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WO2025201277A1 - Method for treating dyslipidemia with angiopoietin-like 3 (angptl3) -targeted rnai agents - Google Patents

Method for treating dyslipidemia with angiopoietin-like 3 (angptl3) -targeted rnai agents

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Publication number
WO2025201277A1
WO2025201277A1 PCT/CN2025/084552 CN2025084552W WO2025201277A1 WO 2025201277 A1 WO2025201277 A1 WO 2025201277A1 CN 2025084552 W CN2025084552 W CN 2025084552W WO 2025201277 A1 WO2025201277 A1 WO 2025201277A1
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WO
WIPO (PCT)
Prior art keywords
subject
angptl3
administered
fixed dose
disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/084552
Other languages
French (fr)
Inventor
Dongxu Shu
Pengcheng Patrick Shao
Yuqiong Li
Yafeng He
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Argo Biopharmaceutical Co Ltd
Original Assignee
Shanghai Argo Biopharmaceutical Co Ltd
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Filing date
Publication date
Application filed by Shanghai Argo Biopharmaceutical Co Ltd filed Critical Shanghai Argo Biopharmaceutical Co Ltd
Publication of WO2025201277A1 publication Critical patent/WO2025201277A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the invention relates to compositions and methods that can be used to inhibit angiopoietin-like 3 (ANGPTL3) protein expression.
  • the present invention relates to methods for reducing serum levels of ANGPTL3 and thus reducing serum levels of LDL-C, TG and non-high-density lipoprotein cholesterol (non-HDL-C) and methods for the treatment of dyslipidemia, e.g., mixed dyslipidemia, hypercholesterolemia and hypertriglyceridemia.
  • dyslipidemia e.g., mixed dyslipidemia, hypercholesterolemia and hypertriglyceridemia.
  • Angiopoietin-like protein3 governs LDL-cholesterol levels through endothelial lipase-dependent VLDL clearance. J Lipid Res 2020; 61: 1271–1286) . It is also noteworthy that current LDL-C lowering therapies, such as statins and PCSK9 inhibitors are LDL-R dependent, and are not effective for patients with low or no residue LDL-R activity. LDL-C lowering through inhibition of ANGPTL3 is LDL-R independent, which could be an effective therapeutic approach to manage lipids for patients with low or no LDL-R activity.
  • ANGPTL3 has emerged as a promising drug target for treating diseases caused by hyperlipidemia with therapeutic modalities including antibody, antisense oligonucleotide (ASO) and siRNA agent in development.
  • siRNA agent particularly GalNAc-conjugated siRNA agent has been shown to be safe, effective and with long during of activity.
  • the present invention provides methods for inhibiting expression of an ANGPTL3 gene in a subject and methods for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of an ANGPTL3 gene, e.g., a disorder mediated by ANGPTL3 expression, such as a hyperlipidemia, e.g., hypercholesterolemia, using RNAi agent compositions which effect the RNA-induced silencing complex (RISC) -mediated cleavage of RNA transcripts of an ANGPTL3 gene.
  • RISC RNA-induced silencing complex
  • the methods of the present invention for inhibiting expression of an ANGPTL3 gene in a subject and methods for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of an ANGPTL3 gene, e.g., a disorder mediated by ANGPTL3 expression, such as dyslipidemia, e.g., hypertriglyceridemia and hypercholesterolemia include administering to a subject a fixed dose of about 50 mg to about 600 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1 or 3 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3
  • RNAi double-strand
  • the present invention provides methods of inhibiting the expression of an ANGPTL3 gene in a subject.
  • the methods include comprising administering to the subject a fixed dose of about 50 mg to about 600 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, the antisense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 0, 1, 2, 3 nucleotides from the complement of nucleotides 1350-1418 of the nucleotide sequence of SEQ ID NO: 1, and optionally, the sense strand is conjugated to a ligand attached at the 5′-terminus, thereby inhibiting the expression of the ANGPTL3 gene in the subject.
  • RNAi double-stranded ribonucleic acid
  • the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand of dsRNA comprising a nucleotide sequence II: 5’ -z 1 UAGAGUAUAACCUUCCz 2 -3’ , wherein z 1 is selected from C, G, A or U, z 2 is a nucleotide sequence IV, wherein the sense strand of dsRNA comprising a nucleotide sequence III: 5’ -z 3 GGAAGGUUAUACUCUAz 4 -3’ , wherein z 3 is a nucleotide sequence V, z 4 is selected from C, G, A or U.
  • z 1 is U.
  • the nucleotide sequence IV is 0-15 nucleotides in length. In certain embodiments, the nucleotide sequence IV is selected from A, AU, AA, AC, AG, AUU, AUA, AUC, AUG, AUUG, AUUU, AUUA, AUUC, AUUUU, AUUUUG, AUUCUU, AUUCGA, AUUUUGA, AUUUUGAG, AUUUUGAGA or AUUUUGAGACUUCCA. In certain embodiments, the nucleotide sequence IV is 1, 2, 3 or 4 nucleotides in length.
  • the nucleotide sequence IV is selected from A, AU, AA, AC, AG, AUU, AUA, AUC, AUG, AUUG, AUUU, AUUA or AUUC.
  • the antisense strand of dsRNA comprising a nucleotide sequence II’ : 5’ -z 1 UAGAGUAUAACCUUCCAz 2’ -3’ , wherein z 1 is selected from C, G, A or U, z 2’ is a nucleotide sequence IV’ .
  • z 1 is U.
  • the nucleotide sequence IV’ is 0-15 nucleotides in length.
  • the nucleotide sequence IV’ is 1, 2, 3 or 4 nucleotides in length. In certain embodiments, the nucleotide sequence IV’ is selected from U, A, C, G, UU, UA, UC, UG, UUG, UUU, UUA or UUC. In certain embodiments, z 4 is A. In certain embodiments, the nucleotide sequence V is 0-15 nucleotides in length.
  • the nucleotide sequence V is selected from U, AU, UU, GU, CU, AAU, UAU, GAU, CAU, GAAU, CAAU, AAAU, UAAU, AAAAU, CAAAAU, UCAAAAU, CUCAAAAU, UCUCAAAAU or UGGAAGUCUCAAAAU. In certain embodiments, the nucleotide sequence V is 1, 2, 3 or 4 nucleotides in length. In certain embodiments, the nucleotide sequence V is selected from U, AU, UU, GU, CU, AAU, UAU, GAU, CAU, GAAU, CAAU, AAAU or UAAU.
  • the sense strand of dsRNA comprising a nucleotide sequence III’ : 5’ -z 3’ UGGAAGGUUAUACUCUAz 4 -3’ , wherein z 3’ is a nucleotide sequence V’ , z 4 is selected from C, G, A or U. In certain embodiments, z 4 is A. In certain embodiments, the nucleotide sequence V’ is 1, 2, 3 or 4 nucleotides in length. In certain embodiments, the nucleotide sequence V’ is selected from A, U, G, C, AA, UA, GA, CA, GAA, CAA, AAA or UAA.
  • the dsRNA agent includes a sense strand and an antisense strand, wherein the sense strand of dsRNA consists of the nucleotide sequence III and the antisense strand of dsRNA consists of the nucleotide sequence II, wherein the nucleotide sequence II and III are as described above.
  • the dsRNA agent includes a sense strand and an antisense strand, wherein the sense strand of dsRNA consists of the nucleotide sequence III’ and the antisense strand of dsRNA consists of the nucleotide sequence II’ , wherein the nucleotide sequence II’ and III’ are as described above.
  • the antisense strand comprises the nucleotide sequence 5′-UUAGAGUAUAACCUUCCAUUC-3′ (SEQ ID NO: 40) .
  • the sense strand comprises the nucleotide sequence 5′-GAAUGGAAGGUUAUACUCUAA-3′ (SEQ ID NO: 5) .
  • the double-stranded RNAi agent targets nucleotides 1358-1378 of SEQ ID NO: 1.
  • the double-stranded RNAi agent targeting nucleotides 1358-1378 of SEQ ID NO: 1 is AD00042. um.
  • the double-stranded ribonucleic acid RNAi agent comprises at least one modified nucleotide.
  • nucleotides of the antisense strand are modified nucleotides. In some embodiments, all or substantially all of the nucleotides of the sense strand and the antisense strand are modified nucleotides.
  • the antisense strand comprises the nucleotide sequence of 5′-u*Uf*agagUfauaaCfcUfuCfcau*u*c-3′ (SEQ ID NO: 41) .
  • n is independently selected from 1 or 2.
  • the disorder that would benefit from reduction in ANGPTL3 expression is dyslipidemia.
  • the disorder is one or more of: hypertriglyceridemia, severe hypertriglyceridemia (SHTG) , familial chylomicronemia syndrome, mixed dyslipidemia, hypercholesterolemia, homozygous familial hypercholesterolemia (HoFH) , heterozygous familial hypercholesterolemia (HeFH) , abnormal lipid and/or cholesterol metabolism, statin resistant hypercholesterolemia, cardiometabolic disease, obesity, atherosclerosis, type II diabetes mellitus, cardiovascular disease, coronary artery disease, cerebrovascular disease, peripheral artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis caused by hypertriglyceridemia.
  • hypertriglyceridemia severe hypertriglyceridemia (SHTG)
  • familial chylomicronemia syndrome mixed dyslipidemia
  • hypercholesterolemia homozygous familial hypercholesterolemia (HoFH)
  • HeFH heterozygous familial hypercholesterolemia
  • the fixed dose may be administered to the subject at an interval of once a quarter, bianually or every nine months.
  • the subject is administered a fixed dose of about 50 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 150 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 200 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 300 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 600 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 50 mg biannually (i.e., twice a year) . In some embodiment, the subject is administered a fixed dose of about 150 mg biannually. In some embodiment, the subject is administered a fixed dose of about 200 mg biannually.
  • the subject is administered a fixed dose of about 300 mg biannually. In some embodiment, the subject is administered a fixed dose of about 600 mg biannually. In some embodiment, the subject is administered a fixed dose of about 50 mg every nine months. In some embodiment, the subject is administered a fixed dose of about 150 mg every nine months. In some embodiment, the subject is administered a fixed dose of about 200 mg every nine months. In some embodiment, the subject is administered a fixed dose of about 300 mg every nine months. In some embodiment, the subject is administered a fixed dose of about 600 mg every nine months.
  • the disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 150 mg biannually.
  • the disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 200 mg biannually.
  • the disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 300 mg biannually.
  • the disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 600 mg biannually.
  • the disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 150 mg once a quarter.
  • the disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 200 mg once a quarter.
  • the disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 300 mg once a quarter.
  • the disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 600 mg once a quarter.
  • the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about one month to about three months later, and thereafter for subsequent doses there is about six months or nine months between dose administrations.
  • the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about one month to about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
  • the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about one month to about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
  • the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
  • the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
  • the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
  • the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 200 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
  • the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 300 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
  • the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 300 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
  • the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
  • the second dose is equal to the initial dose.
  • the RNAi agent is administered in a dosing regimen that includes a loading phase and a maintenance phase.
  • the loading phase comprises administering a fixed dose of about 50 mg of the RNAi agent to the subject
  • the maintenance phase comprises administering a fixed dose of about 50 mg of the RNAi agent to the subject about every nine months.
  • the loading phase comprises administering a fixed dose of about 150 mg of the RNAi agent to the subject
  • the maintenance phase comprises administering a fixed dose of about 150 mg of the RNAi agent to the subject about every nine months.
  • the loading phase comprises administering a fixed dose of about 900 mg of the RNAi agent to the subject
  • the maintenance phase comprises administering a fixed dose of about 900 mg of the RNAi agent to the subject about every nine months.
  • the subject is a human.
  • ANGPTL3 expression is inhibited by at least about 40%.
  • ANGPTL3 expression is inhibited by at least about 60%.
  • ANGPTL3 expression is inhibited by at least about 90%.
  • administering the double-stranded RNAi agent results in a decrease in serum lipid in the subject and/or a decrease in ANGPTL3 protein.
  • FIG. 1 is Mean ( ⁇ SD) Percentage Change from Baseline Serum ANGPTL3 Level over Time
  • compositions comprising ANGPTL3 single-stranded (ssRNA) and dsRNA agents to inhibit ANGPTL3 gene expression, as well as compositions and methods for treating diseases and conditions caused by or modulated by ANGPTL3 gene expression.
  • ssRNA single-stranded
  • dsRNA agents to inhibit ANGPTL3 gene expression
  • RNAi As used herein, the term “RNAi” , which is known in the art, and may be referred to as “siRNA agent” , “double stranded RNAi agent, “ “double-stranded RNA (dsRNA) molecule, " “dsRNA agent, “ or “dsRNA” refers to an agent that comprises RNA and mediates targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • an RNAi target region refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene, including messenger RNA (mRNA) that is a product of RNA processing of a primary transcription product.
  • mRNA messenger RNA
  • a “dsRNA agent” may mean a composition that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner.
  • dsRNA agents of the invention may operate through the RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) of mammalian cells) , or by any alternative mechanism (s) or pathway (s) .
  • DsRNA agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: short interfering RNAs (siRNA agents) , RNAi agents, micro RNAs (mRNAi agents) , short hairpin RNAs (shRNA) , and dicer substrates.
  • siRNA agents short interfering RNAs
  • mRNAi agents micro RNAs
  • shRNA short hairpin RNAs
  • dicer substrates The antisense strand of the dsRNA agents described herein is at least partially complementary to the mRNA being targeted. It is understood in the art that different lengths of dsRNA duplex structure can be used to inhibit target gene expression.
  • non-HDL-C levels are decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 8
  • VLDL-C levels are decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%
  • the present invention also provides methods for lowering a subject’s plasma lipid level, a subject’s TG level, a subject’s LDL level, a subject’s VLDL level, a subject’s non-HDL level, a subject’s ApoB level, a subject’s TC level, a subject’s LDL: HDL ratio, etc.
  • ANGPTL3 gene expression is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 81%, 8
  • the double-stranded RNAi agent is administered to a subject as a fixed dose.
  • a “fixed dose” e.g., a dose in mg
  • an RNAi agent of the invention is administered to a subject as a weight-based dose.
  • a “weight-based dose” e.g., a dose in mg/kg
  • the administration may be repeated, for example, on a regular basis.
  • the fixed dose may be administered to the subject at an interval of once a week, once every two weeks, once a month, once a quarter, or biannually for six months or a year or longer, i.e., chronic administration.
  • the present invention provides methods of decreasing the level of TG (triglyceride) , LDL-C (low-density lipoprotein-cholesterol) , ApoB (apolipoprotein B) , TC (total cholesterol) , VLDL-C (very low-density lipoprotein cholesterol) and non-HDL-C (non-high-density lipoprotein-cholesterol) in a subject.
  • TG triglyceride
  • LDL-C low-density lipoprotein-cholesterol
  • ApoB apolipoprotein B
  • TC total cholesterol
  • VLDL-C very low-density lipoprotein cholesterol
  • non-HDL-C non-high-density lipoprotein-cholesterol
  • the administration of the RNAi agents to a subject may be repeated on a regular basis, for example, at an interval of once a week, once every two weeks, once a month, once a quarter, biannually or every nine months.
  • the RNAi agent is administered in a dosing regimen that includes a “loading phase” of closely spaced administrations that may be followed by a “maintenance phase” , in which the RNAi agent is administered at longer spaced intervals. For example, after administration weekly or biweekly for one month, administration can be repeated once per month, for six months or a year or longer, i.e., chronic administration.
  • Any of these schedules may optionally be repeated for one or more iterations.
  • the number of iterations may depend on the achievement of a desired effect, e.g., the suppression of an ANGPTL3 gene, and/or the achievement of a therapeutic or prophylactic effect, e.g., reducing plasma lipid levels or reducing a symptom of hyperlipidemia.
  • the patient can be monitored for changes in his/her condition.
  • the dosage of the RNAi agent may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
  • the present invention provides methods of inhibiting the expression of an ANGPTL3 gene in a subject.
  • the methods include administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen that includes a loading phase followed by a maintenance phase, wherein the loading phase comprises administering a fixed dose of about 50 mg to about 900 mg of the RNAi agent to the subject, and wherein the maintenance phase comprises administering a fixed dose of about 50 mg to about 900 mg of the RNAi agent to the subject about every six months or every nine months, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, the antisense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 0, 1, 2, 3 nucleotides from the complement of nucleotides 1350-1418 of the nucleotide sequence of SEQ ID NO: 1, thereby inhibiting
  • the methods include administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen that includes a loading phase followed by a maintenance phase, wherein the loading phase comprises administering to the subject a fixed dose of about 50 mg to about 900 mg of the RNAi agent, and wherein the maintenance phase comprises administering to the subject a fixed dose of about 50 mg to about 900 mg of the RNAi agent every six months or every nine months, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, the antisense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 0, 1, 2, 3 nucleotides from the complement of nucleotides 1350-1418 of the nucleotide sequence of SEQ ID NO: 1.
  • RNAi ribonucleic acid
  • the methods and uses of the invention include administering a composition described herein such that expression of the target ANGPTL3 gene is decreased, for an extended period of time, such as, for about 80 days, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
  • a reduction in the expression of ANGPTL3 may be determined by determining the mRNA expression level of ANGPTL3 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR, by determining the protein level of ANGPTL3 using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques, and/or by determining a biological activity of ANGPTL3, such as the effect on one or more plasma lipid parameters, such as, for example, total cholesterol levels, high density lipoprotein cholesterol (HDL) levels, non-HDL levels, low density lipoprotein cholesterol (LDL) levels, very low density lipoprotein cholesterol (VLDL) levels, ApoB (apolipoprotein B) , triglyceride levels, and lipoprotein particle size, etc.
  • HDL high density lipoprotein cholesterol
  • LDL low density lipoprotein cholesterol
  • VLDL very low density lipoprotein cholesterol
  • ApoB apolipoprotein B
  • the reduction can be, for example, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,
  • a treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated.
  • a favorable change of at least 10%in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50%or more can be indicative of effective treatment.
  • Efficacy for a given RNAi agent drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.
  • administration can be provided when Low Density Lipoprotein cholesterol (LDL-C) levels reach or surpass a predetermined minimal level, such as greater than 70 mg/dL, 100 mg/dL, 130 mg/dL, 160 mg/dL, 190 mg/dL, 300 mg/dL, 400 mg/dL, 500 mg/dL, or 1000 mg/dL.
  • LDL-C Low Density Lipoprotein cholesterol
  • administration can be provided when triglyceride (TG) levels reach or surpass a predetermined minimal level, such as greater than 150 mg/dL, 200 mg/dL, 500 mg/dL or 880mg/dL.
  • TG triglyceride
  • ANGPTL3 expression is decreased for an extended duration, e.g., at least one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four month, six months or longer.
  • expression of the ANGPTL3 gene is suppressed by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,
  • the ANGPTL3 gene is suppressed by at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%by administration of the RNAi agent agent.
  • the administration is via a pump.
  • the pump may be an external pump or a surgically implanted pump.
  • the pump is a subcutaneously implanted osmotic pump.
  • the pump is an infusion pump.
  • An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions.
  • the infusion pump is a subcutaneous infusion pump.
  • the pump is a surgically implanted pump that delivers the RNAi agent to the liver.
  • modes of administration include epidural, intracerebral, intracerebroventricular, nasal administration, intraarterial, intracardiac, intraosseous infusion, intrathecal, and intravitreal, and pulmonary.
  • the mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated.
  • the route and site of administration may be chosen to enhance targeting.
  • the RNAi agent can be administered by intravenous infusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about a 25 minute period.
  • the administration may be repeated, for example, on a regular basis, such as weekly, biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer.
  • the treatments can be administered on a less frequent basis. For example, after administration weekly or biweekly for three months, administration can be repeated once per month, for six months or a year or longer.
  • RNAi agent Before administration of a full dose of the RNAi agent, patients can be administered a smaller dose, such as a 5%infusion, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.
  • cytokine e.g., TNF-alpha or INF-alpha
  • RNAi agent of the invention may be administered in “naked” form, or as a “free RNAi agent. ”
  • a naked RNAi agent is administered in the absence of a pharmaceutical composition.
  • the naked RNAi agent may be in a suitable buffer solution.
  • the buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.
  • the buffer solution is phosphate buffered saline (PBS) .
  • PBS phosphate buffered saline
  • an RNAi agent of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.
  • Exemplary fibrates include, e.g., bezafibrate (e.g., Roche's Kissei's Bezatol) , clofibrate (e.g., Wyeth's ) , fenofibrate (e.g., Fournier's Lipidil/Lipantil, Abbott's Takeda's Lipantil, generics) , gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate (Sanofi-Synthelabo's ) .
  • bezafibrate e.g., Roche's Kissei's Bezatol
  • clofibrate e.g., Wyeth's
  • fenofibrate e.g., Fournier's Lipidil/Lipantil, Abbott's Takeda's Lipantil, generics
  • gemfibrozil e.g., Pf
  • antiplatelet agents include, e.g., aspirin (e.g., Bayer's aspirin) , clopidogrel (Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix) , and ticlopidine (e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine) .
