturated brine (1 L), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate = 20:1 to 5:1). Compound I was obtained as a colorless oil (12.1 g, 78.4% yield).

[0193]

number

[0194] A dried hydrogenation bath was purged with argon three times. Pd / C (200 mg, 10%) was added, followed by MeOH (5 mL), followed by a solution of compound I (1.00 g, 1.97 mmol) in MeOH (5 mL). The reaction mixture was degassed under vacuum and refilled with H. This process was repeated three times. The reaction mixture was stirred under H (15 psi) at 25 °C for 12 h. LCMS showed that compound I was completely consumed. The reaction mixture was filtered under reduced pressure under N. The filtrate was concentrated under reduced pressure to give compound II (655 mg, 79.7% yield) as a yellow oil, which was used in the next step without further purification. 1 H NMR (400MHz, CDCl3): δppm 3.44(s,4H),3.31(s,2H),2.78(t,J=7.1Hz,2H),2.68(t,J=6.9Hz,2H),1.88(br s,1H),1.69(quin,J=7.03Hz,2H),1.44-1.50(s,27H).

[0195] A mixture of compound II (655 mg, 1.57 mmol), 6-hydroxyhexanoic acid (249 mg, 1.89 mmol), DIEA (1.02 g, 7.86 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 904 mg, 4.72 mmol), and 1-hydroxybenzotriazole (HOBt, 637 mg, 4.72 mmol) in DMF (6 mL) was degassed and purged with N three times, then stirred under N at 5 °C for 3 h. LCMS showed the desired product. The reaction mixture was diluted with HO (10 mL) and extracted with 20 mL of EtOAc (10 mL × 2). The combined organics were washed with saturated brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether: ethyl acetate = 5:1 to 1:1) to give compound III (650 mg, 77.8% yield) as a yellow oil. 1H NMR(400MHz,CDCl3):δppm 3.90-3.95(s,2H),3.63(t,J=6.40Hz,2H),3.38-3.45(m,6H),2.72(t,J =6.65Hz,2H),2.40(t,J=7.28Hz,2H),1.55-1.75(m,8H),1.44(s,27H).C 27 H 50 Calculated mass of N2O8: 530.36; Found: 531.3 (M+H + ).

[0196] A mixture of compound III (5.5 g, 10.3 mmol) in 2 M HCl / dioxane (55 mL) was stirred at 25 °C for 3 h. LCMS showed that compound III was completely consumed. The reaction mixture was filtered, washed with EtOAc (50 mL), and dried under reduced pressure to give a crude product, which was dissolved in CH3CN (50 mL) and the volatiles removed in vacuo. This process was repeated three times to give compound IV (2.05 g, 54.5% yield) as a white solid. 1 H NMR(400MHz,D2O) :δppm 4.21(s,1H),4.07(d,J=4.5Hz,4H),3.99(s,1H),3.45-3.52(m,3H),3. 42(t,J=6.5Hz,1H),3.32-3.38(m,1H),3.24-3.31(m,1H),2.37(t,J=7 .4Hz,1H),2.24(t,J=7.4Hz,1H),1.99(dt,J=15.5,7.53Hz,1H),1.85-1.94(m,1H),1.85-1.94(m,1H),1.39-1.56(m,4H),1.19-1.31(m,2H).

[0197] A mixture of compound IV (500 mg, 1.05 mmol), intermediate A (2.02 g, 3.67 mmol), DIEA (813 mg, 6.30 mmol), EDCI (704 mg, 3.67 mmol), and HOBt (496 mg, 3.67 mmol) in DMF (10 mL) was degassed and purged with N three times. The mixture was then stirred under a N atmosphere at 25 °C for 3 h. LCMS showed the desired product. The reaction mixture was quenched by the addition of H2O (10 mL) and extracted with DCM (10 mL x 2). The combined organics were extracted with 10% citric acid (20 mL). The aqueous phase was neutralized with saturated NaHCO3 solution and re-extracted with DCM (10 mL x 2). The organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give compound Va (570 mg, 0.281 mmol, 26.8% yield) as a white solid. 1 H NMR:(400MHz,CDCl3)ppmδ7.84-8.12(m,3H),6.85-7.15(m,2H),6.66-6.81(m,1H),5.36(br d,J=2.7Hz,3H),5.11-5.27(m,3H),4.63-4.85(m,3H),3.90-4.25(m,18H),3.37-3.75(m,28H),3.15-3.28(m,4H),2.64(br d,J=6.53Hz,2H),2.30-2.46(m,2H),2.13-2.18(m,9H),2.05(s,9H),1.94-2.03(m,18H),1.68(br s,2H),1.45(br s,2H),1.12(br t,J=7.0Hz,2H).

[0198] To a solution of compound Va (260 mg, 0.161 mmol) in anhydrous DCM (5 mL) was added diisopropylammonium tetrazolide (30.3 mg, 0.177 mmol) under a nitrogen atmosphere at room temperature, followed by the dropwise addition of 3-bis(diisopropylamino)phosphanyloxypropanenitrile (194 mg, 0.645 mmol). The reaction mixture was stirred at 20–25°C for 2 h. LCMS indicated complete consumption of compound Va. After cooling to −20°C, the mixture was added to a stirring solution of brine / saturated aqueous NaHCO3 (1:1, 5 mL) at 0°C. After stirring for 1 min, DCM (5 mL) was added. The layers were separated. The organics were washed with brine / saturated aqueous NaHCO3 (1:1, 5 mL), dried over Na2SO4, filtered, and concentrated to a volume of approximately 1 mL. The residual solution was added dropwise to 20 mL of methyl tert-butyl ether (MTBE) with stirring. This resulted in the precipitation of a white solid. The mixture was centrifuged, and the solid was collected. The solid was redissolved in 1 mL of DCM, and MTBE (20 mL) was added to precipitate. The solid was again separated by centrifugation. The collected solid was dissolved in anhydrous CH3CN. The volatiles were removed. This process was repeated two more times to give the GalNAc ligand phosphoramidite compound GLPA 1 (153 mg, 84.4 μmol) as a white solid. 1 H NMR (400MHz, CDCl3):ppmδ7.71-8.06(m,2H),6.60-7.06(m,3H),5.37(br d,J=3.0Hz,3H),5.18-5.32(m,3H),4.70-4.86(m,3H),3.92-4.25(m,18H),3.42-3.85(m,30H),3.25(m ,4H),2.59-2.75(m,4H),2.27-2.44(m,2H),2.15-2.20(s,9H)2.07(s,9H),1.96-2.03(m,18H),1.65(br s,4H),1.44(br d,J=7.28Hz,2H),1.14-1.24(m,12H). 31 P NMR(CDCl3): ppm δ 147.15.

[0199] GalNAc ligand phosphoramidite compound GLPA2 was synthesized in a similar manner except that intermediate B was used. 1 H NMR (400MHz, CDCl3):ppmδ7.94-8.18(m,1H),7.69(brs,1H),6.66-7.10(m,3H),5.35(d, J=3.5Hz,3H),5.07-5.25(m,3H),4.76-4.86(m,3H),4.01-4.31(m,10H),3.91-4.01(m,8H ),3.74-3.86(m,4H),3.52-3.71(m,30H),3.42-3.50(m,6H),3.15-3.25(m,4H),2.52-2.7 0(m,4H),2.22-2.45(m,2H),2.15-2.22(s,9H),2.06(s,9H),1.95-2.03(m,18H),1.77(br s,2H),1.58-1.66(m,4H),1.40(m,2H),1.08-1.24(m,12H). 31 P NMR(CDCl3): ppm δ 147.12.

[0200] GLPA15 was prepared according to Scheme 3 below.

[0201] [ka]

[0202] Starting from secondary amine compound I (compound II in Scheme 2), a Cbz protecting group was introduced to give compound II. The tert-butyl group of compound II was removed by treatment with acid to give tricarbamic acid compound III. Compound IV was obtained by amide coupling of compound III with intermediate A. The Cbz protecting group of compound IV was removed by hydrogenation to give secondary amine compound V, which was then reacted with glutaric anhydride to give carboxyl compound VI. Compound VI was reacted with piperidin-4-ol under amide coupling reaction conditions to give compound VII. Compound VII was treated with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole to synthesize the phosphoramidite compound GLPA15. 1 H NMR (in 400MHz DMSO-d6): δppm 8.05(br d,J=6.50Hz,2H),7.81(br d,J=9.01Hz,3H),5.22(d,J=3.25Hz,3H),4.98(dd,J=11.26,3.25Hz,3H),4.55(br d,J=8.50Hz,3H),4.03(s,9H),3.64-3.97(m,12H),3.55-3.63(m,6H),3.50(br s,5H),3.40(br d,J=6.13Hz,6H),3.17-3.30(m,9H),3.07(br d,J=14.26Hz,4H),2.76(t,J=5.82Hz,2H),2.18-2.47(m,6H),2.10(s,9H),1 .99(s,9H),1.89(s,9H),1.78(s,9H),1.52-1.74(m,6H),1.12-1.19(m,12H). 31 P NMR(DMSO-d6): ppm δ 145.25.