  • aspirin e.g., Bayer's aspirin
  • clopidogrel Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix
  • ticlopidine e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine
  • Other aspirin-like compounds useful in combination with a dsRNA targeting ANGPTL3 include, e.g., Asacard (slow-release aspirin, by Pharmacia) and Pamicogrel (K
  • Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe (Merck/Schering-Plough Pharmaceuticals ) and Pamaqueside (Pfizer) .
  • Exemplary CETP inhibitors include, e.g., Torcetrapib (also called CP-529414, Pfizer) , JTT-705 (Japan Tobacco) , and CETi-I (Avant Immunotherapeutics) .
  • Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, e.g., implitapide (Bayer) , R-103757 (Janssen) , and CP-346086 (Pfizer) .
  • Other exemplary cholesterol modulators include, e.g., NO-1886 (Otsuka/TAP Pharmaceutical) , CI-1027 (Pfizer) , and WAY-135433 (Wyeth-Ayerst) .
  • Exemplary bile acid modulators include, e.g., HBS-107 (Hisamitsu/Banyu) , Btg-511 (British Technology Group) , BARI-1453 (Aventis) , S-8921 (Shionogi) , SD-5613 (Pfizer) , and AZD-7806 (AstraZeneca) .
  • Exemplary peroxisome proliferation activated receptor (PPAR) agonists include, e.g., tesaglitazar (AZ-242) (AstraZeneca) , Netoglitazone (MCC-555) (Mitsubishi/Johnson &Johnson) , GW-409544 (Ligand Pharniaceuticals/GlaxoSmithKline) , GW-501516 (Ligand Pharmaceuticals/GlaxoSmithKline) , LY-929 (Ligand Pharmaceuticals and Eli Lilly) , LY-465608 (Ligand Pharmaceuticals and Eli Lilly) , LY-518674 (Ligand Pharmaceuticals and Eli Lilly) , and MK-767 (Merck and Kyorin) .
  • tesaglitazar AZ-242
  • MCC-555 Netoglitazone
  • MCC-555 Netoglitazone
  • GW-409544 Liigand Pharniaceuticals/GlaxoSmithKline
  • Exemplary gene-based therapies include, e.g., AdGWEGF 121.10 (GenVec) , ApoAl (UCB Pharma/Groupe Fournier) , EG-004 (Trinam) (Ark Therapeutics) , and ATP-binding cassette transporter-A1 (ABCA1) (CV Therapeutics/Incyte, Aventis, Xenon) .
  • Exemplary Glycoprotein IIb/IIIa inhibitors include, e.g., roxifiban (also called DMP754, Bristol-Myers Squibb) , Gantofiban (Merck KGaA/Yamanouchi) , and Cromafiban (Millennium Pharmaceuticals) .
  • Exemplary squalene synthase inhibitors include, e.g., BMS-1884941 (Bristol-Myers Squibb) , CP-210172 (Pfizer) , CP-295697 (Pfizer) , CP-294838 (Pfizer) , and TAK-475 (Takeda) .
  • An exemplary MCP-I inhibitor is, e.g., RS-504393 (Roche Bioscience) .
  • the anti-atherosclerotic agent BO-653 Choi Pharmaceuticals
  • the nicotinic acid derivative Nyclin Yamanouchi Pharmaceuticals
  • Exemplary combination therapies suitable for administration with a dsRNA targeting ANGPTL3 include, e.g., advicor (Niacin/lovastatin from Kos Pharmaceuticals) , amlodipine/atorvastatin (Pfizer) , and ezetimibe/simvastatin (e.g., 10/10, 10/20, 10/40, and 10/80 tablets by Merck/Schering-Plough Pharmaceuticals) .
  • advicor Niacin/lovastatin from Kos Pharmaceuticals
  • Amlodipine/atorvastatin Pfizer
  • ezetimibe/simvastatin e.g., 10/10, 10/20, 10/40, and 10/80 tablets by Merck/Schering-Plough Pharmaceuticals
  • an RNAi agent agent is administered in combination with an ezetimibe/simvastatin combination (e.g., (Merck/Schering-Plough Pharmaceuticals) ) .
  • an ezetimibe/simvastatin combination e.g., (Merck/Schering-Plough Pharmaceuticals)
  • the RNAi agent agent is administered to the patient, and then the additional therapeutic agent is administered to the patient (or vice versa) . In another embodiment, the RNAi agent and the additional therapeutic agent are administered at the same time.
  • a healthcare provider such as a doctor, nurse, or family member, can take a family history before prescribing or administering an RNAi agent of the invention.
  • a test may be performed to determine a geneotype or phenotype.
  • a DNA test may be performed on a sample from the patient, e.g., a blood sample, to identify the ANGPTL3 genotype and/or phenotype before an ANGPTL3 dsRNA is administered to the patient.
  • the present invention further provides methods of inhibiting expression of an angiopoietin like 3 (ANGPTL3) in a cell, such as a cell within a subject, e.g., a human subject.
  • ANGPTL3 angiopoietin like 3
  • the present invention provides methods of inhibiting expression of an ANGPTL3 gene in a cell.
  • the methods include contacting a cell with an RNAi agent, e.g., a double stranded RNAi agent, in an amount effective to inhibit expression of the ANGPTL3 gene in the cell, thereby inhibiting expression of the ANGPTL3 in the cell.
  • an RNAi agent e.g., a double stranded RNAi agent
  • RNAi agent may be done in vitro or in vivo.
  • Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting are also possible. Contacting may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art.
  • the targeting ligand is a carbohydrate moiety, e.g., a GalNAc ligand, or any other ligand that directs the RNAi agent to a site of interest, e.g., the liver of a subject.
  • inhibiting as used herein, is used interchangeably with “reducing, ” “silencing, ” “downregulating” and other similar terms, and includes any level of inhibition.
  • ANGPTL3 inhibiting expression of an ANGPTL3
  • any ANGPTL3 gene such as, e.g., a mouse ANGPTL3 gene, a rat ANGPTL3 gene, a monkey ANGPTL3 gene, or a human ANGPTL3 gene
  • the ANGPTL3 gene may be a wild-type ANGPTL3 gene, a mutant ANGPTL3 gene, or a transgenic ANGPTL3 gene in the context of a genetically manipulated cell, group of cells, or organism.
  • “Inhibiting expression of an ANGPTL3 gene” includes any level of inhibition of an ANGPTL3 gene, e.g., at least partial suppression of the expression of an ANGPTL3 gene.
  • the expression of the ANGPTL3 gene may be assessed based on the level, or the change in the level, of any variable associated with ANGPTL3 gene expression, e.g., ANGPTL3 mRNA level, ANGPTL3 protein level, or lipid levels. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.
  • Inhibition of the expression of an ANGPTL3 protein may be manifested by a reduction in the level of the ANGPTL3 protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject) .
  • the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.
  • the level of ANGPTL3 mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing mRNA expression.
  • the level of expression of ANGPTL3 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the ANGPTL3 gene.
  • RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis) , RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland) .
  • Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035) , Northern blotting, in situ hybridization, and microarray analysis.
  • Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction (PCR) analyses and probe arrays.
  • One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to ANGPTL3 mRNA.
  • the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose.
  • the probe (s) are immobilized on a solid surface and the mRNA is contacted with the probe (s) , for example, in an Affymetrix gene chip array.
  • a skilled artisan can readily adapt known mRNA detection methods for use in determining the level of ANGPTL3 mRNA.
  • An alternative method for determining the level of expression of ANGPTL3 in a sample involves the process of nucleic acid amplification and/or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202) , ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189-193) , self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878) , transcriptional amplification system (Kwoh et al. (1989) Proc. Natl.
  • ANGPTL3 mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like) , or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids) . See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5, 445, 934, which are incorporated herein by reference.
  • the determination of ANGPTL3 expression level may also comprise using nucleic acid probes in solution.
  • the level of ANGPTL3 protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC) , thin layer chromatography (TLC) , hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double) , immunoelectrophoresis, Western blotting, radioimmunoassay (RIA) , enzyme-linked immunosorbent assays (ELISAs) , immunofluorescent assays, electrochemiluminescence assays, and the like.
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • hyperdiffusion chromatography fluid or gel precipitin reactions
  • absorption spectroscopy a colorimetric assays
  • sample refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject.
  • biological fluids include blood, serum and serosal fluids, plasma, lymph, urine, cerebrospinal fluid, saliva, ocular fluids, and the like.
  • Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes) .
  • a “sample derived from a subject” refers to blood or plasma drawn from the subject.
  • a “sample derived from a subject” refers to liver tissue derived from the subject.
  • the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject.
  • the inhibition of expression of ANGPTL3 may be assessed using measurements of the level or change in the level of ANGPTL3 mRNA or ANGPTL3 protein in a sample derived from fluid or tissue from the specific site within the subject.
  • the site is the liver.
  • the site may also be a subsection or subgroup of cells from any one of the aforementioned sites.
  • the site may also include cells that express a particular type of receptor.
  • mismatches are tolerated for efficacy in dsRNA, especially the mismatches are within terminal region of dsRNA.
  • Certain mismatches tolerate better, for example mismatches with wobble base pairs G: U and A: C are tolerated better for efficacy (Du et el., A systematic analysis of the silencing effects of an active siRNA agent at all single-nucleotide mismatched target sites.
  • an ANGPTL3 dsRNA agent may contain one or more mismatches to the ANGPTL3 target sequence.
  • ANGPTL3 dsRNA agent of the invention includes no mismatches.
  • ANGPTL3 dsRNA agent of the invention includes no more than 1 mismatch.
  • ANGPTL3 dsRNA agent of the invention includes no more than 2 mismatches.
  • ANGPTL3 dsRNA agent of the invention includes no more than 3 mismatches.
  • an antisense strand of an ANGPTL3 dsRNA agent contains mismatches to an ANGPTL3 target sequence that are not located in the center of the region of complementarity.
  • the antisense strand of the ANGPTL3 dsRNA agent includes 1, 2, 3, 4, or more mismatches that are within the last 5, 4, 3, 2, or 1 nucleotide from one or both of the 5' or 3' end of the region of complementarity.
  • the term “complementary” when used to describe a first nucleotide sequence (e.g., ANGPTL3 dsRNA agent sense strand or targeted ANGPTL3 mRNA) in relation to a second nucleotide sequence (e.g., ANGPTL3 dsRNA agent antisense strand or a single-stranded antisense polynucleotide) means the ability of an oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize [form base pair hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro) ] and form a duplex or double helical structure under certain conditions with an oligonucleotide or polynucleotide including the second nucleotide sequence.
  • Complementary sequences for example, within an ANGPTL3 dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences.
  • Such sequences can be referred to as “fully complementary” with respect to each other herein. It will be understood that in embodiments when two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs are not regarded herein as mismatches with regard to the determination of complementarity.
  • an ANGPTL3 dsRNA agent comprising one oligonucleotide 19 nucleotides in length and another oligonucleotide 20 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 19 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.
  • “fully complementary” means that all (100%) of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide.
  • the contiguous sequence may comprise all or a part of a first or second nucleotide sequence.
  • substantially complementary means that in a hybridized pair of nucleobase sequences, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not all, of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide.
  • substantially complementary can be used in reference to a first sequence with respect to a second sequence if the two sequences include one or more, for example at least 1, 2, 3, 4, or 5 mismatched base pairs upon hybridization for a duplex up to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs (bp) , while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of ANGPTL3 gene expression via a RISC pathway.
  • partially complementary may be used herein in reference to a hybridized pair of nucleobase sequences, in which at least 75%, but not all, of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide.
  • the duplex region can be of any length that permits specific degradation of a desired target ANGPTL3 RNA through a RISC pathway but will typically range from 9 to 30 base pairs in length, e.g., 15-30 base pairs in length.
  • the duplex can be any length in this range, 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, and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20
  • ANGPTL3 dsRNA agents generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 base pairs in length.
  • One strand of the duplex region of an ANGPTL3 dsDNA agent comprises a sequence that is substantially complementary to a region of a target ANGPTL3 RNA.
  • the two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules.
  • the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop” ) between the 3' -end of one strand and the 5'-end of the respective other strand forming the duplex structure.
  • a hairpin look comprises 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.
  • an ANGPTL3 dsRNA agent may include a sense and antisense sequence that have no-unpaired nucleotides or nucleotide analogs at one or both terminal ends of the dsRNA agent.
  • An end with no unpaired nucleotides is referred to as a “blunt end” and as having no nucleotide overhang. If both ends of a dsRNA agent are blunt, the dsRNA is referred to as “blunt ended” .
  • a first end of a dsRNA agent is blunt, in some embodiments a second end of a dsRNA agent is blunt, and in certain embodiments of the invention, both ends of an ANGPTL3 dsRNA agent are blunt.
  • the dsRNA does not have one or two blunt ends.
  • a dsRNA can comprise an overhang of at least 1, 2, 3, 4, 5, 6, or more nucleotides.
  • a nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.
  • nucleotide overhang is on a sense strand of a dsRNA agent, on an antisense strand of a dsRNA agent, or on both ends of a dsRNA agent and nucleotide (s) of an overhang can be present on the 5' end, 3' end or both ends of either an antisense or sense strand of a dsRNA.
  • nucleotides in an overhang is replaced with a nucleoside thiophosphate.
  • antisense strand or “guide strand” refers to the strand of an ANGPTL3 dsRNA agent that includes a region that is substantially complementary to an ANGPTL3 target sequence.
  • sense strand, ” or “passenger strand” refers to the strand of an ANGPTL3 dsRNA agent that includes a region that is substantially complementary to a region of the antisense strand of the ANGPTL3 dsRNA agent.
  • RNA compounds useful in certain embodiments of ANGPTL3 dsRNA agents, ANGPTL3 antisense polynucleotides, and ANGPTL3 sense polynucleotides of the invention include, but are not limited to RNAs comprising modified backbones or no natural internucleoside linkages.
  • RNA molecule can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below, and molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex.
  • DsRNA agents, ANGPTL3 antisense polynucleotides, and/or ANGPTL3 sense polynucleotides of the invention may, in some embodiments comprise one or more independently selected modified nucleotide and/or one or more independently selected non-phosphodiester linkage.
  • independently selected used in reference to a selected element, such as a modified nucleotide, non-phosphodiester linkage, etc., means that two or more selected elements can but need not be the same as each other.
  • ribonucleotide or “nucleotide” may be used herein to refer to an unmodified nucleotide, a modified nucleotide, a nucleotide analog, or a surrogate replacement moiety.
  • guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety.
  • C1-6 alkyl optionally substituted by halogen or cyano means that halogen or cyano may, but not necessarily, be present, including the case where alkyl is substituted by halogen or cyano and the case where alkyl is not substituted by halogen and cyano.
  • modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway.
  • PNAs peptide nucleic acids
  • an ANGPTL3 RNA interference agent includes a single stranded RNA that interacts with a target ANGPTL3 RNA sequence to direct the cleavage of the target ANGPTL3 RNA.
  • Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones siloxane backbones
  • sulfide, sulfoxide and sulfone backbones formacetyl and thioformacetyl backbones
  • methylene formacetyl and thioformacetyl backbones alkene containing backbones
  • sulfamate backbones methyleneimino and methylenehydrazino backbones
  • sulfonate and sulfonamide backbones amide backbones
  • others having mixed N, O, S and CH 2 component parts.
  • Means of preparing modified RNA backbones that do not include a phosphorus atom are routinely practiced 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.
  • RNA mimetics are included in ANGPTL3 dsRNAs, ANGPTL3 antisense polynucleotides, and/or ANGPTL3 sense polynucleotides, such as, but not limited to: replacement of the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units with novel groups.
  • base units are maintained for hybridization with an appropriate ANGPTL3 nucleic acid target compound.
  • Means of preparing RNA mimetics are routinely practiced in the art and such methods can be used to prepare certain modified ANGPTL3 dsRNA agents of the invention.
  • RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones and in particular -CH 2 -NH-CH 2 -, -CH 2 -N (CH 3 ) -O-CH 2 - [known as a methylene (methylimino) or MMI backbone] , -CH 2 -O-N (CH 3 ) -CH 2 -, -CH 2 -N (CH 3 ) -N (CH 3 ) -CH 2 -and -N (CH 3 ) -CH 2 - [wherein the native phosphodiester backbone is represented as -O-P-O-CH 2 -] .
  • RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones are routinely practiced in the art and such methods can be used to prepare certain modified ANGPTL3 dsRNA agents, certain ANGPTL3 antisense polynucleotides, and/or certain ANGPTL3 sense polynucleotides of the invention.
  • Modified RNAs can also contain one or more substituted sugar moieties.
  • ANGPTL3 dsRNAs, ANGPTL3 antisense polynucleotides, and/or ANGPTL3 sense polynucleotides of the invention may comprise one of the following at the 2'position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • dsRNAs include one of the following at the 2'position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an ANGPTL3 dsRNA agent, or a group for improving the pharmacodynamic properties of an ANGPTL3 dsRNA agent, ANGPTL3 antisense polynucleotide, and/or ANGPTL3 sense polynucleotide, and other substitu
  • the modification includes a 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O- (2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504) i.e., an alkoxy-alkoxy group.
  • Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a O (CH 2 ) 2 ON (CH 3 ) 2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE) , i.e., 2'-O-CH 2 -O-CH 2 -N (CH 2 ) 2 .
  • Means of preparing modified RNAs such as those described are routinely practiced in the art and such methods can be used to prepare certain modified ANGPTL3 dsRNA agents of the invention.
  • modifications include 2'-methoxy (2'-OCH 3 ) , 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F) .
  • Similar modifications can also 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 2'-5'linked ANGPTL3 dsRNAs, ANGPTL3 antisense polynucleotides, or ANGPTL3 sense polynucleotides, and the 5'position of 5'terminal nucleotide.
  • ANGPTL3 dsRNA agents, ANGPTL3 antisense polynucleotides, and/or ANGPTL3 sense polynucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • Means of preparing modified RNAs such as those described are routinely practiced 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.
  • An ANGPTL3 dsRNA agent, ANGPTL3 antisense polynucleotide, and/or ANGPTL3 sense polynucleotide may, in some embodiments, include nucleobase (often referred to in the art simply as "base” ) modifications or substitutions.
  • nucleobase often referred to in the art simply as "base”
  • “unmodified” or “natural” nucleobases include the purine bases adenine and guanine, and the pyrimidine bases thymine, cytosine and uracil.
  • nucleobases that may be included in certain embodiments of ANGPTL3 dsRNA agents of the invention are known in the art, see for example: Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. Ed. Wiley-VCH, 2008; The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.L, Ed. John Wiley &Sons, 1990, English et al., Angewandte Chemie, International Edition, 1991, 30, 613, Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., Ed., CRC Press, 1993.
  • an abasic or inverted abasic nucleotide at the end of oligonucleotide enhances stability (Czauderna et al. Structural variations and stabilizing modifications of synthetic siRNA agents in mammalian cells. Nucleic Acids Res. 2003; 31 (11) : 2705-2716. doi: 10.1093/nar/gkg393) .
  • an ANGPTL3 dsRNA compound includes one or more inverted abasic residues (invab) at either 3’ -end or 5’ -end, or both 3’ -end and 5’ -end. Exemplified inverted abasic residues (invab) include, but are not limited to the following:
  • antisense polynucleotides of the invention further comprise a phosphate moiety.
  • a phosphate moiety refers to a phosphate group including phosphates or phosphates mimics that attached to the sugar moiety (e.g., a ribose or deoxyribose or analog thereof) of a nucleotide.
  • a nucleotide comprising a phosphate mimic may also be defined as a phosphonate modified nucleotide.
  • a vinyl phosphonate of the instant disclosure may be attached to either the antisense or the sense strand of a dsRNA of the disclosure.
  • a vinyl phosphonate of the instant disclosure is attached to the antisense strand of a dsRNA, optionally at the 5’ end of the antisense strand of the dsRNA.
  • X is O or S
  • R5'is C (H) -P (O) (OH) 2 and the double bond between the C5'carbon and R5'is in the E or Z orientation (e.g., E orientation) ;
  • B is a nucleobase or a modified nucleobase, optionally where B is adenine, guanine, cytosine, thymine, or uracil.
  • R5'is C (H) -P (O) (OH) 2 and the double bond between the C5’ carbon and R5’ is in the E orientation.
  • Vinyl phosphonate modifications are also contemplated for the dsRNAs, the compositions and methods of the instant disclosure.
  • An exemplary vinyl phosphonate structure is:
  • protecting groups are used during the preparation of the compounds of the invention.
  • the term "protected” means that the indicated moiety has a protecting group appended thereon.
  • compounds contain one or more protecting groups.
  • a wide variety of protecting groups can be employed in the methods of the invention. In general, protecting groups render chemical functionalities inert to specific reaction conditions, and can be appended to and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule.
  • Protecting groups in general and hydroxyl protecting groups in particular are well known in the art (Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley &Sons, New York, 1991) .
  • each 3’ and 5’ terminal end of the sense strand independently includes an inverted abasic residue.
  • the sense strand includes one inverted abasic residues at 3’ and 5’ terminal end respectively and either residue at 3’ or 5’ terminal end is further conjugated to a targeting group, which preferably is aforesaid GLS-15*.
  • the modified nucleotide is a modified nucleotide defined above.