[0203] In certain studies, the method used to attach a GalNAc-containing targeting group (also referred to herein as a GalNAc delivery compound) to the 5' end of the sense strand involves using a GalNAc phosphoramidite (GLPA1) in the final coupling step of a synthetic process, e.g., a process used when performing oligonucleotide chain propagation to add nucleotides to the 5' end of the sense strand.

[0204] In some studies, methods for attaching a GalNAc-containing targeting group to the 3'-end of the sense strand include using a solid support (CPG) containing GLO-n. In some studies, methods for attaching a GalNAc-containing targeting group to the 3'-end of the sense strand include attaching the GalNAc targeting group to a CPG solid support via an ester bond, or using a CPG with a GalNAc targeting group attached during synthesis of the sense strand so that the GalNAc targeting group is attached to the 3'-end of the sense strand.

[0205] Example 4 In vitro screening of ANGPTL3 siRNA duplexes Hep 3B cells were trypsinized to the appropriate density and seeded into 96-well plates. Simultaneously, cells were transfected with test or control siRNA using Lipofectamine RNAiMax (Invitrogen-13778-150) according to the manufacturer's recommended protocol. siRNAs were measured in triplicate at two concentrations (0.2 nM and 1.0 nM), and control siRNA was measured in triplicate at eight 3-fold dilutions ranging from 4.6 pM to 10 nM.

[0206] After transfection, the cells were incubated for 24 hours. The medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using the RNeasy® 96 Kit (QIAGEN-74182) according to the manufacturer's instructions.

[0207] cDNA was synthesized using the FastKing RT Kit (with gDNase, Tiangen-KR116-02) according to the manufacturer's instructions. Human ANGPTL3 cDNA expression was normalized to GAPDH expression (TaqMan Gene Expression Assay, Thermo Scientific, Assay ID: Hs02786624_g) and determined by qPCR using a TaqMan Gene Expression Assay (ANGPTL3, Thermo Scientific, Assay ID: Hs00205581_ml). Percent inhibition was calculated by comparing ANGPTL3 expression with siRNA in PBS-treated samples.

[0208] [Table 7] JPEG2026035574000056.jpg228149 JPEG2026035574000057.jpg39149

[0209] Example 5 In vivo testing of ANGPTL3 siRNA duplexes To evaluate the in vivo activity of ANGPTL3 siRNA, mice (four per group) infected with AAV encoding the human ANGPTL3 gene were used. 14 days before siRNA administration, female C57BL / 6J mice were infected intravenously with 25 μL of a stock solution of an adeno-associated virus 8 (AAV 8) vector encoding the human ANGPTL3 gene. On day 0, mice received a single subcutaneous injection of 3 mg / kg of ANGPTL3 siRNA or PBS. Blood samples were collected on day 0 before siRNA administration and on day 7 at the end of the treatment. Human angiopoietin-like protein 3 (ANGPTL3) concentrations were measured by ELISA assay according to the manufacturer's recommended protocol (R&D Systems, Human Angiopoietin-like 3 Quantikine ELISA Kit). Percent knockdown was calculated by comparing human ANGPTL3 levels in plasma samples from mice treated with siRNA and PBS on day 7. The knockdown activity percentages of compounds AD00112, AD00135, and AD00143 (Table 3) were 91%, 83%, and 84%, respectively. In this example, GLO-0 in compounds AD00112, AD00135, and AD00143 refers to compound GalNAc3 in Jayaprakash et al., (2014) J. Am. Chem. Soc., 136, 16958, 16961.