  • N Z is a vinyl phosphonate modified nucleotide.
  • N Z is Vpu*, which has the structure
  • a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 3 (ANGPTL3) is provided, wherein the dsRNA agent including a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a dsRNA duplex, wherein said sense strand is complementary to the antisense strand, wherein said antisense strand comprises a region of complementarity to an mRNA encoding ANGPTL3, wherein the region of complementarity comprises at least 15 contiguous nucleotides, wherein the dsRNA duplex represented by formula (III) :
  • N Z is a vinyl phosphonate modified nucleotide.
  • the modified nucleotide is a modified nucleotide defined above.
  • the modified nucleotide is a 2’ -OMe modified nucleotide or a 2’ -F modified nucleotide.
  • N M2 , N M3 and N M6 each independently represents a 2'-fluoro-modified nucleotide; in certain embodiment, N M2 , N M3 and N M6 are all 2'-fluoro-modified nucleotides.
  • the antisense strand includes one inverted abasic residue at 3’ -terminal end.
  • the sense strand includes one or two inverted abasic residues and/or one or two imann residues at 3’ or/and 5’ terminal end.
  • each 3’ and 5’ terminal end of the sense strand independently includes an inverted abasic residue.
  • each 3’ and 5’ terminal end of the sense strand independently includes an imann residue.
  • At least one strand includes a 3’ overhang of at least 1 nucleotide. In certain embodiments of Formula (I) , (II) or (III) , at least one strand includes a 3’ overhang of at least 2 nucleotides.
  • N M1 , N M2 , N M3 , N M4 , N M5 , N M6 , N M7 , N M8 , N M9 , N′ N1 , N′ N2 , N′ N3 , N′ N4 , N′ N5 , N′ N6 , N′ L , N L and N z each independently is linked to a neighboring nucleotide via phosphodiester (PO) linkage.
  • PO phosphodiester
  • Formula (I) , (II) or (III) further includes inverted abasic residues, imann residues and/or targeting groups, and the linkage within positions 1-10 of the termini positions of each end of the strand independently comprises 1, 2, 3, 4, 5 or 6 phosphorothioate (PS) linkages.
  • the linkage within positions 1-5 of the termini positions of each end of the strand independently comprises 1, 2 or 3 phosphorothioate (PS) linkages.
  • the linkage within positions 1-3 of the termini positions of each end of the strand independently comprises 1 or 2 phosphorothioate (PS) linkages.
  • compositions Containing ANGPTL3 dsRNA
  • compositions containing an ANGPTL3 dsRNA agent include use of pharmaceutical compositions containing an ANGPTL3 dsRNA agent and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition containing the ANGPTL3 dsRNA agent can be used in methods of the invention to reduce ANGPTL3 gene expression and ANGPTL3 activity in a cell and is useful to treat an ANGPTL3-associated disease or condition.
  • Such pharmaceutical compositions can be formulated based on the mode of delivery.
  • formulations for modes of delivery are: a composition formulated for subcutaneous delivery, a composition formulated for systemic administration via parenteral delivery, a composition formulated for intravenous (IV) delivery, a composition formulated for intrathecal delivery, a composition formulated for direct delivery into brain, etc.
  • An ANGPTL3 dsRNA agent can also be delivered directly to a target tissue, for example directly into the liver, directly into a kidney, etc. It will be understood that “delivering an ANGPTL3 dsRNA agent” into a cell encompasses delivering an ANGPTL3 dsRNA agent, directly as well as expressing an ANGPTL3 dsRNA agent in a cell from an encoding vector that is delivered into a cell, or by any suitable means with which the ANGPTL3 dsRNA becomes present in a cell. Preparation and use of formulations and means for delivering inhibitory RNAs are well known and routinely used in the art.
  • a “pharmaceutical composition” comprises a pharmacologically effective amount of an ANGPTL3 dsRNA agent of the invention and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium.
  • pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives.
  • Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in drug formulations are described further herein below.
  • Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • compositions comprising an ANGPTL3 RNAi construct suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • these preparations are sterile and fluid to the extent that easy injectability exists.
  • Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • compositions for use in the methods of the present invention generally may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with free amino groups) derived from inorganic acids (e.g., hydrochloric or phosphoric acids) , or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like) . Salts formed with the free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like) .
  • inorganic acids e.g., hydrochloric or phosphoric acids
  • organic acids e.g., acetic, oxalic, tartaric, mandelic, and the like
  • Salts formed with the free carboxyl groups can also be derived
  • Sodium salts of the ANGPTL3 RNAi constructs are particularly useful for therapeutic administration to human subjects.
  • the ANGPTL3 RNAi constructs are in the form of a sodium salt.
  • the ANGPTL3 RNAi constructs are in the form of a potassium salt.
  • the solution For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose.
  • aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure.
  • a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion or injection, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580) .
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA standards.
  • a pharmaceutical composition for use in the methods of the invention comprises or consists of a sterile saline solution and an ANGPTL3 RNAi construct described herein.
  • a pharmaceutical composition useful for treating, ameliorating, preventing, or reducing the risk of hyperlipidemia according to the methods of the invention comprises an effective amount of an ANGPTL3 RNAi construct, Ortho-Phosphoric acid, and/or sodium hydroxide.
  • the pharmaceutical composition comprises about 200 mg/mL of an ANGPTL3 RNAi construct, which is calculated based on 100%pure sodium salt, and water for injection as the diluent, and optionally, sodium hydroxide or 85%ortho-phosphoric acid at 0.1N or 1N solution as the pH adjust mentor when needed.
  • Certain embodiments of methods of the invention includes delivery of an ANGPTL3 dsRNA agent into a cell.
  • delivery means facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an ANGPTL3 dsRNA agent can occur through unaided diffusive or active cellular processes, or by use of delivery agents, targeting agents, etc. that may be associated with an ANGPTL3 dsRNA agent of the invention.
  • Delivery means that are suitable for use in methods of the invention include, but are not limited to: in vivo delivery, in which an ANGPTL3 dsRNA agent is in injected into a tissue site or administered systemically. In some embodiments of the invention, an ANGPTL3 dsRNA agent is attached to a delivery agent.
  • Non-limiting examples of methods that can be used to deliver 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 used successfully in the art to deliver therapeutic RNAi agents for treatment of various diseases and conditions, such as but not limited to: neurodegenerative diseases, liver diseases, acute intermittent porphyria (AIP) , hemophilia, pulmonary fibrosis, etc. Details of various delivery means are found in publications such as: Nikam, R.R. &K.R. Gore (2016) Nucleic Acid Ther, 28 (4) , 209-224 Aug 2018; Springer A.D.
  • Some embodiments of the invention comprise use of functional moieties to deliver an ANGPTL3 dsRNA agent of the invention to a cell, tissue, and/or subject.
  • the functional moiety is a hydrophobic moiety.
  • the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins.
  • the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA) .
  • the fatty acid selected from the group consisting of Eicosapentaenoic acid (EPA) , Docosahexaenoic acid (DHA) and Docosanoic acid (DCA) .
  • the functional moieties may comprise one or more ligands tethered to an RNA silencing agent to improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell-type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism.
  • Ligands and associated modifications can also increase sequence specificity and consequently decrease off-site targeting.
  • a tethered ligand can include one or more modified bases or sugars that can function as intercalators. These can be located in an internal region, such as in a bulge of RNA silencing agent/target duplex.
  • the intercalator can be an aromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound.
  • a polycyclic intercalator can have stacking capabilities, and can include systems with 2, 3, or 4 fused rings.
  • the universal bases described herein can be included on a ligand.
  • the ligand can include a cleaving group that contributes to target gene inhibition by cleavage of the target nucleic acid.
  • the cleaving group can be, for example, a bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2) , pyrene, phenanthroline (e.g., O-phenanthroline) , a polyamine, a tripeptide (e.g., lys-tyr-lys tripeptide) , or a metal ion chelating group.
  • a bleomycin e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2
  • pyrene e.g., phenanthroline (e.g., O-phenanthroline)
  • phenanthroline e.g., O-phenanthroline
  • polyamine e.g., a tripeptide (e.g., lys-tyr-lys tripeptide)
  • a metal ion chelating group e.g.
  • the metal ion chelating group can include, e.g., an Lu (III) or EU (III) macrocyclic complex, a Zn (II) 2, 9-dimethylphenanthroline derivative, a Cu (II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the site of the bulge by free metal ions, such as Lu (III) .
  • a peptide ligand can be tethered to a RNA silencing agent to promote cleavage of the target RNA, e.g., at the bulge region.
  • 1, 8-dimethyl-1, 3, 6, 8, 10, 13-hexaazacyclotetradecane can be conjugated to a peptide (e.g., by an amino acid derivative) to promote target RNA cleavage.
  • a tethered ligand can be an aminoglycoside ligand, which can cause an RNA silencing agent to have improved hybridization properties or improved sequence specificity.
  • Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine.
  • Use of an acridine analog can increase sequence specificity.
  • neomycin B has a high affinity for RNA as compared to DNA, but low sequence-specificity.
  • an acridine analog has an increased affinity for the HIV Rev-response element (RRE) .
  • the guanidine analog (the guanidinoglycoside) of an aminoglycoside ligand is tethered to an RNA silencing agent.
  • the amine group on the amino acid is exchanged for a guanidine group.
  • Attachment of a guanidine analog can enhance cell permeability of an RNA silencing agent.
  • a tethered ligand can be a poly-arginine peptide, peptoid or peptidomimetic, which can enhance the cellular uptake of an oligonucleotide agent.
  • Exemplary ligands are coupled, either directly or indirectly, via an intervening tether, to a ligand-conjugated carrier.
  • the coupling is through a covalent bond.
  • the ligand is attached to the carrier via an intervening tether.
  • a ligand alters the distribution, targeting or lifetime of an RNA silencing agent into which it is incorporated.
  • a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.
  • Exemplary ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified RNA silencing agent, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.
  • Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases.
  • General examples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin, diosgenin) , terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid) , vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal) , carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics.
  • steroids e.g., uvaol, hecigenin, diosgenin
  • terpenes e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid
  • vitamins e.g., folic acid, vitamin A, bio
  • Ligands can include a naturally occurring substance, (e.g., human serum albumin (HSA) , low-density lipoprotein (LDL) , or globulin) ; carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid) ; amino acid, or a lipid.
  • HSA human serum albumin
  • LDL low-density lipoprotein
  • globulin carbohydrate
  • carbohydrate e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid
  • amino acid or a lipid.
  • the ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid.
  • polyamino acids examples include polyamino acid is a polylysine (PLL) , poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly (L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N- (2-hydroxypropyl) methacrylamide copolymer (HMPA) , polyethylene glycol (PEG) , polyvinyl alcohol (PVA) , polyurethane, poly (2-ethylacryllic acid) , N-isopropyl acrylamide polymers, or polyphosphazine.
  • PLL polylysine
  • poly L-aspartic acid poly L-glutamic acid
  • styrene-maleic acid anhydride copolymer poly (L-lactide-co-glycolied) copolymer
  • polyamines include: polyethylenimine, polylysine (PLL) , spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
  • Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
  • a cell or tissue targeting agent e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
  • a targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine (GalNAc) or derivatives thereof, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B 12, biotin, or an RGD peptide or RGD peptide mimetic.
  • ligands include dyes, intercalating agents (e.g. acridines and substituted acridines) , crosslinkers (e.g. psoralene, mitomycin C) , porphyrins (TPPC4, texaphyrin, Sapphyrin) , polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrenes) , lys-tyr-lys tripeptide, aminoglycosides, guanidium aminoglycodies, artificial endonucleases (e.g.
  • intercalating agents e.g. acridines and substituted acridines
  • crosslinkers e.g. psoralene, mitomycin C
  • porphyrins TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophen
  • EDTA lipophilic molecules, e.g, cholesterol (and thio analogs thereof) , cholic acid, cholanic acid, lithocholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters, e.g., C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 fatty acids) and ethers thereof, e.g., C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl; e.g., l, 3-bis-O (hexadecyl) glycerol, l, 3-bis-O (octaadecyl) glycerol) , gerany
  • the ligand is GalNAc or a derivative thereof.
  • Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell.
  • Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose.
  • the functional moiety is linked to the 5’ end and/or 3’ end of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5’ end and/or 3’ end of an antisense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5’ end and/or 3’ end of a sense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 3’ end of a sense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5’ end of a sense strand of the RNA silencing agent of the disclosure.
  • the functional moiety is linked to the RNA silencing agent by a linker. In certain embodiments, the functional moiety is linked to the antisense strand and/or sense strand by a linker. In certain embodiments, the functional moiety is linked to the 3’ end of a sense strand by a linker. In certain embodiments, the linker comprises a divalent or trivalent linker. In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.
  • a delivery agent that may be used in embodiments of the invention to deliver an ANGPTL3 dsRNA agent of the invention to a cell, tissue and/or subject is an agent comprising GalNAc that is attached to an ANGPTL3 dsRNA agent of the invention and delivers the ANGPTL3 dsRNA agent to a cell, tissue, and/or subject.
  • agents comprising GalNAc that can be used in certain embodiments of methods and composition of the invention are disclosed in PCT Application: WO2020191183A1 and WO2023045995 (incorporated herein in its entirety) .
  • GalNAc targeting ligand that can be used in compositions and methods of the invention to deliver an ANGPTL3 dsRNA agent to a cell is a targeting ligand cluster.
  • Examples of targeting ligand clusters that are presented herein are referred to as: GalNAc Ligand with phosphodiester link (GLO) and GalNAc Ligand with phosphorothioate link (GLS) .
  • GLX-n may be used herein to indicate the attached GalNAc-containing compound is any one of compounds 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 structure of each of which is shown below, with the below with location of attachment of the GalNAc-targeting ligand to an RNAi agent of the invention at far right of each (shown with ) .
  • in vivo delivery can also be by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety.
  • an ANGPTL3 dsRNA is delivered without a targeting agent. These RNAs may be delivered as “naked” RNA molecules.
  • an ANGPTL3 dsRNA of the invention may be administered to a subject to treat an ANGPTL3-associated disease or condition in the subject, in a pharmaceutical composition comprising the RNAi agent, but not including a targeting agent such as a GalNAc targeting compound.
  • RNAi delivery means such as but not limited to those described herein and those used in the art, can be used in conjunction with embodiments of ANGPTL3 RNAi agents and treatment methods described herein.
  • ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents of the invention can be administered to reduce ANGPTL3 expression and/or activity in one more of in vitro, ex vivo, and in vivo cells.
  • one or more ANGPTL3 dsRNA agents may be administered in formulations, which may be administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • an ANGPTL3 dsRNA agent may be formulated with another therapeutic agent for simultaneous administration.
  • an ANGPTL3 dsRNA agent may be administered in a pharmaceutical composition.
  • a pharmaceutical composition comprises an ANGPTL3 dsRNA agent and optionally, a pharmaceutically-acceptable carrier.
  • Pharmaceutically-acceptable carriers are well-known to those of ordinary skill in the art.
  • a pharmaceutically-acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients, e.g., the ability of the ANGPTL3 dsRNA agent to inhibit ANGPTL3 gene expression in a cell or subject.
  • a pharmaceutically-acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients, e.g., the ability of the ANGPTL3 dsRNA agent to inhibit ANGPTL3 gene expression in a cell or subject.
  • Numerous methods to administer and deliver dsRNA agents s for therapeutic use are known in the art and may be utilized in methods of the invention.
  • Some embodiments of methods of the invention include administering one or more ANGPTL3 dsRNA agents directly to a tissue.
  • the tissue to which the compound is administered is a tissue in which the ANGPTL3-associated disease or condition is present or is likely to arise, non-limiting examples of which are the liver or kidney.
  • Direct tissue administration may be achieved by direct injection or other means.
  • Many orally delivered compounds naturally travel to and through the liver and kidneys and some embodiments of treatment methods of the invention include oral administration of one or more ANGPTL3 dsRNA agents to a subject.
  • ANGPTL3 dsRNA agents either alone or in conjunction with other therapeutic agents, may be administered once, or alternatively they may be administered in a plurality of administrations.
  • the ANGPTL3 dsRNA agent may be administered via different routes.
  • a first (or first several) administrations may be made via subcutaneous means and one or more additional administrations may be oral and/or systemic administrations.
  • the ANGPTL3 dsRNA agent may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with or without an added preservative.
  • ANGPTL3 dsRNA agent formulations (also referred to as pharmaceutical compositions) may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose) , and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day may be used as needed to achieve appropriate systemic or local levels of one or more ANGPTL3 dsRNA agents and to achieve appropriate reduction in ANGPTL3 protein activity.
  • methods of the invention include use of a delivery vehicle such as biocompatible microparticle, nanoparticle, or implant suitable for implantation into a recipient, e.g., a subject.
  • a delivery vehicle such as biocompatible microparticle, nanoparticle, or implant suitable for implantation into a recipient, e.g., a subject.
  • exemplary bioerodible implants that may be useful in accordance with this method are described in PCT Publication No. WO 95/24929 (incorporated by reference herein) , which describes a biocompatible, biodegradable polymeric matrix for containing a biological macromolecule.
  • a matrix may be biodegradable.
  • Matrix polymers may be natural or synthetic polymers.
  • a polymer can be selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months can be used.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90%of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers.
  • ANGPTL3 dsRNA agents may be delivered in some embodiments of the invention using the bioerodible implant by way of diffusion, or by degradation of the polymeric matrix.
  • Exemplary synthetic polymers for such use are well known in the art.
  • Biodegradable polymers and non-biodegradable polymers can be used for delivery of ANGPTL3 dsRNA agents s using art-known methods.
  • Bioadhesive polymers such as bioerodible hydrogels (see H.S. Sawhney, C.P. Pathak and J.A.
  • Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated by reference herein) may also be used to deliver ANGPTL3 dsRNA agents for treatment of an ANGPTL3-associated disease or condition.
  • Additional suitable delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of an ANGPTL3 dsRNA agent, increasing convenience to the subject and the medical care professional.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. (See for example: U.S. Pat. Nos.
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • Long-term sustained release implant may be suitable for prophylactic treatment of subjects and for subjects at risk of developing a recurrent ANGPTL3-associated disease or condition.
  • Long-term release means that the implant is constructed and arranged to deliver a therapeutic level of an ANGPTL3 dsRNA agent for at least up to 10 days, 20 days, 30 days, 60 days, 90 days, six months, a year, or longer.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • Therapeutic formulations of ANGPTL3 dsRNA agents may be prepared for storage by mixing the molecule or compound having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers [Remington's Pharmaceutical Sciences 21st edition, (2006) ] , in the form of lyophilized formulations or aqueous solutions.
  • ANGPTL3-associated diseases and conditions in which a decrease in a level and/or activity of ANGPTL3 polypeptide is effective to treat the disease or condition can be treated using methods and ANGPTL3 dsRNA agents of the invention to inhibit ANGPTL3 expression.
  • diseases and conditions that may be treated with an ANGPTL3 dsRNA agent of the invention and a treatment method of the invention, include, but are not limited to: hyperlipidemia, hypertriglyceridemia, severe hypertriglyceridemia (SHTG) , familial chylomicronemia syndrome, mixed dyslipidemia, hypercholesterolemia, homozygous familial hypercholesterolemia (HoFH) , heterozygous familial hypercholesterolemia (HeFH) , abnormal lipid and/or cholesterol metabolism, statin resistant hypercholesterolemia, cardiometabolic disease, obesity, atherosclerosis, type II diabetes mellitus, cardiovascular disease, coronary artery disease, cerebrovascular disease, peripheral artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis caused by hypertriglyceridemia.
  • Such diseases and conditions may be referred to herein as “ANGPTL3-associated diseases and conditions” and “diseases and conditions caused and/or modulated by
  • a subject may be administered an ANGPTL3 dsRNA agent of the invention at a time that is one or more of before or after diagnosis of an ANGPTL3-associated disease or condition.
  • a subject is at risk of having or developing an ANGPTL3-associated disease or condition.
  • a subject at risk of developing an ANGPTL3-associated disease or condition is one who has an increased probability of developing the ANGPTL3-associated disease or condition, compared to a control risk of developing the ANGPTL3-associated disease or condition.
  • a level of risk may be statistically significant compared to a control level of risk.
  • a subject at risk may include, for instance, a subject who is, or will be, a subject who has a preexisting disease and/or a genetic abnormality that makes the subject more susceptible to an ANGPTL3-associated disease or condition than a control subject without the preexisting disease or genetic abnormality; a subject having a family and/or personal medical history of the ANGPTL3-associated disease or condition; and a subject who has previously been treated for an ANGPTL3-associated disease or condition.
  • an ANGPTL3 dsRNA agent may be administered to a subject based on a medical status of the individual subject.
  • a health-care provided for a subject may assess a lipid level measured in a sample obtained from a subject and determine it is desirable to reduce the subject’s lipid level, by administration of an ANGPTL3 dsRNA agent of the invention.
  • the lipid level may be considered to be a physiological characteristic of an ANGPTL3-associated condition, even if the subject is not diagnosed as having an ANGPTL3-assoicated disease such as one disclosed herein.