[0210] Example 6 In vivo testing of ANGPTL3 siRNA duplexes Female C57BL / 6J mice (4 per group) were infected intravenously with an adeno-associated virus 8 (AAV8) vector encoding human ANGPTL3 and luciferase genes for 14 days prior to siRNA administration. On day 0, mice received a single subcutaneous injection of 3 mg / kg of ANGPTL3 siRNA or PBS. Blood samples were collected on day 0 before siRNA administration and on day 7 at the end of the siRNA administration. Serum samples were isolated and luciferase activity was measured according to the manufacturer's recommended protocol. Because human ANGPTL3 expression levels correlate with luciferase expression levels, luciferase activity measurement served as a surrogate for ANGPTL3 expression. ANGPTL3 persistence was calculated by comparing luciferase activity in samples from each mouse before siRNA treatment (day 0) and after treatment (day 7), and normalized by the change in luciferase activity in samples from control-treated mice over the same period. The results are summarized in Table 8. In this example, GLO-0 in the compounds listed in Table 3 refers to the compound GalNAc 3 in Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958.

[0211] [Table 8]

[0212] Example 7 In vivo testing of ANGPTL3 siRNA duplexes On day 14 before siRNA administration, female C57BL / 6J mice were infected intravenously with an adeno-associated virus 8 (AAV 8) vector encoding human ANGPTL3 and luciferase genes. On day 0, mice were subcutaneously administered a single dose of AD00112-2 or PBS at 1 mg / kg or 10 mg / kg. Blood samples were collected on day 0 before siRNA administration and on day 7 at the end of the study. Serum samples were isolated, and luciferase activity in the serum samples was measured according to the manufacturer's recommended protocol. Because the expression level of human ANGPTL3 correlates with the expression level of luciferase, measuring luciferase activity served as a surrogate for ANGPTL3 expression. The results are summarized in Table 9.

[0213] [Table 9]

[0214] Example 8 In vivo testing of ANGPTL3 siRNA duplexes in an NHP PD model Male cynomolgus monkeys (13-22 years old, weighing 7-9 kg, 4 monkeys per group) were enrolled in this study. Each monkey received a subcutaneous injection of one of the test substances prepared in PBS at 4 mg / kg on Day 1 (before siRNA administration). After an overnight fast, blood samples were collected on Days -7 (before administration), 1 (before administration), 8, 15, 22, 29, 43, and 50. Serum angiopoietin-like protein 3 (ANGPTL3) concentrations were measured by ELISA. The residual ANGPTL3 levels (normalized to Day 1 before siRNA administration) in the groups treated with compounds AD00112, AD00135, and AD00136 are shown in Figure 1. Lipid profiles were also measured. The percent changes in serum HDL, LDL, TC (total cholesterol), and TG (triglyceride) levels (normalized to pre-siRNA administration, day 1) are shown in Figures 2, 3, 4, and 5, respectively. A significant and sustained decrease in serum ANGPTL3 (up to 86% decrease) was observed in all three groups of monkeys administered the siRNA compound. A significant decrease in TG (up to 60% decrease) and moderate decreases in HDL-C and TC were also observed.

[0215] Example 9 In vivo testing of ANGPTL3 siRNA duplexes in NHP disease models Male cynomolgus monkeys (13-21 years old) were screened for baseline lipid profiles, including HDL, LDL, TC (total cholesterol), and TG (triglycerides). Twenty monkeys with elevated baseline LDL (1.03-2.36 mmol / L) and TG (1.42-6.71 mmol / L) levels were selected and randomly divided into two groups. On day 0, they received a single subcutaneous injection of saline or AD00112-2 at 10 mg / kg. After an overnight fast, blood samples were collected on days -10 (pre-dose), -2 (pre-dose), 7, 14, 21, 28, 35, and 42. Serum angiopoietin-like protein 3 (ANGPTL3) levels were measured by ELISA. The residual ANGPTL3 levels (normalized to baseline, averaged over days -10 and -2) in each group administered saline or compound AD00112-2 at 10 mg / kg are shown in Figure 6. Lipid profiles were also measured. The percent changes in serum HDL, LDL, TC (total cholesterol), and TG (triglyceride) levels (normalized to baseline, before siRNA administration) are shown in Figures 7, 8, 9, and 10, respectively. Significant and sustained decreases in ANGPTL3 concentrations and TG levels were observed. Furthermore, slight decreases in HDL-C, LDL-C, and TC levels were also observed.