  • a healthcare provider may monitor changes in the subject’s lipid level, as a measure of efficacy of the administered ANGPTL3 dsRNA agent of the invention.
  • a biological sample such as a blood or serum sample may be obtained from a subject and a lipid level for the subject determined in the sample.
  • An ANGPTL3 dsRNA agent is administered to the subject and a blood or serum sample is obtained from the subject following the administration and the lipid level determined using the sample and the results compared to the results determined in the subject’s pre-administration (prior) sample.
  • a reduction in the subject’s lipid level in the later sample compared to the pre-administration level indicates the administered ANGPTL3 dsRNA agent efficacy in reducing the lipid level in the subject.
  • Certain embodiments of methods of the invention include adjusting a treatment that includes administering an ANGPTL3 dsRNA agent of the invention to a subject based at least in part on assessment of a change in one or more of the subject’s physiological characteristics of an ANGPTL3-associated disease or condition resulting from the treatment.
  • an effect of an administered dsRNA agent of the invention may be determined for a subject and used to assist in adjusting an amount of a dsRNA agent of the invention subsequently administered to the subject.
  • a subject is administered a dsRNA agent of the invention, the subject’s lipid level is determined after the administration, and based at least in part on the determined level, a greater amount of the dsRNA agent is determined to be desirable in order to increase the physiological effect of the administered agent, for example to reduce or further reduce the subject’s lipid level.
  • a subject is administered a dsRNA agent of the invention, the subject’s lipid level is determined after the administration and based at least in part on the determined level, a lower amount of the dsRNA agent is desirable to administer to the subject.
  • some embodiments of the invention include assessing a change in one or more physiological characteristics of resulting from a subject’s previous treatment to adjust an amount of a dsRNA agent of the invention subsequently administered to the subject.
  • Some embodiments of methods of the invention include 1, 2, 3, 4, 5, 6, or more determinations of a physiological characteristic of an ANGPTL3-associated disease or condition to assess and/or monitor the efficacy of an administered ANGPTL3 dsRNA agent of the invention, and optionally using the determinations to adjust one or more of: a dose, administration regimen, and or administration frequency of a dsRNA agent of the invention to treat an ANGPTL3-associated disease or condition in a subject.
  • a desired result of administering an effective amount of a dsRNA agent of the invention to a subject is a reduction of the subject’s lipid level, serum lipid level, LDL level, LDL : HDL ratio, triglyceride level, TC level, fat present in a subject’s liver, etc., as compared to a prior level determined for the subject, or to a control level.
  • the terms “treat” , “treated” , or “treating” when used with respect to an ANGPTL3-associated disease or condition may refer to a prophylactic treatment that decreases the likelihood of a subject developing the ANGPTL3-associated disease or condition, and also may refer to a treatment after the subject has developed an ANGPTL3-associated disease or condition in order to eliminate or reduce the level of the ANGPTL3-associated disease or condition, prevent the ANGPTL3-associated disease or condition from becoming more advanced (e.g., more severe) , and/or slow the progression of the ANGPTL3-associated disease or condition in a subject compared to the subject in the absence of the therapy to reduce activity in the subject of ANGPTL3 polypeptide.
  • the term "patient” as used herein, refers to a mammal, including humans, and can be used interchangeably with the term “subject” . In preferred embodiments, the patient is a human patient.
  • hypolipidemia which can be used interchangeably with “dyslipidemia” , as used herein, means a metabolic disorder characterized by abnormally high or low amounts of any or all lipids (e.g. fats, triglycerides, cholesterol, phospholipids) or lipoproteins in the blood.
  • Dyslipidemia is a risk factor for the development of atherosclerotic cardiovascular diseases (ASCVD) which include coronary artery disease, cerebrovascular disease, and peripheral artery disease.
  • ASCVD atherosclerotic cardiovascular diseases
  • Certain embodiments of agents, compositions, and methods of the invention can be used to inhibit ANGPTL3 gene expression.
  • the terms “inhibit, ” “silence, ” “reduce, ” “down-regulate, ” and “knockdown” mean the expression of the ANGPTL3 gene, as measured by one or more of: a level of RNA transcribed from the gene, a level of activity of ANGPTL3 expressed, and a level of ANGPTL3 polypeptide, protein or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the ANGPTL3 gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is contacted with (e.g., treated with) an ANGPTL3 dsRNA agent of the invention, compared to a control level of RNA transcribed from the ANGPTL3 gene, a level of activity of expressed
  • an ANGPTL3 dsRNA agent is administered to a subject to treat an ANGPTL3-associated disease or condition in conjunction with one or more additional therapeutic regimens for treating the ANGPTL3-associate disease or condition.
  • additional therapeutic regimens are: administering a non-ANGPTL3 dsRNA therapeutic agent, and a behavioral modification.
  • An additional therapeutic regimen may be administered at a time that is one or more of: prior to, simultaneous with, and following administration of an ANGPTL3 dsRNA agent of the invention.
  • An ANGPTL3 dsRNA agent of the invention administered to a subject to treat an ANGPTL3-associated disease or condition may act in a synergistic manner with one or more other therapeutic agents or activities and increase the effectiveness of the one or more therapeutic agents or activities and/or to increase the effectiveness of the ANGPTL3 dsRNA agent at treating the ANGPTL3-associated disease or condition.
  • subsequent dose and “additional dose” may be used interchangeably, meaning the dose after the second dose, and may be administered for once or at regular intervals.
  • Treatment methods of the invention that include administration of an ANGPTL3 dsRNA agent can be used prior to the onset of an ANGPTL3-associated disease or condition and/or when an ANGPTL3-associated disease or condition is present, including at an early stage, mid-stage, and late stage of the disease or condition and all times before and after any of these stages.
  • Methods of the invention may also be to treat subjects who have previously been treated for an ANGPTL3-associated disease or condition with one or more other therapeutic agents and/or therapeutic activities that were not successful, were minimally successful, and/or are no longer successful at treating the ANGPTL3-associated disease or condition in the subject.
  • kits that comprise one or more ANGPTL3 dsRNA agents and/or ANGPTL3 antisense polynucleotide agents and instructions for its use in methods of the invention.
  • Kits of the invention may include one or more of an ANGPTL3 dsRNA agent, ANGPTL3 sense polynucleotide, and ANGPTL3 antisense polynucleotide agent that may be used to treat an ANGPTL3-associated disease or condition.
  • Kits containing one or more ANGPTL3 dsRNA agents, ANGPTL3 sense polynucleotides, and ANGPTL3 antisense polynucleotide agents can be prepared for use in treatment methods of the invention.
  • kits of the invention may be packaged either in aqueous medium or in lyophilized form.
  • a kit of the invention may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means or series of container means such as test tubes, vials, flasks, bottles, syringes, or the like.
  • a first container means or series of container means may contain one or more compounds such as an ANGPTL3 dsRNA agent and/or ANGPTL3 sense or antisense polynucleotide agent.
  • a second container means or series of container means may contain a targeting agent, a labelling agent, a delivery agent, etc. that may be included as a portion of an ANGPTL3 dsRNA agent to be administered in an embodiment of a treatment method of the invention.
  • Example 2 A Phase I, Randomized, Double-Blind, Placebo-Controlled, Single Ascending Dose Study to Evaluate the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Subcutaneously Administered AD00112-2 in Adult Subjects
  • subjects aged > 50 need to have stopped smoking more than 12 months prior to screening; subjects aged ⁇ 50 need to have stopped smoking 30 days prior to screening.
  • Females must be non-pregnant and non-lactating, and either surgically sterile or postmenopausal. Women of child-bearing potential, defined as all women physiologically capable of becoming pregnant, can participate if they are using highly effective methods of contraception from 28 days prior to screening until 24 weeks following administration of the study drug. The dual contraception is required, which is defined as use of a highly effective method of contraception by the female subject combined with use of a condom by the male partner.
  • a condom is not required for a male if in same-sex relationship.
  • the dual contraception is required, which is defined as use of a condom by the male subject combined with a highly effective method of contraception by the female partner.
  • Hormonal methods of contraception including oral contraceptives containing combined estrogen and progesterone, a vaginal ring, injectable and implantable hormonal contraceptives, intrauterine hormone-releasing system (e.g. Mirena) and progestogen-only hormonal contraception associated with inhibition of ovulation
  • hormonal contraceptives should begin at least 1 month prior to screening to ensure contraceptive is in full effect. Subjects will not be allowed to donate ova or sperm during the study.
  • Cohort 1 50 mg AD00112-2 or placebo
  • AD00112-2 Single dose administration of AD00112-2 was evaluated in Cohorts 1 to 4 at dose levels of 50, 150, 300, and 600 mg, respectively.
  • AD00112-2 was provided as a solution for subcutaneous (SC) injection (200 mg/mL [as sodium salt form] , presented as 1 mL extractable volume per vial) .
  • Placebo was provided as sodium chloride injection, 0.9%w/v administered via SC injection.
  • Properties of ANGPTL3 dsRNA agent (AD00112-2) are described in Table 3.
  • a schematic representation of ANGPTL3 dsRNA agent (AD00112-2) is shown in FIG. 8.
  • a sentinel dosing approach was applied in each cohort. Two sentinel subjects (1 received AD00112-2 and 1 received placebo) were dosed first. If deemed safe and tolerated by the Investigator, the remaining subjects in the same cohort were dosed after at least 24 hours of dosing of the 2 nd sentinel subject.
  • BMI Body Mass Index.
  • SD Standard Deviation.
  • ANGPTL3 Angiopoietin-like protein 3.
  • LDL-C Low density lipoprotein cholesterol.
  • HDL-C High density lipoprotein cholesterol.

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Abstract

It relates to methods of inhibiting the expression of an ANGPTL3 gene in a subject, as well as therapeutic and prophylactic methods for treating subjects having a lipid disorder, such as a hyperlipidemia using RNAi agents, e.g., double-stranded RNAi agents, targeting the ANGPTL3 gene.

Description

METHOD FOR TREATING DYSLIPIDEMIA WITH ANGIOPOIETIN-LIKE 3 (ANGPTL3) -TARGETED RNAI AGENTS Field of the Invention
The invention relates to compositions and methods that can be used to inhibit angiopoietin-like 3 (ANGPTL3) protein expression. In particular, the present invention relates to methods for reducing serum levels of ANGPTL3 and thus reducing serum levels of LDL-C, TG and non-high-density lipoprotein cholesterol (non-HDL-C) and methods for the treatment of dyslipidemia, e.g., mixed dyslipidemia, hypercholesterolemia and hypertriglyceridemia.
Background
Angiopoietin-like protein 3 (ANGPTL3) is a secreted protein that is mainly expressed 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) . Acting through inhibition of LPL and EL, ANGPTL3 reduces hydrolysis of triglycerides (TG) , particularly in muscle and fat tissue (Kersten S. Physiological regulation of lipoprotein lipase. Biochem Biophys Acta 2014; 1841: 919–933. Shimamura et al. Angiopoietin-like protein3 regulates plasma HDL cholesterol through suppression of endothelial lipase. Arterioscler. Thromb. Vasc. Biol. 2007; 27: 366–372) . Thus, inhibition of ANGPTL3 disinhibits LPL and EL activity, which results reduction of TG and high-density lipoprotein cholesterol (HDL-C) . Inhibition of ANGPTL3 also leads to reduction of low-density lipoprotein cholesterol (LDL-C) , possibly through EL-mediated processing of VLDL (Adam, et al. Angiopoietin-like protein3 governs LDL-cholesterol levels through endothelial lipase-dependent VLDL clearance. J Lipid Res 2020; 61: 1271–1286) . It is also noteworthy that current LDL-C lowering therapies, such as statins and PCSK9 inhibitors are LDL-R dependent, and are not effective for patients with low or no residue LDL-R activity. LDL-C lowering through inhibition of ANGPTL3 is LDL-R independent, which could be an effective therapeutic approach to manage lipids for patients with low or no LDL-R activity.
Hyperlipidemia is strongly associated with diseases including high blood pressure, atherosclerosis, heart diseases, diabetes, nonalcoholic steatohepatitis (NASH) . Study have shown beneficial effect of loss function mutation of ANGPTL3 in human. Homozygous loss of ANGPTL3 function causes familial combined hypolipidemia characterized by low plasma levels of triglycerides, high-density lipoprotein (HDL) cholesterol, and LDL-C and a decreased risk of coronary artery disease (Romeo et el., 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 antibody, antisense oligonucleotide (ASO) and siRNA agent in development. siRNA agent, particularly GalNAc-conjugated siRNA agent has been shown to be safe, effective and with long during of activity. Thus, there is a need for new ANGPTL3 siRNA agents for treating various diseases and conditions.
Summary of the Invention
In general, the present invention provides methods for inhibiting expression of an ANGPTL3 gene in a subject and methods for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of an ANGPTL3 gene, e.g., a disorder mediated by ANGPTL3 expression, such as a hyperlipidemia, e.g., hypercholesterolemia, using RNAi agent compositions which effect the RNA-induced silencing complex (RISC) -mediated cleavage of RNA transcripts of an ANGPTL3 gene.
According to an aspect of the invention, the methods of the present invention for inhibiting expression of an ANGPTL3 gene in a subject and methods for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of an ANGPTL3 gene, e.g., a disorder mediated by ANGPTL3 expression, such as dyslipidemia, e.g., hypertriglyceridemia and hypercholesterolemia, include administering to a subject a fixed dose of about 50 mg to about 600 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1 or 3 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 2 or 4, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and optionally, wherein the sense strand is conjugated to a ligand attached at the 5′-terminus.
In some embodiments, the present invention provides methods of inhibiting the expression of an ANGPTL3 gene in a subject. The methods include comprising administering to the subject a fixed dose of about 50 mg to about 600 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, the antisense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 0, 1, 2, 3 nucleotides from the complement of nucleotides 1350-1418 of the nucleotide sequence of SEQ ID NO: 1, and optionally, the sense strand is conjugated to a ligand attached at the 5′-terminus, thereby inhibiting the expression of the ANGPTL3 gene in the subject.
In some embodiments, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in ANGPTL3 expression. The methods include administering to the subject a fixed dose of about 50 mg to about 600 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, the antisense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 0, 1, 2, 3 nucleotides from the complement of nucleotides 1350-1418 of the nucleotide sequence of SEQ ID NO: 1, and optionally, the sense strand is conjugated to a ligand attached at the 5′-terminus, thereby treating the subject having a disorder that would benefit from reduction in ANGPTL3 expression.
In some embodiments, the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand of dsRNA comprising a nucleotide sequence II: 5’ -z1UAGAGUAUAACCUUCCz2-3’ , wherein z1 is selected from C, G, A or U, z2 is a nucleotide sequence IV, wherein the sense strand of dsRNA comprising a nucleotide sequence III: 5’ -z3GGAAGGUUAUACUCUAz4-3’ , wherein z3 is a nucleotide sequence V, z4 is selected from C, G, A or U. In certain embodiments, z1 is U. In certain embodiments, the nucleotide sequence IV is 0-15 nucleotides in length. In certain embodiments, the nucleotide sequence IV is selected from A, AU, AA, AC, AG, AUU, AUA, AUC, AUG, AUUG, AUUU, AUUA, AUUC, AUUUU, AUUUUG, AUUCUU, AUUCGA, AUUUUGA, AUUUUGAG, AUUUUGAGA or AUUUUGAGACUUCCA. In certain embodiments, the nucleotide sequence IV is 1, 2, 3 or 4 nucleotides in length. In certain embodiments, the nucleotide sequence IV is selected from A, AU, AA, AC, AG, AUU, AUA, AUC, AUG, AUUG, AUUU, AUUA or AUUC. In certain embodiments, the antisense strand of dsRNA comprising a nucleotide sequence II’ : 5’ -z1UAGAGUAUAACCUUCCAz2’ -3’ , wherein z1 is selected from C, G, A or U, z2’ is a nucleotide sequence IV’ . In certain embodiments, z1 is U. In certain embodiments, the nucleotide sequence IV’ is 0-15 nucleotides in length. In certain embodiments, the nucleotide sequence IV’ is 1, 2, 3 or 4 nucleotides in length. In certain embodiments, the nucleotide sequence IV’ is selected from U, A, C, G, UU, UA, UC, UG, UUG, UUU, UUA or UUC. In certain embodiments, z4 is A. In certain embodiments, the nucleotide sequence V is 0-15 nucleotides in length. In certain embodiments, the nucleotide sequence V is selected from U, AU, UU, GU, CU, AAU, UAU, GAU, CAU, GAAU, CAAU, AAAU, UAAU, AAAAU, CAAAAU, UCAAAAU, CUCAAAAU, UCUCAAAAU or UGGAAGUCUCAAAAU. In certain embodiments, the nucleotide sequence V is 1, 2, 3 or 4 nucleotides in length. In certain embodiments, the nucleotide sequence V is selected from U, AU, UU, GU, CU, AAU, UAU, GAU, CAU, GAAU, CAAU, AAAU or UAAU. In certain embodiments, the sense strand of dsRNA comprising a nucleotide sequence III’ : 5’ -z3’ UGGAAGGUUAUACUCUAz4-3’ , wherein z3’ is a nucleotide sequence V’ , z4 is selected from C, G, A or U. In certain embodiments, z4 is A. In certain embodiments, the nucleotide sequence V’ is 1, 2, 3 or 4 nucleotides in length. In certain embodiments, the nucleotide sequence V’ is selected from A, U, G, C, AA, UA, GA, CA, GAA, CAA, AAA or UAA. In certain embodiments, z1 is a nucleotide complementary to z4. In certain embodiments, z2 is a nucleotide sequence complementary to z3. In certain embodiments, z2’ is a nucleotide sequence complementary to z3’ . In certain embodiments, the dsRNA agent includes a sense strand and an antisense strand, wherein the antisense strand of dsRNA consists of the nucleotide sequence II or II'a s described above, wherein the sense strand is no more than 30 nucleotides in length comprising a region of complementarity to the antisense strand including at least 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the dsRNA agent includes a sense strand and an antisense strand, wherein the sense strand of dsRNA consists of the nucleotide sequence III and the antisense strand of dsRNA consists of the nucleotide sequence II, wherein the nucleotide sequence II and III are as described above. In some embodiments, the dsRNA agent includes a sense strand and an antisense strand, wherein the sense strand of dsRNA consists of the nucleotide sequence III’ and the antisense strand of dsRNA consists of the nucleotide sequence II’ , wherein the nucleotide sequence II’ and III’ are as described above.
In some embodiments, the antisense strand comprises the nucleotide sequence 5′-UUAGAGUAUAACCUUCCAUUC-3′ (SEQ ID NO: 40) .
In some embodiments, the sense strand comprises the nucleotide sequence 5′-GAAUGGAAGGUUAUACUCUAA-3′ (SEQ ID NO: 5) .
In some embodiments, the double-stranded RNAi agent targets nucleotides 1358-1378 of SEQ ID NO: 1.
In some embodiments, the double-stranded RNAi agent targeting nucleotides 1358-1378 of SEQ ID NO: 1 is AD00042. um.
In some embodiments, the double-stranded ribonucleic acid RNAi agent comprises at least one modified nucleotide.
In some embodiments, all or substantially all of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, all or substantially all of the nucleotides of the sense strand and the antisense strand are modified nucleotides. In some embodiments, at least one of the modified nucleotides comprises: 2’ -O-methyl nucleotide, 2’ -fluoro nucleotide, 2’ -deoxy nucleotide, 2’ 3’ -seco nucleotide mimic, locked nucleotide, unlocked nucleic acid nucleotide (UNA) , glycol nucleic acid nucleotide (GNA) , 2’ -F-Arabino nucleotide, 2’ -methoyxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted abasic nucleotide, inverted 2’ -OMe nucleotide, inverted 2’ -deoxy nucleotide, isomannide nucleotide, 2’ -amino-modified nucleotide, 2’ -alkyl-modified nucleotide, mopholino nucleotide, and 3’ -OMe nucleotide, a nucleotide including a 5’ -phosphorothioate group, a 5'-phosphonate modified nucleotide, or a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2’ -amino-modified nucleotide, a phosphoramidite, or a non-natural base including nucleotide.
In some embodiments, the sense strand comprises the nucleotide sequence of 5′-g*a*auggaaGfgUfuAfuacucua*a-3′ (SEQ ID NO: 6) .
In some embodiments, the antisense strand comprises the nucleotide sequence of 5′-u*Uf*agagUfauaaCfcUfuCfcau*u*c-3′ (SEQ ID NO: 41) .
In some embodiments, the double-stranded ribonucleic acid RNAi agent further comprises a ligand.
In some embodiments, the ligand is conjugated to the 5′end of the sense strand of the double-stranded ribonucleic acid RNAi agent.
In some embodiments, the ligand is an N-acetylgalactosamine (GalNAc) derivative.
In some embodiments, the ligand has a structure as Formula (X) :
Each n" is independently selected from 1 or 2.