[0216] Equivalent aspects While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision numerous other means and / or structures for performing the several functions and / or obtaining the results and / or one or more of the advantages described herein. Such variations and / or modifications are deemed to be within the scope of the present invention. More generally, those skilled in the art will readily recognize that all parameters, dimensions, materials, and configurations described herein are exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application or specific use to which the teachings of the present invention are put. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Accordingly, it will be understood that the foregoing embodiments are merely illustrative and that, within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and protected. The present invention relates to each of the features, systems, articles, materials, and / or methods described herein. Also, any combination of two or more such features, systems, articles, materials and / or methods, to the extent that such combinations are not mutually inconsistent, is within the scope of the present invention.

[0217] All definitions defined and used herein should be understood to supersede dictionary definitions, definitions in documents incorporated by reference, and / or common meanings of the defined terms.

[0218] As used in this specification and claims, nouns not modified by numerals will be understood to mean "at least one or one," unless expressly indicated to the contrary.

[0219] The term "and / or" as used in this specification and in the claims should be understood to mean "either or both" of the elements so connected; that is, the elements are present together in some instances and separately in other instances. Unless expressly indicated otherwise, elements other than those expressly identified by the "and / or" phrase may optionally be present, whether related or unrelated to those expressly identified elements.

[0220] All references, patents and patent applications and publications cited or referred to in this application are incorporated herein by reference in their entirety.

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of angiopoietin-like 3 (ANGPTL3), wherein the dsRNA agent comprises a sense strand and an antisense strand; The antisense strand of the dsRNA has the following nucleotide sequence II:5'-z １ uagaguauaaccuuccz ２ -3', z １ is selected from c, g, a, or u; z ２ is nucleotide sequence IV, wherein said nucleotide sequence IV is 0 to 15 nucleotides in length.

2. z １ is u or the nucleotide sequence IV is selected from a, au, aa, ac, ag, auu, aua, auc, aug, auug, auuu, auuas, auuc, auuuuu, auuuuug, auucuu, auucga, auuuuuga, auuuuugag, auuuuugaga, or auuuuugagacuucca; said nucleotide sequence IV is 1, 2, 3 or 4 nucleotides in length; or 2. The dsRNA agent of claim 1, wherein the nucleotide sequence IV is selected from a, au, aa, ac, ag, auu, aua, auc, aug, auug, auuu, auua, or auuc.

3. The sense strand of the dsRNA has the nucleotide sequence III:5'-z ３ ggaagguuauacucuaz ４ -3', z ３ is a nucleotide sequence V, and z ４ 2. The dsRNA agent of claim 1, wherein V is selected from c, g, a, or u, and the nucleotide sequence V is 0 to 15 nucleotides in length.

4. z ４ is a or the nucleotide sequence V is selected from u, au, uu, gu, cu, aau, uau, gau, cau, gaau, caau, aaau, uaau, aaaau, caaaau, ucaaau, cucaaaau, ucucaaau, or uggaagucucaaaau; the nucleotide sequence V is 1, 2, 3 or 4 nucleotides in length; or 4. The dsRNA agent of claim 3, wherein the nucleotide sequence V is selected from u, au, uu, gu, cu, aau, uau, gau, cau, gaau, caau, aaau, or uaau.

5. the dsRNA agent comprises at least one modified nucleotide; Optionally, the antisense strand comprises all or substantially all nucleotides that are modified nucleotides; 2. The dsRNA agent of claim 1, optionally wherein the at least one modified nucleotide comprises a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2'-3'-seconucleotide mimic, a locked nucleotide, an unlocked nucleic acid (UNA) nucleotide, a glycol nucleic acid (GNA) nucleotide, a 2'-F-arabino nucleotide, a 2'-methoxyethyl nucleotide, an abasic nucleotide, ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-OMe nucleotide, an inverted 2'-deoxy nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, and a 3'-OMe nucleotide, a 5'-phosphorothioate group-containing nucleotide, or a terminal nucleotide linked with a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, a phosphoramidate, or a non-natural base-containing nucleotide.

6. an E-vinylphosphonate nucleotide at the 5' end of the antisense strand; Optionally, the dsRNA agent comprises at least one phosphorothioate internucleoside linkage; Optionally, the sense strand comprises at least one phosphorothioate internucleoside linkage; Optionally, the antisense strand comprises at least one phosphorothioate internucleoside linkage; Optionally, the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages; Optionally, the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages; Optionally, the sense strand and the antisense strand have all or substantially all modified nucleotides.