In some embodiments, the double-stranded ribonucleic acid RNAi agent is conjugated to any one of the ligands as shown in the following:




In some embodiments, the sense and antisense strands of the dsRNA comprise nucleotide sequences and modification selected from the group consisting of
5’ -g*a*auggaaGfgUfuAfuacucua*a (GLO-0) -3’ (SEQ ID NO: 37)
5’ -u*Uf*agagUfauaaCfcUfuCfcau*u*c -3’ (SEQ ID NO: 72)
5’ - (GLS-5*) (invab*) gaauggaaGfgUfuAfuacucuaa* (invab) -3’ (SEQ ID NO: 38)
5’ -u*Uf*agagUfauaaCfcUfuCfcau*u*c -3’ (SEQ ID NO: 73)
5’ - (GLS-15*) (invab*) gaauggaaGfgUfuAfuacucuaa* (invab) -3’ (SEQ ID NO: 39)
5’ -u*Uf*agagUfauaaCfcUfuCfcau*u*c -3’ (SEQ ID NO: 74) .
In some embodiments, the disorder that would benefit from reduction in ANGPTL3 expression is dyslipidemia.
In some embodiments, the disorder is one or more of: hypertriglyceridemia, severe hypertriglyceridemia (SHTG) , familial chylomicronemia syndrome, mixed dyslipidemia, hypercholesterolemia, homozygous familial hypercholesterolemia (HoFH) , heterozygous familial hypercholesterolemia (HeFH) , abnormal lipid and/or cholesterol metabolism, statin resistant hypercholesterolemia, cardiometabolic disease, obesity, atherosclerosis, type II diabetes mellitus, cardiovascular disease, coronary artery disease, cerebrovascular disease, peripheral artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis caused by hypertriglyceridemia.
In some embodiments, the fixed dose may be administered to the subject at an interval of once a quarter, bianually or every nine months.
In some embodiments, the subject is administered a fixed dose of about 50 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 150 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 200 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 300 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 600 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 50 mg biannually (i.e., twice a year) . In some embodiment, the subject is administered a fixed dose of about 150 mg biannually. In some embodiment, the subject is administered a fixed dose of about 200 mg biannually. In some embodiment, the subject is administered a fixed dose of about 300 mg biannually. In some embodiment, the subject is administered a fixed dose of about 600 mg biannually. In some embodiment, the subject is administered a fixed dose of about 50 mg every nine months. In some embodiment, the subject is administered a fixed dose of about 150 mg every nine months. In some embodiment, the subject is administered a fixed dose of about 200 mg every nine months. In some embodiment, the subject is administered a fixed dose of about 300 mg every nine months. In some embodiment, the subject is administered a fixed dose of about 600 mg every nine months.
In some embodiments, the disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 150 mg biannually.
In some embodiments, the disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 200 mg biannually.
In some embodiments, the disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 300 mg biannually.
In some embodiments, the disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 600 mg biannually.
In some embodiments, the disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 150 mg once a quarter.
In some embodiments, the disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 200 mg once a quarter.
In some embodiments, the disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 300 mg once a quarter.
In some embodiments, the disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 600 mg once a quarter.
In some embodiments, the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about one month to about three months later, and thereafter for subsequent doses there is about six months or nine months between dose administrations.
In some embodiments, the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about one month to about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about one month to about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of about 50 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of about 50 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of about 150 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of about 150 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of about 200 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of about 200 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of about 300 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of about 300 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of about 600 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of about 600 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is mixed dyslipidemia and the human subject is administered a fixed dose of about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of between about 50 mg and about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 50 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 50 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 150 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 150 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 200 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 200 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 300 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 300 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 600 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 600 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about six months between dose administrations.
In some embodiments, the disorder is severe hypertriglyceridemia and the human subject is administered a fixed dose of about 900 mg, and the initial dose is followed by a second dose about three months later, and thereafter for subsequent doses there is about nine months between dose administrations.
In some embodiments, the second dose is equal to the initial dose.
In some embodiments, the subsequent dose is equal to the initial dose.
In some embodiments, the RNAi agent is administered in a dosing regimen that includes a loading phase and a maintenance phase.
In some embodiments, the methods include administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen that includes a loading phase followed by a maintenance phase, wherein the loading phase comprises administering a fixed dose of about 50 mg to about 900 mg of the RNAi agent to the subject, and wherein the maintenance phase comprises administering a fixed dose of about 50 mg to about 900 mg of the RNAi agent to the subject about once a quarter, biannually or every nine months.
In some embodiments, the methods include administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen that includes a loading phase followed by a maintenance phase, wherein the loading phase comprises administering a fixed dose of about 50 mg to about 900 mg of the RNAi agent to the subject, and wherein the maintenance phase comprises administering a fixed dose of about 50 mg to about 900 mg of the RNAi agent to the subject about once a quarter, biannually or every nine months.
In some embodiments, the loading phase comprises administering a fixed dose of about 50 mg of the RNAi agent to the subject, and the maintenance phase comprises administering a fixed dose of about 50 mg of the RNAi agent to the subject about every nine months.
In some embodiments, the loading phase comprises administering a fixed dose of about 150 mg of the RNAi agent to the subject, and the maintenance phase comprises administering a fixed dose of about 150 mg of the RNAi agent to the subject about every nine months.
In some embodiments, the loading phase comprises administering a fixed dose of about 200 mg of the RNAi agent to the subject, and the maintenance phase comprises administering a fixed dose of about 200 mg of the RNAi agent to the subject about every nine months.
In some embodiments, the loading phase comprises administering a fixed dose of about 300 mg of the RNAi agent to the subject, and the maintenance phase comprises administering a fixed dose of about 300 mg of the RNAi agent to the subject about every nine months.
In some embodiments, the loading phase comprises administering a fixed dose of about 600 mg of the RNAi agent to the subject, and the maintenance phase comprises administering a fixed dose of about 600 mg of the RNAi agent to the subject about every nine months.
In some embodiments, the loading phase comprises administering a fixed dose of about 900 mg of the RNAi agent to the subject, and the maintenance phase comprises administering a fixed dose of about 900 mg of the RNAi agent to the subject about every nine months.
In some embodiments, the double stranded RNAi agent may be administered to the subject subcutaneously, e.g., by subcutaneous injection.
In some embodiments, the subject is a human.
In some embodiments, ANGPTL3 expression is inhibited by at least about 30%.
In some embodiments, ANGPTL3 expression is inhibited by at least about 40%.
In some embodiments, ANGPTL3 expression is inhibited by at least about 50%.
In some embodiments, ANGPTL3 expression is inhibited by at least about 60%.
In some embodiments, ANGPTL3 expression is inhibited by at least about 70%.
In some embodiments, ANGPTL3 expression is inhibited by at least about 80%.
In some embodiments, ANGPTL3 expression is inhibited by at least about 90%.
In some embodiments, ANGPTL3 expression is inhibited by at least about 95%.
In some embodiments, ANGPTL3 expression is inhibited by at least about 99%.
In some embodiments, administering the double-stranded RNAi agent results in a decrease in serum lipid in the subject and/or a decrease in ANGPTL3 protein.
In some embodiments, the methods of the invention further comprise determining the serum ANGPTL3 or lipid level in the subject.
In some embodiments, the methods of the invention further comprise administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is one of more of: (i) a statin; (ii) one or more of PCSK9 antibody, PCSK9 antisense oligonucleotide (ASO) , and PCSK9 siRNA agent molecule 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.
In some embodiments, the RNAi agent is administered as a pharmaceutical composition. The RNAi agent may be administered in an unbuffered solution, such as saline or water, or administered with a buffer solution. In some embodiments, the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. In some embodiments, the buffer solution is phosphate buffered saline (PBS) .
In one aspect, the present invention provides kits for performing the method of the invention. The kits include the RNAi agent, and instructions for use, and optionally, means for administering the RNAi agent to the subject.
The present invention is further illustrated by the following detailed description and drawings.
Brief Description of the Sequences
SEQ ID NO: 1 and SEQ ID NO: 2 (reverse complement) are Homo sapiens angiopoietin like 3 (ANGPTL3) mRNA [NCBI Reference Sequence: NM_014495.4] .
SEQ ID NO: 3 and SEQ ID NO: 4 (reverse complement) are Predicted Macaca fascicularis angiopoietin like 3 (ANGPTL3) mRNA [NCBI Reference Sequence: XM_005543185.2] .
Brief Description of the Drawings
FIG. 1 is Mean (± SD) Percentage Change from Baseline Serum ANGPTL3 Level over Time
FIG. 2 is Mean (± SD) Percentage Change in LDL-C from Baseline over Time
FIG. 3 is Mean (± SD) Percentage Change in Triglyceride from Baseline over Time
FIG. 4 is Mean (± SD) Percentage Change in non-HDL-C from Baseline over Time
FIG. 5 is Mean (± SD) Percentage Change in ApoB from Baseline over Time
FIG. 6 is Mean (± SD) Percentage Change in TC from Baseline over Time
FIG. 7 is Mean (± SD) Percentage Change in VLDL-C from Baseline over Time
FIG. 8 is the chemical structure of AD00112-2 in sodium salt form.
Detailed Description
The present invention provides methods for inhibiting expression of an ANGPTL3 gene in a subject and methods for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of an ANGPTL3 gene, e.g., a disorder mediated by ANGPTL3 expression, such as a hyperlipidemia, e.g., hypercholesterolemia, using RNAi agent compositions which effect the RNA-induced silencing complex (RISC) -mediated cleavage of RNA transcripts of an ANGPTL3 gene.
The following describes how to make and use compositions comprising ANGPTL3 single-stranded (ssRNA) and dsRNA agents to inhibit ANGPTL3 gene expression, as well as compositions and methods for treating diseases and conditions caused by or modulated by ANGPTL3 gene expression.
As used herein, "angiopoietin-like 3" used interchangeably with the term "ANGPTL3" refers to the naturally occurring gene that encodes an angiopoietin-like 3 from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless specified otherwise. The term also refers to fragments and variants of native ANGPTL3 that maintain at least one in vivo or in vitro activity of a native ANGPTL3. The amino acid and complete coding sequences of the reference sequence of the human ANGPTL3 gene may be found in, for example, GenBank Ref Seq Accession No. NM_013913.4 (SEQ ID NO: 1 and SEQ ID NO: 2) . Mammalian orthologs of the human ANGPTL3 gene may be found in, for example, GenBank Ref Seq Accession No. XM_005543185.2, Cynomolgus monkey (SEQ ID NO: 3 and SEQ ID NO: 4) . Additional examples of ANGPTL3 mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, Ensembl and OMIM. It is understood by a skilled artisan that a substitution of a T with a U in any target sequences or mRNA transcripts (e.g., SEQ ID NO: 1 to SEQ ID NO: 4) does not count as a difference.
As used herein, "G, " "C, " "A" and "U" each generally stands for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person understands that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.
As used herein, the term “RNAi” , which is known in the art, and may be referred to as “siRNA agent” , "double stranded RNAi agent, " "double-stranded RNA (dsRNA) molecule, " "dsRNA agent, " or "dsRNA" refers to an agent that comprises RNA and mediates targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. As is known in the art, an RNAi target region refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene, including messenger RNA (mRNA) that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion. A target sequence may be from 8-30 nucleotides long (inclusive) , from 10-30 nucleotides long (inclusive) , from 12-25 nucleotides long (inclusive) , from 15-23 nucleotides long (inclusive) , from 16 -23 nucleotides long (inclusive) , or from 18-23 nucleotides long (inclusive) , including all shorter lengths within each stated range. In some embodiments of the invention, a target sequence is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides long. In certain embodiment a target sequence is between 9 and 26 nucleotides long (inclusive) , including all sub-ranges and integers there between. For example, though not intended to be limiting, in certain embodiments of the invention a target sequence is 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 long, with the sequence fully or at least substantially complementary to at least part of an RNA transcript of an ANGPTL3 gene. Some aspects of the invention include pharmaceutical compositions comprising one or more ANGPTL3 dsRNA agents and a pharmaceutically acceptable carrier. In certain embodiments of the invention, an ANGPTL3 RNAi as described herein inhibits expression of ANGPTL3 protein.
As used herein, a “dsRNA agent” may mean a composition that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner. Although not wishing to be limited to a particular theory, dsRNA agents of the invention may operate through the RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) of mammalian cells) , or by any alternative mechanism (s) or pathway (s) . Methods for silencing genes in plant, invertebrate, and vertebrate cells are well known in the art [see, for example, (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 disclosure of each of which is incorporated herein by reference in its entirety. ] . Art-known gene silencing procedures can be used in conjunction with the disclosure provided herein to inhibit expression of ANGPTL3.
DsRNA agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: short interfering RNAs (siRNA agents) , RNAi agents, micro RNAs (mRNAi agents) , short hairpin RNAs (shRNA) , and dicer substrates. The antisense strand of the dsRNA agents described herein is at least partially complementary to the mRNA being targeted. It is understood in the art that different lengths of dsRNA duplex structure can be used to inhibit target gene expression. For example, dsRNAs having a duplex structure of 19, 20, 21, 22, and 23 base pairs are known to be effective to induce RNA interference (Elbashir et al., EMBO 2001, 20: 6877-6888) . It is also known in the art that shorter or longer RNA duplex structures are also effective to induce RNA interference. In some embodiments, the sense strand and the antisense strand may be the same length or different lengths. In some embodiments, each strand is no more than 40 nucleotides in length. In some embodiments, each strand is no more than 30 nucleotides in length. In some embodiments, each strand is no more than 25 nucleotides in length. In some embodiments, each strand is no more than 23 nucleotides in length. In some embodiments, each strand is no more than 21 nucleotides in length. In some embodiments, the sense and antisense strands of the RNAi agents can each be 15 to 49 nucleotides in length. In some embodiments, the antisense strand is independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the length of the sense strand is independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides. As used herein, the terms “double stranded region” , “duplex region” and “the region of complementarity” can be used interchangeably, and refer to the region that the sense strand is complementary or substantially complementary to the antisense strand as is known in the art. In some embodiments, the sense strand and the antisense strand are both 21 nucleotides in length. In some embodiments, the sense strand is complementary or substantially complementary to the antisense strand, and the region of complementarity is between 15 and 23 nucleotides in length. In some embodiments, the region of complementarity is 19-21 nucleotides in length. In some embodiments, the region of complementarity is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
As used herein the term “independently selected” means each of two or more like elements can be selected independent of the selection of the other elements. As described herein, it will be understood that the sense and antisense strands in a duplex of the invention may be independently selected.
Methods of the Invention
The present invention provides methods of inhibiting the expression of an angiopoietin like 3 (ANGPTL3) gene in a subject. The present invention also provides therapeutic and prophylactic methods for treating or preventing diseases and conditions that can be modulated by down regulating ANGPTL3 gene expression. For example, the compositions described herein can be used to treat dyslipidemia, e.g., a hyperlipidemia and other forms of lipid imbalance such as hypercholesterolemia, hypertriglyceridemia and the pathological conditions associated with these disorders such as heart and circulatory diseases. Other diseases and conditions that can be modulated by down regulating ANGPTL3 gene expression include lysosomal storage diseases including, but not limited to, severe hypertriglyceridemia (SHTG) , familial chylomicronemia syndrome, mixed dyslipidemia, hypercholesterolemia, homozygous familial hypercholesterolemia (HoFH) , heterozygous familial hypercholesterolemia (HeFH) , abnormal lipid and/or cholesterol metabolism, statin resistant hypercholesterolemia, cardiometabolic disease, obesity, atherosclerosis, type II diabetes mellitus, cardiovascular disease, coronary artery disease, cerebrovascular disease, peripheral artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis caused by hypertriglyceridemia. The methods include administering to the subject a therapeutically effective amount or prophylactically effective amount of an RNAi agent of the invention.
As ANGPTL3 is a key regulator for human plasma lipid, the effect of the decreased expression of an ANGPTL3 gene preferably results in a decrease in TG (triglyceride) , LDL-C (low-density lipoprotein-cholesterol) , non-HDL-C (non-high-density lipoprotein-cholesterol) levels, ApoB (apolipoprotein B) , TC (total cholesterol) and VLDL-C (very low-density lipoprotein cholesterol) in the blood, and more particularly in the serum of the mammal. In some embodiments, TG levels are decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%or more, as compared to pretreatment levels. In some embodiments, LDL-C levels are decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%or more, as compared to pretreatment levels. In some embodiments, non-HDL-C levels are decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%or more, as compared to pretreatment levels. In some embodiments, ApoB levels are decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%or more, as compared to pretreatment levels. In some embodiments, TC levels are decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%or more, as compared to pretreatment levels. In some embodiments, VLDL-C levels are decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%or more, as compared to pretreatment levels. Accordingly, the present invention also provides methods for lowering a subject’s plasma lipid level, a subject’s TG level, a subject’s LDL level, a subject’s VLDL level, a subject’s non-HDL level, a subject’s ApoB level, a subject’s TC level, a subject’s LDL: HDL ratio, etc.
In some embodiments of the invention, ANGPTL3 gene expression is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%or more by administration of an ANGPTL3 dsRNA agent of the invention, as compared to pretreatment levels. In some embodiments, ANGPTL3 gene expression is reduced 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%by administration of an ANGPTL3 dsRNA agent of the invention. In some embodiments of the invention, ANGPTL3 gene expression is reduced by at between 5%and 10%, 5%and 25%, 10%and 50%, 10%and 75%, 25%and 75%, 25%and 100%, or 50%and 100%by administration of an ANGPTL3 dsRNA agent of the invention.
In some embodiments of the invention, the double-stranded RNAi agent is administered to a subject as a fixed dose. A “fixed dose” (e.g., a dose in mg) means that one dose of an RNAi agent agent is used for all subjects regardless of any specific subject-related factors, such as weight. In other embodiments, an RNAi agent of the invention is administered to a subject as a weight-based dose. A “weight-based dose” (e.g., a dose in mg/kg) is a dose of the RNAi agent agent that will change depending on the subject's weight.
In some embodiments of the invention, the RNAi agent is administered to the subject as a fixed dose of about 50 mg to about 900 mg, about 100 mg to about 900 mg, about 150 mg to about 900 mg, about 200 mg to about 900 mg, about 250 mg to about 900 mg, about 300 mg to about 900 mg, about 350 mg to about 900 mg, about 400 mg to about 900 mg, about 450 mg to about900 mg, about 500 mg to about 900 mg, about 550 mg to about 900 mg, about 600 to about 900 mg, about 650 to about 900 mg, about 100 mg to about 650 mg, about 150 mg to about 650 mg, about 200 mg to about 650 mg, about 250 mg to about 650 mg, about 300 mg to about 650 mg, about 350 mg to about 650 mg, about 400 mg to about 650 mg, about 450 mg to about 650 mg, about 500 mg to about 650 mg, about 550 mg to about 650 mg, about 600 mg to about 650 mg, about 50 mg to about 600 mg, about 100 mg to about 600 mg, about 150 mg to about 600 mg, about 200 mg to about 600 mg, about 250 mg to about 600 mg, about 300 mg to about 600 mg, about 350 mg to about 600 mg, about 400 mg to about 600 mg, about 450 mg to about 600 mg, about 500 mg to about 600 mg, about 550 mg to about 600 mg, about 100 mg to about 550 mg, about 150 mg to about 550 mg, about 200 mg to about 550 mg, about 250 mg to about 550 mg, about 300 mg to about 550 mg, about 350 mg to about 550 mg, about 400 mg to about 550 mg, about 450 mg to about 550 mg, about 500 mg to about 550 mg, about 100 mg to about 500 mg, about 150 mg to about 500 mg, about 200 mg to about 500 mg, about 250 mg to about 500 mg, about 300 mg to about 500 mg, about 350 mg to about 500 mg, about 400 mg to about 500 mg, or about 450 mg to about 500 mg, e.g., a fixed dose of about 100 mg, about 125 mg, about 150 mg, about 175 mg, 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, or about 700 mg. Values and ranges intermediate to the foregoing recited values are also intended to be part of this invention.
The administration may be repeated, for example, on a regular basis. For example, the fixed dose may be administered to the subject at an interval of once a week, once every two weeks, once a month, once a quarter, or biannually for six months or a year or longer, i.e., chronic administration.
In some embodiments, the subject is administered a fixed dose of about 50 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 150 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 200 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 300 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 600 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 900 mg once a quarter. In some embodiment, the subject is administered a fixed dose of about 50 mg biannually (i.e., twice a year) . In some embodiment, the subject is administered a fixed dose of about 150 mg biannually. In some embodiment, the subject is administered a fixed dose of about 200 mg biannually. In some embodiment, the subject is administered a fixed dose of about 300 mg biannually. In some embodiment, the subject is administered a fixed dose of about 600 mg biannually. In some embodiment, the subject is administered a fixed dose of about 900 mg biannually. In some embodiment, the subject is administered a fixed dose of about 50 mg every nine months. In some embodiment, the subject is administered a fixed dose of about 150 mg every nine months. In some embodiment, the subject is administered a fixed dose of about 200 mg every nine months. In some embodiment, the subject is administered a fixed dose of about 300 mg every nine months. In some embodiment, the subject is administered a fixed dose of about 600 mg every nine months. In some embodiment, the subject is administered a fixed dose of about 900 mg every nine months.
In some embodiments, the present invention provides methods of inhibiting the expression of an ANGPTL3 gene in a subject. The methods include administering to the subject a double-stranded ribonucleic acid (RNAi) agent, e.g., a dsRNA, of the invention (e.g., a pharmaceutical composition comprising a dsRNA of the invention) , wherein a total of about 50 mg to about 900 mg of the double-stranded RNAi agent is administered to the subject every quarter (every 3 month) , biannually (twice a year) or every nine months, and wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, the antisense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 0, 1, 2, 3 nucleotides from the complement of nucleotides 1350-1418 of the nucleotide sequence of SEQ ID NO: 1.
In some embodiments, the present invention provides methods of decreasing the level of TG (triglyceride) , LDL-C (low-density lipoprotein-cholesterol) , ApoB (apolipoprotein B) , TC (total cholesterol) , VLDL-C (very low-density lipoprotein cholesterol) and non-HDL-C (non-high-density lipoprotein-cholesterol) in a subject. The methods include administering to the subject a double-stranded ribonucleic acid (RNAi) agent, wherein a total of about 50 mg to about 900 mg of the double-stranded RNAi agent is administered to the subject every quarter, biannually or every nine months, and wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, the antisense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 0, 1, 2, 3 nucleotides from the complement of nucleotides 1350-1418 of the nucleotide sequence of SEQ ID NO: 1.
In some embodiments, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in ANGPTL3 expression, such as a hyperlipidemia, e.g., hypertriglyceridemia. The methods include administering to the subject a double-stranded ribonucleic acid (RNAi) agent, e.g., a dsRNA, of the invention (e.g., a pharmaceutical composition comprising a dsRNA of the invention) , wherein a total of about 50 mg to about 900 mg of the double-stranded RNAi agent is administered to the subject every quarter, biannually or every nine months , and wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, the antisense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 0, 1, 2, 3 nucleotides from the complement of nucleotides 1350-1418 of the nucleotide sequence of SEQ ID NO: 1.
As indicated above, the administration of the RNAi agents to a subject may be repeated on a regular basis, for example, at an interval of once a week, once every two weeks, once a month, once a quarter, biannually or every nine months.
Accordingly, in some embodiments, the RNAi agent is administered in a dosing regimen that includes a “loading phase” of closely spaced administrations that may be followed by a “maintenance phase” , in which the RNAi agent is administered at longer spaced intervals. For example, after administration weekly or biweekly for one month, administration can be repeated once per month, for six months or a year or longer, i.e., chronic administration.
In one embodiment, the loading phase comprises a single administration of the RNAi agent during the first quarter. In another embodiment, the loading phase comprises a single administration of the RNAi agent during the first month.
Any of these schedules may optionally be repeated for one or more iterations. The number of iterations may depend on the achievement of a desired effect, e.g., the suppression of an ANGPTL3 gene, and/or the achievement of a therapeutic or prophylactic effect, e.g., reducing plasma lipid levels or reducing a symptom of hyperlipidemia. Following treatment, the patient can be monitored for changes in his/her condition. The dosage of the RNAi agent may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
In some embodiments, the present invention provides methods of inhibiting the expression of an ANGPTL3 gene in a subject. The methods include administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen that includes a loading phase followed by a maintenance phase, wherein the loading phase comprises administering a fixed dose of about 50 mg to about 900 mg of the RNAi agent to the subject, and wherein the maintenance phase comprises administering a fixed dose of about 50 mg to about 900 mg of the RNAi agent to the subject about every six months or every nine months, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, the antisense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 0, 1, 2, 3 nucleotides from the complement of nucleotides 1350-1418 of the nucleotide sequence of SEQ ID NO: 1, thereby inhibiting the expression of the ANGPTL3 gene in the subject.
In another aspect, the present invention provides methods of decreasing the level of TG (triglyceride) , LDL-C (low-density lipoprotein-cholesterol) , ApoB (apolipoprotein B) , TC (total cholesterol) , VLDL-C (very low-density lipoprotein cholesterol) and non-HDL-C (non-high-density lipoprotein-cholesterol) in a subject. The methods include administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen that includes a loading phase followed by a maintenance phase, wherein the loading phase comprises administering to the subject a fixed dose of about 50 mg to about 900 mg of the RNAi agent, and wherein the maintenance phase comprises administering to the subject a fixed dose of about 50 mg to about 900 mg of the RNAi agent every six months or every nine months, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, the antisense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 0, 1, 2, 3 nucleotides from the complement of nucleotides 1350-1418 of the nucleotide sequence of SEQ ID NO: 1.
In some embodiments, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in ANGPTL3 expression, such as a hyperlipidemia, e.g., hypertriglyceridemia. The methods include administering to the subject a double-stranded ribonucleic acid (RNAi) agent in a dosing regimen that includes a loading phase followed by a maintenance phase, wherein the loading phase comprises administering to the subject a fixed dose of about 50 mg to about 900 mg of the RNAi agent, and wherein the maintenance phase comprises administering to the subject a fixed dose of about 50 mg to about 900 mg of the RNAi agent every six months or every nine month, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, the antisense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 0, 1, 2, 3 nucleotides from the complement of nucleotides 1350-1418 of the nucleotide sequence of SEQ ID NO: 1, thereby treating the subject having a disorder that would benefit from reduction in ANGPTL3 expression.
In some embodiments, the double-stranded RNAi agent targets nucleotides 1358-1378 of SEQ ID NO: 1.
In some embodiments, the double-stranded ribonucleic acid (RNAi) agent for use in the methods of the present invention comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence 5′-UUAGAGUAUAACCUUCCAUUC-3′ (SEQ ID NO: 40) and the sense strand comprises the nucleotide sequence 5′-GAAUGGAAGGUUAUACUCUAA -3′ (SEQ ID NO: 5) , wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides.
As used herein, a “subject” includes a human or non-human animal, preferably a vertebrate, and more preferably a mammal. A subject may include a transgenic organism. Most preferably, the subject is a human, such as a human suffering from or predisposed to developing an ANGPTL3-associated disease.
The methods and uses of the invention include administering a composition described herein such that expression of the target ANGPTL3 gene is decreased, for an extended period of time, such as, for about 80 days, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, about 180 days, about nine months or one year, or longer.
Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of ANGPTL3 may be determined by determining the mRNA expression level of ANGPTL3 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR, by determining the protein level of ANGPTL3 using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques, and/or by determining a biological activity of ANGPTL3, such as the effect on one or more plasma lipid parameters, such as, for example, total cholesterol levels, high density lipoprotein cholesterol (HDL) levels, non-HDL levels, low density lipoprotein cholesterol (LDL) levels, very low density lipoprotein cholesterol (VLDL) levels, ApoB (apolipoprotein B) , triglyceride levels, and lipoprotein particle size, etc.
Administration of the dsRNA according to the methods and uses of the invention may result in a reduction of the severity, signs, symptoms, and/or markers of such diseases or disorders in a patient with a disorder that would benefit from reduction in ANGPTL3 expression. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.
Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, plasma lipid levels (e.g., LDL-C levels) , quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of a hyperlipidemia may be assessed, for example, by periodic monitoring of LDL-C levels. Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters.
A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10%in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50%or more can be indicative of effective treatment. Efficacy for a given RNAi agent drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.
Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale. Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using an RNAi agent or RNAi agent formulation as described herein.
In some embodiment, administration can be provided when Low Density Lipoprotein cholesterol (LDL-C) levels reach or surpass a predetermined minimal level, such as greater than 70 mg/dL, 100 mg/dL, 130 mg/dL, 160 mg/dL, 190 mg/dL, 300 mg/dL, 400 mg/dL, 500 mg/dL, or 1000 mg/dL.
In some embodiment, administration can be provided when triglyceride (TG) levels reach or surpass a predetermined minimal level, such as greater than 150 mg/dL, 200 mg/dL, 500 mg/dL or 880mg/dL.
In some embodiments of the methods of the invention, ANGPTL3 expression is decreased for an extended duration, e.g., at least one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four month, six months or longer. For example, in certain instances, expression of the ANGPTL3 gene is suppressed by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%by administration of an RNAi agent agent described herein. In some embodiments, the ANGPTL3 gene is suppressed by at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%by administration of the RNAi agent agent.
The RNAi agents of the invention may be administered to a subject using any mode of administration known in the art, including, but not limited to subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combinations thereof. In preferred embodiments, the agents are administered subcutaneously.
In some embodiments, the administration is via a depot injection. A depot injection may release the RNAi agent in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of ANGPTL3, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.
In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi agent to the liver.
Other modes of administration include epidural, intracerebral, intracerebroventricular, nasal administration, intraarterial, intracardiac, intraosseous infusion, intrathecal, and intravitreal, and pulmonary. The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.
The RNAi agent can be administered by intravenous infusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about a 25 minute period. The administration may be repeated, for example, on a regular basis, such as weekly, biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration weekly or biweekly for three months, administration can be repeated once per month, for six months or a year or longer.
Administration of the RNAi agent can reduce ANGPTL3 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%or more.
Before administration of a full dose of the RNAi agent, patients can be administered a smaller dose, such as a 5%infusion, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.
Owing to the inhibitory effects on ANGPTL3 expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life. An RNAi agent of the invention may be administered in “naked” form, or as a “free RNAi agent. ” A naked RNAi agent is administered in the absence of a pharmaceutical composition. The naked RNAi agent may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS) . The pH and osmolarity of the buffer solution containing the RNAi agent can be adjusted such that it is suitable for administering to a subject.
Alternatively, an RNAi agent of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.
The invention further provides methods and uses for the use of an RNAi agent or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction and/or inhibition of ANGPTL3 expression, e.g., a subject having hyperlipidemia, e.g., hypercholesterolemia, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. The siRNA agent and an additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.
Examples of additional therapeutic agents include those known to treat a lipid disorders, such as hypercholesterolemia, atherosclerosis or dyslipidemia. For example, a siRNA agent featured in the invention can be administered with, e.g., an HMG-CoA reductase inhibitor (e.g., a statin) , a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartan potassium, such as Merck &Co. 's ) , an acylCoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol modulator, a bile acid modulator, a peroxisome proliferation activated receptor (PPAR) agonist, a gene-based therapy, a composite vascular protectant (e.g., AGI-1067, from Atherogenics) , a glycoprotein IIb/IIIa inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor (e.g., S-8921, from Shionogi) , a squalene synthase inhibitor, or a monocyte chemoattractant protein (MCP) -I inhibitor. Exemplary HMG-CoA reductase inhibitors include atorvastatin (Pfizer's ) , pravastatin (Bristol-Myers Squibb's Pravachol, Sankyo's Mevalotin/Sanaprav) , simvastatin (Merck's Boehringer Ingelheim's Denan, Banyu's Lipovas) , lovastatin (Merck's Mevacor/Mevinacor, Bexal's Lovastatina, Cepa; Schwarz Pharma's Liposcler) , fluvastatin (Novartis'Fujisawa's Cranoc, Solvay's Digaril) , cerivastatin (Bayer's Lipobay/GlaxoSmithKline's Baycol) , rosuvastatin (AstraZeneca's ) , and pitivastatin (itavastatin/risivastatin) (Nissan Chemical, Kowa Kogyo, Sankyo, and Novartis) . Exemplary fibrates include, e.g., bezafibrate (e.g., Roche's Kissei's Bezatol) , clofibrate (e.g., Wyeth's ) , fenofibrate (e.g., Fournier's Lipidil/Lipantil, Abbott's Takeda's Lipantil, generics) , gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate (Sanofi-Synthelabo's ) . Exemplary bile acid sequestrants include, e.g., cholestyramine (Bristol-Myers Squibb's and Questran LightTM) , colestipol (e.g., Pharmacia's Colestid) , and colesevelam (Genzyme/Sankyo's WelCholTM) . Exemplary niacin therapies include, e.g., immediate release formulations, such as Aventis'Nicobid, Upsher-Smith's Niacor, Aventis'Nicolar, and Sanwakagaku's Perycit. Niacin extended release formulations include, e.g., Kos Pharmaceuticals'Niaspan and Upsher-Smith's SIo-Niacin. Exemplary antiplatelet agents include, e.g., aspirin (e.g., Bayer's aspirin) , clopidogrel (Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix) , and ticlopidine (e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine) . Other aspirin-like compounds useful in combination with a dsRNA targeting ANGPTL3 include, e.g., Asacard (slow-release aspirin, by Pharmacia) and Pamicogrel (Kanebo/Angelini Ricerche/CEPA) . Exemplary angiotensin-converting enzyme inhibitors include, e.g., ramipril (e.g., Aventis'A ltace) and enalapril (e.g., Merck &Co. 's Vasotec) . Exemplary acyl CoA cholesterol acetyltransferase (AC AT) inhibitors include, e.g., avasimibe (Pfizer) , eflucimibe (BioMsrieux Pierre Fabre/Eli Lilly) , CS-505 (Sankyo and Kyoto) , and SMP-797 (Sumito) . Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe (Merck/Schering-Plough Pharmaceuticals) and Pamaqueside (Pfizer) . Exemplary CETP inhibitors include, e.g., Torcetrapib (also called CP-529414, Pfizer) , JTT-705 (Japan Tobacco) , and CETi-I (Avant Immunotherapeutics) . Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, e.g., implitapide (Bayer) , R-103757 (Janssen) , and CP-346086 (Pfizer) . Other exemplary cholesterol modulators include, e.g., NO-1886 (Otsuka/TAP Pharmaceutical) , CI-1027 (Pfizer) , and WAY-135433 (Wyeth-Ayerst) .
Exemplary bile acid modulators include, e.g., HBS-107 (Hisamitsu/Banyu) , Btg-511 (British Technology Group) , BARI-1453 (Aventis) , S-8921 (Shionogi) , SD-5613 (Pfizer) , and AZD-7806 (AstraZeneca) . Exemplary peroxisome proliferation activated receptor (PPAR) agonists include, e.g., tesaglitazar (AZ-242) (AstraZeneca) , Netoglitazone (MCC-555) (Mitsubishi/Johnson &Johnson) , GW-409544 (Ligand Pharniaceuticals/GlaxoSmithKline) , GW-501516 (Ligand Pharmaceuticals/GlaxoSmithKline) , LY-929 (Ligand Pharmaceuticals and Eli Lilly) , LY-465608 (Ligand Pharmaceuticals and Eli Lilly) , LY-518674 (Ligand Pharmaceuticals and Eli Lilly) , and MK-767 (Merck and Kyorin) . Exemplary gene-based therapies include, e.g., AdGWEGF 121.10 (GenVec) , ApoAl (UCB Pharma/Groupe Fournier) , EG-004 (Trinam) (Ark Therapeutics) , and ATP-binding cassette transporter-A1 (ABCA1) (CV Therapeutics/Incyte, Aventis, Xenon) . Exemplary Glycoprotein IIb/IIIa inhibitors include, e.g., roxifiban (also called DMP754, Bristol-Myers Squibb) , Gantofiban (Merck KGaA/Yamanouchi) , and Cromafiban (Millennium Pharmaceuticals) . Exemplary squalene synthase inhibitors include, e.g., BMS-1884941 (Bristol-Myers Squibb) , CP-210172 (Pfizer) , CP-295697 (Pfizer) , CP-294838 (Pfizer) , and TAK-475 (Takeda) . An exemplary MCP-I inhibitor is, e.g., RS-504393 (Roche Bioscience) . The anti-atherosclerotic agent BO-653 (Chugai Pharmaceuticals) , and the nicotinic acid derivative Nyclin (Yamanouchi Pharmaceuticals) are also appropriate for administering in combination with a dsRNA featured in the invention. Exemplary combination therapies suitable for administration with a dsRNA targeting ANGPTL3 include, e.g., advicor (Niacin/lovastatin from Kos Pharmaceuticals) , amlodipine/atorvastatin (Pfizer) , and ezetimibe/simvastatin (e.g., 10/10, 10/20, 10/40, and 10/80 tablets by Merck/Schering-Plough Pharmaceuticals) . Agents for treating hypercholesterolemia, and suitable for administration in combination with a dsRNA targeting ANGPTL3 include, e.g., lovastatin, niacinExtended-Release Tablets (Andrx Labs) , lovastatinTablets (Pfizer) , amlodipine besylate, atorvastatin calciumTablets (AstraZeneca) , rosuvastatin calciumCapsules (Novartis) , fluvastatin sodium (Reliant, Novartis) , fluvastatin sodiumTablets (Parke-Davis) , atorvastatin calciumCapsules (Gate) , Niaspan Extended-Release Tablets (Kos) , niacin Pravachol Tablets (Bristol-Myers Squibb) , pravastatin sodiumTablets (Abbott) , fenofibrate10/10 Tablets (Merck/Schering-Plough Pharmaceuticals) , ezetimibe, simvastatin WelCholTM Tablets (Sankyo) , colesevelam hydrochlorideTablets (Schering) , ezetimibeTablets (Merck/Schering-Plough Pharmaceuticals) , and ezetimibeTablets (Merck) .
In some embodiments, an RNAi agent agent is administered in combination with an ezetimibe/simvastatin combination (e.g.,  (Merck/Schering-Plough Pharmaceuticals) ) .
In some embodiments, an RNAi agent agent is administered in combination with an anti-ANGPTL3 antibody. Exemplary anti-ANGPTL3 antibodies for use in the combination therapies of the invention include, for example, alirocumab (Praluent) , evolocumab (Repatha) , bococizumab (PF-04950615, RN316, RN-316, L1L3; Pfizer, Rinat) , lodelcizumab (LFU720, pJG04; Novartis) , ralpancizumab (RN317, PF-05335810; Pfizer, Rinat) , RG7652 (MPSK3169A, YW508.20.33b; Genentech) , LY3015014 (Lilly) , LPD1462 (h1F11; Schering-Plough) , AX1 (AX189, 1B20, 1D05; Merck &Co) , ALD306 (Alder) ; mAb1 (Boehringer) , and Ig1-PA4 (Nanjing Normal U. ) .
In one embodiment, the RNAi agent agent is administered to the patient, and then the additional therapeutic agent is administered to the patient (or vice versa) . In another embodiment, the RNAi agent and the additional therapeutic agent are administered at the same time.
In another aspect, the invention features, a method of instructing an end user, e.g., a caregiver or a subject, on how to administer an RNAi agent described herein. The method includes, optionally, providing the end user with one or more doses of the RNAi agent, and instructing the end user to administer the RNAi agent on a regimen described herein, thereby instructing the end user.
A healthcare provider, such as a doctor, nurse, or family member, can take a family history before prescribing or administering an RNAi agent of the invention. In addition, a test may be performed to determine a geneotype or phenotype. For example, a DNA test may be performed on a sample from the patient, e.g., a blood sample, to identify the ANGPTL3 genotype and/or phenotype before an ANGPTL3 dsRNA is administered to the patient.
The present invention further provides methods of inhibiting expression of an angiopoietin like 3 (ANGPTL3) in a cell, such as a cell within a subject, e.g., a human subject.
Accordingly, the present invention provides methods of inhibiting expression of an ANGPTL3 gene in a cell. The methods include contacting a cell with an RNAi agent, e.g., a double stranded RNAi agent, in an amount effective to inhibit expression of the ANGPTL3 gene in the cell, thereby inhibiting expression of the ANGPTL3 in the cell.
Contacting of a cell with a double stranded RNAi agent may be done in vitro or in vivo. Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting are also possible. Contacting may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc ligand, or any other ligand that directs the RNAi agent to a site of interest, e.g., the liver of a subject.
The term “inhibiting, ” as used herein, is used interchangeably with “reducing, ” “silencing, ” “downregulating” and other similar terms, and includes any level of inhibition.
The phrase “inhibiting expression of an ANGPTL3” is intended to refer to inhibition of expression of any ANGPTL3 gene (such as, e.g., a mouse ANGPTL3 gene, a rat ANGPTL3 gene, a monkey ANGPTL3 gene, or a human ANGPTL3 gene) as well as variants or mutants of an ANGPTL3 gene. Thus, the ANGPTL3 gene may be a wild-type ANGPTL3 gene, a mutant ANGPTL3 gene, or a transgenic ANGPTL3 gene in the context of a genetically manipulated cell, group of cells, or organism.
“Inhibiting expression of an ANGPTL3 gene” includes any level of inhibition of an ANGPTL3 gene, e.g., at least partial suppression of the expression of an ANGPTL3 gene. The expression of the ANGPTL3 gene may be assessed based on the level, or the change in the level, of any variable associated with ANGPTL3 gene expression, e.g., ANGPTL3 mRNA level, ANGPTL3 protein level, or lipid levels. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.
Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with ANGPTL3 expression compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control) .
Inhibition of the expression of an ANGPTL3 gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which an ANGPTL3 gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with an RNAi agent of the invention, or by administering an RNAi agent of the invention to a subject in which the cells are or were present) such that the expression of an ANGPTL3 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell (s) ) .
Alternatively, inhibition of the expression of an ANGPTL3 gene may be assessed in terms of a reduction of a parameter that is functionally linked to ANGPTL3 gene expression, e.g., ANGPTL3 protein expression, such as lipid levels, cholesterol levels, e.g., LDL-C levels. ANGPTL3 gene silencing may be determined in any cell expressing ANGPTL3, either constitutively or by genomic engineering, and by any assay known in the art. The liver is the major site of ANGPTL3 expression. Other significant sites of expression include the pancreas, kidney, and intestines.