7. the sense strand is complementary or substantially complementary to the antisense strand; Optionally, each strand has a length of 30 nucleotides or less; Optionally, each strand has a length of 25 nucleotides or less; Optionally, the dsRNA agent of claim 1, wherein each strand has a length of 23 nucleotides or less.

8. The dsRNA agent is selected from the group consisting of SEQ ID NO: 328, SEQ ID NO: 576, SEQ ID NO: 577, SEQ ID NO: 578, SEQ ID NO: 580, SEQ ID NO: 581, SEQ ID NO: 582, SEQ ID NO: 583, SEQ ID NO: 584, SEQ ID NO: 585, SEQ ID NO: 586, SEQ ID NO: 587, SEQ ID NO: 588, SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, SEQ ID NO: 597, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, SEQ ID NO: 606, SEQ ID NO: 607, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 611, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 619, SEQ ID NO: 619, SEQ ID NO: 620, SEQ ID NO: 621, SEQ ID NO: 622, SEQ ID NO: 623, SEQ ID NO: 62 2. The dsRNA agent of claim 1, comprising an antisense strand selected from the group consisting of the sequences shown in SEQ ID NO: 595, SEQ ID NO: 596, SEQ ID NO: 597, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604 and SEQ ID NO:

605.

9. The dsRNA agent is selected from the group consisting of SEQ ID NO: 278 and SEQ ID NO: 328; SEQ ID NO: 546 and SEQ ID NO: 576; SEQ ID NO: 547 and SEQ ID NO: 577; SEQ ID NO: 548 and SEQ ID NO: 578; SEQ ID NO: 550 and SEQ ID NO: 580; SEQ ID NO: 551 and SEQ ID NO: 581; SEQ ID NO: 552 and SEQ ID NO: 582; SEQ ID NO: 553 and SEQ ID NO: 583; SEQ ID NO: 554 and SEQ ID NO: 584; SEQ ID NO: 555 and SEQ ID NO: 585; SEQ ID NO: 556 and SEQ ID NO: 586; SEQ ID NO: 557 and SEQ ID NO: 587; SEQ ID NO: 558 and SEQ ID NO: 588; SEQ ID NO: 559 and SEQ ID NO: 589; SEQ ID NO: 560 and SEQ ID NO: 590; SEQ ID NO: 561 and SEQ ID NO: 591; SEQ ID NO: 562 and SEQ ID NO: 592; SEQ ID NO: 563 and SEQ ID NO: 593; SEQ ID NO: 564 and SEQ ID NO: 594; SEQ ID NO: 565 and SEQ ID NO: 595; SEQ ID NO: 566 and SEQ ID NO: 596; SEQ ID NO: 567 and SEQ ID NO: 597; SEQ ID NO: 568 and SEQ ID NO: 598; SEQ ID NO: 569 and SEQ ID NO: 599; SEQ ID NO: 570 and SEQ ID NO: 600; SEQ ID NO: 571 and SEQ ID NO: 601; SEQ ID NO: 572 and SEQ ID NO: 602; SEQ ID NO: 573 and SEQ ID NO: 603; SEQ ID NO: 574 and SEQ ID NO: 604; and 2. The dsRNA agent of claim 1, comprising a sequence selected from the group consisting of the double-stranded sequences shown in SEQ ID NO: 575 and SEQ ID NO:

605.

10. The dsRNA agent is selected from the group consisting of SEQ ID NO: 401 and SEQ ID NO: 428; and 2. The dsRNA agent of claim 1, comprising a sequence selected from the group consisting of the double-stranded sequences shown in SEQ ID NO: 606 and SEQ ID NO:

611.

11. The dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting groups or one or more linking groups; Optionally, the one or more targeting groups or one or more linking groups are conjugated to the sense strand; Optionally, the targeting group or linking group comprises N-acetylgalactosamine (Gal NAc); Optionally, the targeting group has any of the following structures: 【Chemistry 1】 【change】 【change】 【change】

12. the dsRNA agent comprises a targeting group conjugated to the 5' end of the sense strand; and / or 10. The dsRNA agent of claim 1, wherein the dsRNA agent comprises a targeting group conjugated to the 3' end of the sense strand.