Inhibition of the expression of an ANGPTL3 protein may be manifested by a reduction in the level of the ANGPTL3 protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject) . As explained above for the assessment of mRNA suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.
A control cell or group of cells that may be used to assess the inhibition of the expression of an ANGPTL3 gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the invention. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent.
The level of ANGPTL3 mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of ANGPTL3 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the ANGPTL3 gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis) , RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland) . Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035) , Northern blotting, in situ hybridization, and microarray analysis.
In one embodiment, the level of expression of ANGPTL3 is determined using a nucleic acid probe. The term “probe” , as used herein, refers to any molecule that is capable of selectively binding to a specific ANGPTL3. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.
Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to ANGPTL3 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe (s) are immobilized on a solid surface and the mRNA is contacted with the probe (s) , for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of ANGPTL3 mRNA.
An alternative method for determining the level of expression of ANGPTL3 in a sample involves the process of nucleic acid amplification and/or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202) , ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189-193) , self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878) , transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173-1177) , Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6: 1197) , rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, the level of expression of ANGPTL3 is determined by quantitative fluorogenic RT-PCR (i.e., the TaqManTM System) .
The expression levels of ANGPTL3 mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like) , or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids) . See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5, 445, 934, which are incorporated herein by reference. The determination of ANGPTL3 expression level may also comprise using nucleic acid probes in solution.
In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR) . The use of these methods is described and exemplified in the Examples presented herein.
The level of ANGPTL3 protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC) , thin layer chromatography (TLC) , hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double) , immunoelectrophoresis, Western blotting, radioimmunoassay (RIA) , enzyme-linked immunosorbent assays (ELISAs) , immunofluorescent assays, electrochemiluminescence assays, and the like.
The term “sample” as used herein refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, lymph, urine, cerebrospinal fluid, saliva, ocular fluids, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes) . In preferred embodiments, a “sample derived from a subject” refers to blood or plasma drawn from the subject. In further embodiments, a “sample derived from a subject” refers to liver tissue derived from the subject.
In some embodiments of the methods of the invention, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. The inhibition of expression of ANGPTL3 may be assessed using measurements of the level or change in the level of ANGPTL3 mRNA or ANGPTL3 protein in a sample derived from fluid or tissue from the specific site within the subject. In preferred embodiments, the site is the liver. The site may also be a subsection or subgroup of cells from any one of the aforementioned sites. The site may also include cells that express a particular type of receptor.
Mismatches
It is known to skilled in art, mismatches are tolerated for efficacy in dsRNA, especially the mismatches are within terminal region of dsRNA. Certain mismatches tolerate better, for example mismatches with wobble base pairs G: U and A: C are tolerated better for efficacy (Du et el., A systematic analysis of the silencing effects of an active siRNA agent at all single-nucleotide mismatched target sites. Nucleic Acids Res. 2005 Mar 21; 33 (5) : 1671-7. Doi: 10.1093/nar/gki312. Nucleic Acids Res. 2005; 33 (11) : 3698) . Some embodiments of methods and compounds of the invention an ANGPTL3 dsRNA agent may contain one or more mismatches to the ANGPTL3 target sequence. In some embodiments, ANGPTL3 dsRNA agent of the invention includes no mismatches. In certain embodiments, ANGPTL3 dsRNA agent of the invention includes no more than 1 mismatch. In some embodiments, ANGPTL3 dsRNA agent of the invention includes no more than 2 mismatches. In certain embodiments, ANGPTL3 dsRNA agent of the invention includes no more than 3 mismatches. In some embodiments of the invention, an antisense strand of an ANGPTL3 dsRNA agent contains mismatches to an ANGPTL3 target sequence that are not located in the center of the region of complementarity. In some embodiments, the antisense strand of the ANGPTL3 dsRNA agent includes 1, 2, 3, 4, or more mismatches that are within the last 5, 4, 3, 2, or 1 nucleotide from one or both of the 5' or 3' end of the region of complementarity. Methods described herein and/or methods known in the art can be used to determine whether an ANGPTL3 dsRNA agent containing a mismatch to an ANGPTL3 target sequence is effective in inhibiting the expression of the ANGPTL3 gene.
Complementarity
As used herein, unless otherwise indicated, the term “complementary” when used to describe a first nucleotide sequence (e.g., ANGPTL3 dsRNA agent sense strand or targeted ANGPTL3 mRNA) in relation to a second nucleotide sequence (e.g., ANGPTL3 dsRNA agent antisense strand or a single-stranded antisense polynucleotide) , means the ability of an oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize [form base pair hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro) ] and form a duplex or double helical structure under certain conditions with an oligonucleotide or polynucleotide including the second nucleotide sequence. Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. A skilled artisan will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, at least to the extent that the above hybridization requirements are fulfilled. Sequence identity or complementarity is independent of modification.
Complementary sequences, for example, within an ANGPTL3 dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. It will be understood that in embodiments when two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs are not regarded herein as mismatches with regard to the determination of complementarity. For example, an ANGPTL3 dsRNA agent comprising one oligonucleotide 19 nucleotides in length and another oligonucleotide 20 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 19 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein. Thus, as used herein, “fully complementary” means that all (100%) of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.
The term “substantially complementary” as used herein means that in a hybridized pair of nucleobase sequences, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not all, of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The term “substantially complementary” can be used in reference to a first sequence with respect to a second sequence if the two sequences include one or more, for example at least 1, 2, 3, 4, or 5 mismatched base pairs upon hybridization for a duplex up to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs (bp) , while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of ANGPTL3 gene expression via a RISC pathway.
The term, “partially complementary” may be used herein in reference to a hybridized pair of nucleobase sequences, in which at least 75%, but not all, of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. In some embodiments, “partially complementary” means 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 will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide.
The terms “complementary, ” “fully complementary, ” “substantially complementary, ” and “partially complimentary” are used herein in reference to the base matching between the sense strand and the antisense strand of an ANGPTL3 dsRNA agent, between the antisense strand of an ANGPTL3 dsRNA agent and a sequence of a target ANGPTL3 mRNA, or between a single-stranded antisense oligonucleotide and a sequence of a target ANGPTL3 mRNA. It will be understood that the term “antisense strand of an ANGPTL3 dsRNA agent” may refer to the same sequence of an “ANGPTL3 antisense polynucleotide agent” .
As used herein, the term “substantially identical” or “substantial identity” used in reference to a nucleic acid sequence means a nucleic acid sequence comprising a sequence with at least about 85%sequence identity or more, 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%, compared to a reference sequence. 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 at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
As used herein, the term “strand comprising a sequence” means an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature. The term “double-stranded RNA” or “dsRNA, ” as used herein, refers to an RNAi that includes an RNA molecule or complex of molecules having a hybridized duplex region comprising two anti-parallel and substantially or fully complementary nucleic acid strands, which are referred to as having “sense” and “antisense” orientations with respect to a target ANGPTL3 RNA. The duplex region can be of any length that permits specific degradation of a desired target ANGPTL3 RNA through a RISC pathway but will typically range from 9 to 30 base pairs in length, e.g., 15-30 base pairs in length. Considering a duplex between 9 and 30 base pairs, the duplex can be any length in this range, 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, and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. ANGPTL3 dsRNA agents generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 base pairs in length. One strand of the duplex region of an ANGPTL3 dsDNA agent comprises a sequence that is substantially complementary to a region of a target ANGPTL3 RNA. The two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules. Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop” ) between the 3' -end of one strand and the 5'-end of the respective other strand forming the duplex structure. In some embodiments of the invention, a hairpin look comprises 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. Where the two substantially complementary strands of an ANGPTL3 dsRNA agent are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a “linker. ” The term “siRNA agent” is also used herein to refer to a dsRNA agent as described herein.
In some embodiments of the invention an ANGPTL3 dsRNA agent may include a sense and antisense sequence that have no-unpaired nucleotides or nucleotide analogs at one or both terminal ends of the dsRNA agent. An end with no unpaired nucleotides is referred to as a “blunt end” and as having no nucleotide overhang. If both ends of a dsRNA agent are blunt, the dsRNA is referred to as “blunt ended” . In some embodiments of the invention, a first end of a dsRNA agent is blunt, in some embodiments a second end of a dsRNA agent is blunt, and in certain embodiments of the invention, both ends of an ANGPTL3 dsRNA agent are blunt.
In some embodiments of dsRNA agents of the invention, the dsRNA does not have one or two blunt ends. In such instances there is at least one unpaired nucleotide at the end of a strand of a dsRNA agent. For example, when a 3' -end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least 1, 2, 3, 4, 5, 6, or more nucleotides. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. It will be understood that in some embodiments a nucleotide overhang is on a sense strand of a dsRNA agent, on an antisense strand of a dsRNA agent, or on both ends of a dsRNA agent and nucleotide (s) of an overhang can be present on the 5' end, 3' end or both ends of either an antisense or sense strand of a dsRNA. In certain embodiments of the invention, one or more of the nucleotides in an overhang is replaced with a nucleoside thiophosphate.
As used herein, the term “antisense strand” or “guide strand” refers to the strand of an ANGPTL3 dsRNA agent that includes a region that is substantially complementary to an ANGPTL3 target sequence. As used herein the term “sense strand, ” or “passenger strand” refers to the strand of an ANGPTL3 dsRNA agent that includes a region that is substantially complementary to a region of the antisense strand of the ANGPTL3 dsRNA agent.
Modifications
In some embodiments of the invention the RNA of an ANGPTL3 RNAi agent is chemically modified to enhance stability and/or one or more other beneficial characteristics. Nucleic acids in certain embodiments of the invention may be synthesized and/or modified by methods well established in the art, for example, those described in “Current protocols in Nucleic Acid Chemistry, " Beaucage, S. L. et al. (Eds. ) , John Wiley &Sons, Inc., New York, N. Y., USA, which is incorporated herein by reference. Modifications that can be present in certain embodiments of ANGPTL3 dsRNA agents of the invention include, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation, conjugation, inverted linkages, etc. ) 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc. ) , (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides) , or conjugated bases, (c) sugar modifications (e.g., at the 2'position or 4'position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in certain embodiments of ANGPTL3 dsRNA agents, ANGPTL3 antisense polynucleotides, and ANGPTL3 sense polynucleotides of the invention include, but are not limited to RNAs comprising modified backbones or no natural internucleoside linkages. As a non-limiting example, an RNA having a modified backbone may not have a phosphorus atom in the backbone. RNAs that do not have a phosphorus atom in their internucleoside backbone may be referred to as oligonucleosides. In certain embodiments of the invention, a modified RNA has a phosphorus atom in its internucleoside backbone.
It will be understood that the term “RNA molecule” or “RNA” or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. The terms “ribonucleoside” and “ribonucleotide” , “nucleoside” and “nucleotide” may be used interchangeably herein. An RNA molecule can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below, and molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. As non-limiting examples, an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2'-O-methyl modified nucleoside, a nucleoside comprising a 5'phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino-modified nucleoside, 2'-alkyl-modified nucleoside, a 5'-phosphonate modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. In some embodiments of the invention, an 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 the full length of the ANGPTL3 dsRNA agent molecule’s ribonucleosides that are modified ribonucleosides. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule.
DsRNA agents, ANGPTL3 antisense polynucleotides, and/or ANGPTL3 sense polynucleotides of the invention may, in some embodiments comprise one or more independently selected modified nucleotide and/or one or more independently selected non-phosphodiester linkage. As used herein the term “independently selected” used in reference to a selected element, such as a modified nucleotide, non-phosphodiester linkage, etc., means that two or more selected elements can but need not be the same as each other.
As used herein, a “nucleotide base, ” “nucleotide, ” or “nucleobase” is a heterocyclic pyrimidine or purine compound, which is a standard constituent of all nucleic acids, and includes the bases that form the nucleotides adenine, guanine, cytosine, thymine, and uracil. A nucleobase may further be modified to include, though not intended to be limiting: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. The term “ribonucleotide” or “nucleotide” may be used herein to refer to an unmodified nucleotide, a modified nucleotide, a nucleotide analog, or a surrogate replacement moiety. Those in the art will recognize that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety.
As used herein, "optionally" or "optionally" means that the event or environment described later may, but need not, occur, including where the event or environment occurred or did not occur. For example, "C1-6 alkyl optionally substituted by halogen or cyano" means that halogen or cyano may, but not necessarily, be present, including the case where alkyl is substituted by halogen or cyano and the case where alkyl is not substituted by halogen and cyano.
As used herein, in the chemical structures of the compounds of the present disclosure, the bondrepresents an unspecified configuration, i.e., if a chiral isomer is present in the chemical structure, the bondcan beor bothtwo configurations. Although some of the above structural formulas are depicted as some isomeric forms for simplicity, the present disclosure may include all isomers, such as tautomers, rotamers, and mixtures thereof. Suitable chiral compounds include: geometric isomers, diastereomers, racemates and enantiomers.
As used herein, used in the chemical formulas of the present disclosure may be attached to any one or more groups according to the scope of the invention described herein.
In one embodiment, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway. In certain embodiments of the invention, an ANGPTL3 RNA interference agent includes a single stranded RNA that interacts with a target ANGPTL3 RNA sequence to direct the cleavage of the target ANGPTL3 RNA.
Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3' -alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3' -amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3' -5' linkages, 2'-5'linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3' -5'to 5'-3' or 2'-5'to 5'-2'. Various salts, mixed salts and free acid forms are also included. Means of preparing phosphorus-containing linkages are routinely practiced 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.
Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside) ; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. Means of preparing modified RNA backbones that do not include a phosphorus atom are routinely practiced 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.
In certain embodiments of the invention, RNA mimetics are included in ANGPTL3 dsRNAs, ANGPTL3 antisense polynucleotides, and/or ANGPTL3 sense polynucleotides, such as, but not limited to: replacement of the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units with novel groups. In such embodiments, base units are maintained for hybridization with an appropriate ANGPTL3 nucleic acid target compound. Means of preparing RNA mimetics are routinely practiced in the art and such methods can be used to prepare certain modified ANGPTL3 dsRNA agents of the invention.
Some embodiments of the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH2-NH-CH2-, -CH2-N (CH3) -O-CH2- [known as a methylene (methylimino) or MMI backbone] , -CH2-O-N (CH3) -CH2-, -CH2-N (CH3) -N (CH3) -CH2-and -N (CH3) -CH2- [wherein the native phosphodiester backbone is represented as -O-P-O-CH2-] . Means of preparing RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones are routinely practiced in the art and such methods can be used to prepare certain modified ANGPTL3 dsRNA agents, certain ANGPTL3 antisense polynucleotides, and/or certain ANGPTL3 sense polynucleotides of the invention.
Modified RNAs can also contain one or more substituted sugar moieties. ANGPTL3 dsRNAs, ANGPTL3 antisense polynucleotides, and/or ANGPTL3 sense polynucleotides of the invention may comprise one of the following at the 2'position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. 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 from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2'position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an ANGPTL3 dsRNA agent, or a group for improving the pharmacodynamic properties of an ANGPTL3 dsRNA agent, ANGPTL3 antisense polynucleotide, and/or ANGPTL3 sense polynucleotide, and other substituents having similar properties. In some embodiments, the modification includes a 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O- (2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a O (CH2) 2ON (CH3) 2 group, also known as 2'-DMAOE, as described in examples herein below, 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. Means of preparing modified RNAs such as those described are routinely practiced in the art and such methods can be used to prepare certain modified ANGPTL3 dsRNA agents of the invention.
Other modifications include 2'-methoxy (2'-OCH3) , 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F) . Similar modifications can also 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 2'-5'linked ANGPTL3 dsRNAs, ANGPTL3 antisense polynucleotides, or ANGPTL3 sense polynucleotides, and the 5'position of 5'terminal nucleotide. ANGPTL3 dsRNA agents, ANGPTL3 antisense polynucleotides, and/or ANGPTL3 sense polynucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Means of preparing modified RNAs such as those described are routinely practiced 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.
An ANGPTL3 dsRNA agent, ANGPTL3 antisense polynucleotide, and/or ANGPTL3 sense polynucleotide may, in some embodiments, include nucleobase (often referred to in the art simply as "base" ) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine and guanine, and the pyrimidine bases thymine, cytosine and uracil. Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-Me-C) , 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil) , 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, 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-daazaadenine and 3-deazaguanine and 3-deazaadenine. Additional nucleobases that may be included in certain embodiments of ANGPTL3 dsRNA agents of the invention are known in the art, see for example: Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. Ed. Wiley-VCH, 2008; The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.L, Ed. John Wiley &Sons, 1990, English et al., Angewandte Chemie, International Edition, 1991, 30, 613, Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., Ed., CRC Press, 1993. Means of preparing dsRNAs, ANGPTL3 antisense strand polynucleotides and/or ANGPTL3 sense strand polynucleotides that comprise nucleobase modifications and/or substitutions such as those described herein are routinely practiced in the art and such methods can be used to prepare certain modified ANGPTL3 dsRNA agents, ANGPTL3 sense polynucleotides, and/or ANGPTL3 antisense polynucleotides of the invention.
Certain embodiments of ANGPTL3 dsRNA agents, ANGPTL3 antisense polynucleotides, and/or ANGPTL3 sense polynucleotides of the invention include RNA modified to include one or more locked nucleic acids (LNA) . A locked nucleic acid is a nucleotide with a modified ribose moiety comprising an extra bridge connecting the 2'a nd 4'carbons. This structure effectively “locks” the ribose in the 3' -endo structural conformation. The addition of locked nucleic acids in an ANGPTL3 dsRNA agent, ANGPTL3 antisense polynucleotides, and/or ANGPTL3 sense polynucleotides of the invention may increase stability in serum, and to 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) . Means of preparing dsRNA agents, ANGPTL3 antisense polynucleotides, and/or ANGPTL3 sense polynucleotides that comprise locked nucleic acid (s) are routinely practiced in the art and such methods can be used to prepare certain modified ANGPTL3 dsRNA agents of the invention.
Certain embodiments of ANGPTL3 dsRNA compounds, sense polynucleotides, and/or antisense polynucleotides of the invention, include at least one modified nucleotide, wherein the at least one modified nucleotide comprises: a 2’ -O-methyl nucleotide, 2’ -Fluoro nucleotide, 2’ -deoxy nucleotide, 2’ 3’ -seco nucleotide mimic, locked nucleotide, 2’ -F-Arabino nucleotide, 2’ -methoyxyethyl nucleotide, 2’ -amino-modified nucleotide, 2’ -alkyl-modified nucleotide, mopholino nucleotide, and 3’ -OMe nucleotide, a nucleotide comprising a 5’ -phosphorothioate group, a nucleotide comprising vinyl phosphonate, a nucleotide comprising adenosine-glycol nucleic acid (GNA) , a nucleotide comprising thymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising 2-hydroxymethyl-tetrahydrofuran-5-phosphate, a nucleotide comprising 2’ -deoxythymidine-3’ -phosphate, a nucleotide comprising 2’ -deoxyguanosine-3’ -phosphate, a nucleotide comprising 2’ -deoxyadenosine-3’ -phosphate, a nucleotide comprising 2’ -deoxycytidine-3’-phosphate, a nucleotide comprising 2’ -deoxyuridine-3’ -phosphate, or a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2’ -amino-modified nucleotide, a phosphoramidate, or a non-natural base comprising nucleotide. In some embodiments, an ANGPTL3 dsRNA compound includes an E-vinylphosphonate nucleotide at the 5′-end of the antisense strand, also referred to herein as the guide strand.
Certain embodiments of ANGPTL3 dsRNA compounds, 3’ and 5’ end of sense polynucleotides, and/or 3’ end of antisense polynucleotides of the invention, include at least one modified nucleotide, wherein the at least one modified nucleotide comprises: abasic nucleotide, ribitol, inverted nucleotide, inverted abasic nucleotide, inverted 2’ -OMe nucleotide, inverted 2’ -deoxy nucleotide. It is known to skilled in art, including an abasic or inverted abasic nucleotide at the end of oligonucleotide enhances stability (Czauderna et al. Structural variations and stabilizing modifications of synthetic siRNA agents in mammalian cells. Nucleic Acids Res. 2003; 31 (11) : 2705-2716. doi: 10.1093/nar/gkg393) . In some embodiments, an ANGPTL3 dsRNA compound includes one or more inverted abasic residues (invab) at either 3’ -end or 5’ -end, or both 3’ -end and 5’ -end. Exemplified inverted abasic residues (invab) include, but are not limited to the following:
Certain embodiments of ANGPTL3 dsRNA compounds, antisense polynucleotides of the invention, include at least one modified nucleotide, wherein the at least one modified nucleotide comprises unlocked nucleic acid nucleotide (UNA) or/and glycol nucleic acid nucleotide (GNA) . It is known to skilled in art, UNA and GNA are thermally destabilizing chemical modifications, can significantly improves the off-target profile of a siRNA agent compound (Janas, et al., Selection of GalNAc-conjugated siRNA agents 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 agent performance in vitro and in vivo. Mol BioSyst. 2010; 6: 862–70) .