13. the antisense strand comprises one inverted abasic residue at the 3' end; Optionally, the sense strand comprises one or two inverted abasic residues at the 3' and / or 5' end.

14. the dsRNA agent has two blunt ends, or 10. The dsRNA agent of claim 1, wherein at least one strand comprises a 3' overhanging end having at least one or two nucleotides.

15. A composition comprising the dsRNA agent of any one of claims 1 to 14, optionally further comprising a pharmaceutically acceptable carrier; optionally further comprising one or more additional therapeutic agents; Optionally, the composition is packaged in a kit, container, pack, dispenser, pre-filled syringe, or vial; Optionally, the composition is prepared for subcutaneous or intravenous (IV) administration.

16. 1. A method for inhibiting expression of the ANGPTL3 gene in a cell in vitro, comprising: (i) preparing a cell containing an effective amount of the double-stranded ribonucleic acid (dsRNA) agent of any one of claims 1 to 14; Optionally, the method further comprises the step (ii) of inhibiting expression of the ANGPTL3 gene in the cells by maintaining the cells prepared in step (i) for a time sufficient to obtain degradation of mRNA transcripts of the ANGPTL3 gene.

17. 1. A method for inhibiting expression of the ANGPTL3 gene in a cell in vitro, comprising: (i) preparing cells containing an effective amount of the composition of claim 15; Optionally, the method further comprises the step (ii) of inhibiting expression of the ANGPTL3 gene in the cells by maintaining the cells prepared in step (i) for a time sufficient to obtain degradation of mRNA transcripts of the ANGPTL3 gene.

18. 15. A double-stranded ribonucleic acid (dsRNA) agent according to any one of claims 1 to 14 for use in the treatment of a disease or disorder associated with the presence of ANGPTL3 protein, comprising: Optionally, the disease or disorder is one or more selected from the group consisting of hyperlipidemia, hypertriglyceridemia, dyslipidemia and / or cholesterol metabolism disorders, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, cardiometabolic disease, obesity, atherosclerosis, type 2 diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis due to hypertriglyceridemia.

19. 16. The composition of claim 15 for use in the treatment of a disease or disorder associated with the presence of an ANGPTL3 protein, comprising: Optionally, the disease or disorder is one or more selected from the group consisting of hyperlipidemia, hypertriglyceridemia, dyslipidemia and / or cholesterol metabolism disorders, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, cardiometabolic disease, obesity, atherosclerosis, type 2 diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis due to hypertriglyceridemia; Optionally, the composition further comprises one or more ANGPTL3 antisense polynucleotides or non-ANGPTL3 dsRNA therapeutic agents; Optionally, the non-ANGPTL3 dsRNA therapeutic agent is one or more selected from the group consisting of: (i) a statin; (ii) one or more of a PCSK9 siRNA molecule, an antibody, and an antisense oligonucleotide (ASO) capable of reducing PCSK9 expression; (iii) a therapeutic agent capable of reducing lipid accumulation in a subject; and (iv) a therapeutic agent capable of reducing cholesterol levels and / or accumulation in a subject.

20. to reduce the level of angiopoietin-like protein 3 (ANGPTL3) in a subject compared to the baseline pre-treatment level of angiopoietin-like protein 3 (ANGPTL3) in said subject; or 15. The double-stranded ribonucleic acid (dsRNA) agent of any one of claims 1 to 14, for use in altering physiological characteristics of an ANGPTL3-associated disease or disorder in a subject compared to baseline pre-treatment physiological characteristics of the ANGPTL3-associated disease or disorder in the subject, Optionally, the physiological characteristic is one or more selected from the group consisting of the subject's serum lipid level, the subject's HDL level, the subject's HDL / LDL ratio, the subject's serum triglyceride level, and the subject's liver fat content.

21. to reduce the level of angiopoietin-like protein 3 (ANGPTL3) in a subject compared to the baseline pre-treatment level of angiopoietin-like protein 3 (ANGPTL3) in said subject; or 16. The composition of claim 15, for use in altering physiological characteristics of an ANGPTL3-associated disease or disorder in a subject compared to baseline pre-treatment physiological characteristics of the ANGPTL3-associated disease or disorder in the subject, comprising: Optionally, the physiological characteristic is one or more selected from the group consisting of the subject's serum lipid level, the subject's HDL level, the subject's HDL / LDL ratio, the subject's serum triglyceride level, and the subject's liver fat content.

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