Certain embodiments of ANGPTL3 dsRNA compounds, antisense polynucleotides of the invention further comprise a phosphate moiety. As used herein, a phosphate moiety refers to a phosphate group including phosphates or phosphates mimics that attached to the sugar moiety (e.g., a ribose or deoxyribose or analog thereof) of a nucleotide. A nucleotide comprising a phosphate mimic may also be defined as a phosphonate modified nucleotide.
In some embodiments, the phosphate mimic is a 5’ -vinyl phosphonate (VP) . In exemplary embodiments, a vinyl phosphonate of the disclosure has the following structure:
A vinyl phosphonate of the instant disclosure may be attached to either the antisense or the sense strand of a dsRNA of the disclosure. In certain preferred embodiments, a vinyl phosphonate of the instant disclosure is attached to the antisense strand of a dsRNA, optionally at the 5’ end of the antisense strand of the dsRNA.
In certain embodiments, a vinyl phosphonate modified nucleotide of the disclosure has the structure of formula (IV) :
wherein X is O or S;
R is hydrogen, hydroxy, fluoro, or C1-20 alkoxy (e.g., methoxy or n-hexadecyloxy) ;
R5'is =C (H) -P (O) (OH) 2 and the double bond between the C5'carbon and R5'is in the E or Z orientation (e.g., E orientation) ; and
B is a nucleobase or a modified nucleobase, optionally where B is adenine, guanine, cytosine, thymine, or uracil.
In certain embodiments, R5'is =C (H) -P (O) (OH) 2 and the double bond between the C5’ carbon and R5’ is in the E orientation. In certain embodiments, R is methoxy and R5'is =C (H) -P (O) (OH) 2 and the double bond between the C5’ carbon and R5’ is in the E orientation. In certain embodiments, X is S, R is methoxy, and R5'is =C (H) -P (O) (OH) 2 and the double bond between the C5’ carbon and R5’ is in the E orientation.
Vinyl phosphonate modifications are also contemplated for the dsRNAs, the compositions and methods of the instant disclosure. An exemplary vinyl phosphonate structure is:
In many cases, protecting groups are used during the preparation of the compounds of the invention. As used herein, the term "protected" means that the indicated moiety has a protecting group appended thereon. In some embodiments of the invention, compounds contain one or more protecting groups. A wide variety of protecting groups can be employed in the methods of the invention. In general, protecting groups render chemical functionalities inert to specific reaction conditions, and can be appended to and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule. Protecting groups in general and hydroxyl protecting groups in particular are well known in the art (Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley &Sons, New York, 1991) .
As used herein, examples of protecting groups (e.g., hydroxyl protecting groups) include, but are not limited to, methyl, ethyl, benzyl (Bn) , phenyl, isopropyl, tert-butyl, acetyl, chloroacetyl, trichloro acetyl, trifluoroacetyl, pivaloyl, tert-butoxymethyl, methoxymethyl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, allyl, cyclohexyl, 9-fluorenylmethoxycarbonyl (Fmoc) , methanesulfonate, toluenesulfonate, triflate, benzoyl, benzoylformate , p-phenylbenzoyl, 4-methoxybenzyl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 4-chlorobenzyl, 4-nitrobenzyl, 2, 4-dinitrophenyl, 4-acyloxybenzyl, 2-methylphenyl, 2, 6-dimethylphenyl, 2-chlorophenyl, 2, 6-dichlorobenzyl, diphenylmethyl, triphenylmethyl, 4-methylthio-1-butyl, S-acetylthioacetate (SATA) , 2-cyanoethyl, 2-cyanol, 1-dimethylethyl (CDM) , 4-cyano-2-butenyl, 2- (trimethylsilyl) ethyl (TSE) , 2- (phenylthio) ethyl, 2- (triphenylsilyl) ethyl, 2- (benzylsulfonyl) ethyl, 2, 2, 2-trichloroethyl, 2, 2, 2-tribromoethyl, 2, 3-dibromopropyl, 2, 2, 2-trifluoroethyl, phenylthio, 2-chloro-4-tritylphenyl, 2-bromophenyl, 2- [N-isopropyl-N- (4-methoxybenzoyl) amino] ethyl, 4- (N-trifluoroacetylamino) butyl, 4-oxopentyl, 4-tritylaminophenyl, 4-benzyl aminophenyl, tetrahydropyranyl, morpholino, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triphenyl Silyl, triisopropylsilyl, pivaloyloxymethyl (POM) and 9-phenylxanthine-9-yl.
As used herein, examples of amino protecting groups include, but are not limited to, carbamate protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc) , 1-methyl-1- (4-biphenyl) ethoxycarbonyl (Bpoc) , tert-butyloxycarbonyl (BOC) , allyloxycarbonyl (Alloc) , 9-fluorenyl-methoxycarbonyl (Fmoc) , benzyloxycarbonyl (Cbz) ; amide protecting groups, such as formyl, acetyl, pivaloyl, trihaloacetyl, benzoyl, 2-nitrobenzenesulfonyl; and imine and cyclic imide protecting groups, such as phthalimido and dithiasuccinoyl. Equivalents of these amino-protecting groups are also encompassed by the compounds and methods of the invention.
Another modification that may be included in the RNA of certain embodiments of ANGPTL3 dsRNA agents, ANGPTL3 antisense polynucleotides, and/or ANGPTL3 sense polynucleotides of the invention, comprises chemically linking to the RNA one or more ligands, moieties or conjugates that enhance one or more characteristics of the ANGPTL3 dsRNA agent, ANGPTL3 antisense polynucleotide, and/or ANGPTL3 sense polynucleotide, respectively. Non-limiting examples of characteristics that may be enhanced are: ANGPTL3 dsRNA agent, ANGPTL3 antisense polynucleotide, and/or ANGPTL3 sense polynucleotide activity, cellular distribution, delivery of an ANGPTL3 dsRNA agent, pharmacokinetic properties of an ANGPTL3 dsRNA agent, and cellular uptake of the ANGPTL3 dsRNA agent. In some embodiments of the invention, an ANGPTL3 dsRNA agent comprises one or more targeting groups or linking groups, which in certain embodiments of ANGPTL3 dsRNA agents of the invention are conjugated to the sense strand. A non-limiting example of a targeting group is a compound comprising N-acetyl-galactosamine (GalNAc) . The terms “targeting group” , “targeting agent” , “linking agent” , “targeting compound” , “delivery molecule” , “delivery compound” and “targeting ligand” may be used interchangeably herein. In certain embodiments of the invention an ANGPTL3 dsRNA agent comprises a targeting compound that is conjugated to the 5'-terminal end of the sense strand. In certain embodiments of the invention an ANGPTL3 dsRNA agent comprises a targeting compound that is conjugated to the 3' -terminal end of the sense strand. In some embodiments of the invention, an ANGPTL3 dsRNA agent comprises a targeting group that comprises GalNAc. In certain embodiments of the invention an ANGPTL3 dsRNA agent does not include a targeting compound conjugated to one or both of the 3' -terminal end and the 5'-terminal end of the sense strand. In certain embodiments of the invention an ANGPTL3 dsRNA agent does not include a GalNAc containing targeting compound conjugated to one or both of the 5'-terminal end and the 3' -terminal end of the sense strand.
In some embodiments, a ligand attached to an ANGPTL3 dsRNA agent of the invention functions as a pharmacokinetic (PK) modulator. An example of a PK modulator that may be used in compositions and methods of the invention includes but is not limited to: a lipophiles, a bile acid, a steroid, a phospholipid analogue, a peptide, a protein binding agent, PEG, a vitamin, cholesterol, a fatty acid, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, a phospholipid, a sphingolipid, naproxen, ibuprofen, vitamin E, biotin, an aptamer that binds a serum protein, etc. Oligonucleotides comprising a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone may also be used in compositions and/or methods of the invention as ligands.
In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from a 2’ -O-methyl nucleotide, a 2’ -fluoro nucleotide and optionally an UNA modified nucleotide, wherein less than 6 modified nucleotides are 2’ -fluoro nucleotides. In some embodiments, the antisense strand comprises 3 or 5 2’ -fluoro nucleotides, preferably, the antisense strand comprises 5 2’ -fluoro nucleotides. In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from a 2’ -O-methyl nucleotide and a 2’ -fluoro nucleotide, wherein less than 4 modified nucleotides are 2’ -fluoro nucleotides. In certain embodiments, the sense strand comprises 3 2’ -fluoro nucleotides. In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from a 2’ -O-methyl nucleotide and a 2’ -fluoro nucleotide, wherein at least 14 modified nucleotides are 2’ -O-methyl nucleotides and the nucleotides at positions 2, 7, 12, 14 and/or 16 counting from the first matching position from the 5’ end of the antisense strand is independently a 2’ -fluoro nucleotide. In some embodiments, the antisense strand comprises at least one UNA modified nucleotide and 5 2’ -fluoro nucleotides. In some embodiments, the antisense strand comprises one UNA modified nucleotide at position 7 and 4 2’ -fluoro nucleotides at positions 2, 12, 14 and 16 counting from the first matching position from the 5’ end, and the rest are 2’ -O-methyl nucleotides. In some embodiments, the antisense strand comprises 5 2’ -fluoro nucleotides at positions 2, 7, 12, 14 and 16 counting from the first matching position from the 5’ end, and the rest are 2’ -O-methyl nucleotides. In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from a 2’ -O-methyl nucleotide and a 2’ -fluoro nucleotide. In some embodiments, at least 18 modified nucleotides are 2’ -O-methyl nucleotides and the nucleotides at positions 9, 11 and/or 13 counting from the first matching position from the 3’ end of the sense strand are 2’ -fluoro nucleotides.
In some embodiments, the antisense strand includes one inverted abasic residue (invab) at 3’ -terminal end. In certain embodiments, the sense strand includes one or two inverted abasic residues at 3’ or/and 5’ terminal end. In certain embodiments, each end of the sense strand includes one inverted abasic residue respectively. In some embodiments, the dsRNA agent has two blunt ends. In some embodiments, at least one strand includes a 3’ overhang of at least 1 nucleotide. In some embodiments, at least one strand includes a 3’ overhang of at least 2 nucleotides.
In some embodiments, at least one linkage of the sense strand and/or the antisense strand is a phosphodiester (PO) linkage. In some embodiments, at least one linkage of the sense strand and/or the antisense strand is a modified linkage. In some embodiments, at least one linkage of the sense strand and/or the antisense strand is a phosphorothioate (PS) linkage. In some embodiments, the dsRNA agent includes at least one phosphorothioate internucleoside linkage. In some embodiments, the sense strand includes at least one phosphorothioate internucleoside linkage. In some embodiments, the antisense strand includes at least one phosphorothioate internucleoside linkage. In some embodiments, the sense strand includes 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand includes 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In some embodiments, at least one phosphorothioate (PS) linkage is introduced at the 5’ -end, 3’ -end or both ends of the sense strand and/or the antisense strand. In some embodiments, 1, 2, 3, 4, 5, or 6 phosphorothioate (PS) linkages are introduced at the 5’ -end, 3’ -end or both ends of the sense strand and/or the antisense strand independently. In some embodiments, at least the terminal two modified or unmodified nucleotides at one end or both ends of the antisense strand are linked through phosphorothioate linkages. In some embodiments, the terminal three modified or unmodified nucleotides at one end or both ends of the antisense strand are linked through phosphorothioate linkages. In some embodiments, at least the terminal two modified or unmodified nucleotides at one end or both ends of the sense strand are linked through phosphorothioate linkages. In some embodiments, the terminal three modified or unmodified nucleotides at one end or both ends of the sense strand are linked through phosphorothioate linkages. In some embodiments, the terminal three modified or unmodified nucleotides at 5’ end of the sense strand are linked through phosphorothioate linkages and the terminal two modified or unmodified nucleotides at 3’ end of the sense strand are linked through phosphorothioate linkages. In some embodiments, one or more inverted abasic residues or one or more imann residues conjugate to either end or both ends of the sense strand via phosphorothioate linkages. In some embodiments, the targeting group further conjugates to either end of the sense strand via a phosphorothioate linkage. In some embodiments, the targeting group further conjugates to 5’ -end of the sense strand via a phosphorothioate linkage.
In some embodiments, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 3 (ANGPTL3) is provided, wherein the dsRNA agent including a sense strand and an antisense strand, wherein the sense strand is complementary to the antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding ANGPTL3, wherein each strand is about 14 to about 30 nucleotides in length, wherein the sense strand sequence may be represented by formula (I) :
5′- (N′L) n′N′LN′L N′N1 N′N2 N′N3 N′N4 N′L N′F N′L N′N5N′N6 N′L N′L N′L (N′L) m′-3′ (I)
wherein:
each N′F represents a 2'-fluoro-modified nucleotide; each N′N1, N′N2, N′N3, N′N4, N′N5, and N′N6 independently represents a modified or unmodified nucleotide; each N′L independently represents a modified or unmodified nucleotide but not a 2'-fluoro-modified nucleotide, and m′and n′are each independently an integer of 0 to 7.
In some embodiments, the modified nucleotide is a modified nucleotide defined above.
In some embodiments, the modified nucleotide is a 2’ -OMe modified nucleotide or a 2’ -F modified nucleotide.
In some embodiments, N′N4 and N′N5 each independently represents a 2'-fluoro-modified nucleotide.
In some embodiments, N′N2 and N′N4 each independently represents a 2'-fluoro-modified nucleotide.
In some embodiments, m′is 4 and n′is 3.
In some embodiments, m′is 2 and n′is 3, or m′is 2 and n′is 4, or m′is 2 and n′is 5.
In some embodiments, the dsRNA agent includes a targeting group that is conjugated to the 5’ -terminal end of the sense strand. In some embodiments, the targeting group is any one selected from aforesaid GLO-1 through GLO-16 and GLS-1*through GLS-16*, more preferably, the targeting group is aforesaid GLS-15*. In certain embodiments, the dsRNA agent includes a targeting group that is conjugated to the 3' -terminal end of the sense strand. In certain embodiments, the antisense strand includes one inverted abasic residue at 3’ -terminal end. In certain embodiments, the sense strand includes one or two inverted abasic residues at 3’ or/and 5’ terminal end. In certain embodiments, each 3’ and 5’ terminal end of the sense strand independently includes an inverted abasic residue. In certain embodiments, the sense strand includes one inverted abasic residues at 3’ and 5’ terminal end respectively and either residue at 3’ or 5’ terminal end is further conjugated to a targeting group, which preferably is aforesaid GLS-15*.
In some embodiments, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 3 (ANGPTL3) is provided, wherein the dsRNA agent including a sense strand and an antisense strand, wherein the sense strand is complementary to the antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding ANGPTL3, wherein each strand is about 18 to about 30 nucleotides in length, wherein the antisense strand sequence may be represented by formula (II) :
3′- (NL) n NM1 NL NM2 NL NF NL NM3 NM9 NM4 NL NM5 NM6 NL NM7 NM8 NL NF NZ-5′ (II)
wherein:
each NF represents a 2'-fluoro-modified nucleotide; each NM1, NM2, NM3, NM4, NM5, NM6, NM7, NM8 and NM9 independently represents a modified or unmodified nucleotide; each of NL and NZ independently represents a modified or unmodified nucleotide but not a 2'-fluoro-modified nucleotide; and n is an integer of 0 to 7.
In some embodiments, the modified nucleotide is a modified nucleotide defined above.
In some embodiments, the modified nucleotide is a 2’ -OMe modified nucleotide, or a 2’ -F modified nucleotide.
In some embodiments, NZ represents a 5'-phosphonate modified nucleotide.
In some embodiments, NZ is a vinyl phosphonate modified nucleotide.
In some embodiments, NZ is Vpu*, which has the structure
In some embodiments, NM2, NM3 and NM6 each independently represents a 2'-fluoro-modified nucleotide.
In some embodiments, NM2 and NM3 each independently represents a 2'-fluoro-modified nucleotide and NM6 represents an UNA modified nucleotide;
In some embodiments, n is 1, or n is 2, or n is 3.
In some embodiments, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 3 (ANGPTL3) is provided, wherein the dsRNA agent including a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a dsRNA duplex, wherein said sense strand is complementary to the antisense strand, wherein said antisense strand comprises a region of complementarity to an mRNA encoding ANGPTL3, wherein the region of complementarity comprises at least 15 contiguous nucleotides, wherein the dsRNA duplex represented by formula (III) :
sense: 5′- (N′L) n′N′LN′L N′N1 N′N2 N′N3 N′N4 N′L N′F N′L N′N5N′N6 N′L N′L N′L (N′L) m′-3′
antisense: 3′- (NL) n NM1 NL NM2 NL NF NL NM3 NM9 NM4 NL NM5 NM6 NL NM7 NM8 NL NF Nz-5′ (III)
wherein:
each strand is about 17 to about 30 nucleotides in length;
each NF and N′F independently represents a 2'-fluoro-modified nucleotide; NM1, NM2, NM3, NM4, NM5, NM6, NM7, NM8, NM9, N′N1, N′N2, N′N3, N′N4, N′N5, and N′N6 each independently represents a modified or unmodified nucleotide; each Nz, NL and N′L independently represents a modified or unmodified nucleotide but not a 2'-fluoro-modified nucleotide, and m′, n′and n are each independently an integer of 0 to 7.
In some embodiments, NZ represents a 5'-phosphonate modified nucleotide.
In some embodiments, NZ is a vinyl phosphonate modified nucleotide.
In some embodiments, NZ is Vpu*, which has the structure
In some embodiments, n′is 1 and m′is 2, n′is 2 and m′is 2, or n′is 1 and m′is 4, or n′is 3 and m′is 2, or n′is 3 and m′is 4, or n′is 4 and m′is 2, or n′is 5 and m′is 2.
In some embodiments, the modified nucleotide is a modified nucleotide defined above.
In some embodiments, the modified nucleotide is a 2’ -OMe modified nucleotide or a 2’ -F modified nucleotide.
In some embodiments, N′N2 and N′N4 each independently represents a 2'-fluoro-modified nucleotide.
In some embodiments, N′N4 and N′N5 each independently represents a 2'-fluoro-modified nucleotide.
In some embodiments, n is 1, or n is 2, or n is 3.
In some embodiments, NM2, NM3 and NM6 each independently represents a 2'-fluoro-modified nucleotide; in certain embodiment, NM2, NM3 and NM6 are all 2'-fluoro-modified nucleotides.
In some embodiments, NM2 and NM3 each independently represents a 2'-fluoro-modified nucleotide and NM6 represents an UNA modified nucleotide.
In some embodiments of Formula (II) or (III) , the antisense strand includes one inverted abasic residue at 3’ -terminal end. In some embodiments of Formula (I) or (III) , the sense strand includes one or two inverted abasic residues and/or one or two imann residues at 3’ or/and 5’ terminal end. In certain embodiments, each 3’ and 5’ terminal end of the sense strand independently includes an inverted abasic residue. In some embodiments of Formula (I) or (III) , each 3’ and 5’ terminal end of the sense strand independently includes an imann residue. In some embodiments of Formula (I) or (III) , the sense strand includes one inverted abasic residues at 3’ and 5’ terminal end respectively and either residue at 3’ or 5’ terminal end is further conjugated to a targeting group which mediates delivery to a liver tissue, which optionally is GLS-15*defined in the invention. In some embodiments of Formula (I) or (III) , the sense strand includes one imann residues at 3’ and 5’ terminal end respectively and either residue at 3’ or 5’ terminal end is further conjugated to a targeting group which mediates delivery to a liver tissue, which optionally is GLS-15*defined in the invention. In certain embodiments, the aforesaid dsRNA agent has two blunt ends. In certain embodiments of Formula (I) , (II) or (III) , at least one strand includes a 3’ overhang of at least 1 nucleotide. In certain embodiments of Formula (I) , (II) or (III) , at least one strand includes a 3’ overhang of at least 2 nucleotides.
In some embodiments of Formula (I) , (II) or (III) , NM1, NM2, NM3, NM4, NM5, NM6, NM7, NM8, NM9, N′N1, N′N2, N′N3, N′N4, N′N5, N′N6, N′L, NL and Nz each independently is linked to a neighboring nucleotide via phosphodiester (PO) linkage. In some embodiments of Formula (I) , (II) or (III) , at least one of NM1, NM2, NM3, NM4, NM5, NM6, NM7, NM8, NM9, N′N1, N′N2, N′N3, N′N4, N′N5, N′N6, N′L, NL and Nz is linked to a neighboring nucleotide via phosphorothioate (PS) linkage. In some embodiments of aforesaid Formula (I) , (II) or (III) further includes inverted abasic residues, imann residues and/or targeting groups, and the linkage within positions 1-10 of the termini positions of each end of the strand independently comprises 1, 2, 3, 4, 5 or 6 phosphorothioate (PS) linkages. In some embodiments of aforesaid Formula (I) , (II) or (III) including inverted abasic residues, imann residues and/or targeting groups, the linkage within positions 1-5 of the termini positions of each end of the strand independently comprises 1, 2 or 3 phosphorothioate (PS) linkages. In some embodiments of aforesaid Formula (I) , (II) or (III) including inverted abasic residues, imann residues and/or targeting groups, the linkage within positions 1-3 of the termini positions of each end of the strand independently comprises 1 or 2 phosphorothioate (PS) linkages.
Pharmaceutical Compositions Containing ANGPTL3 dsRNA
Certain embodiments of the invention include use of pharmaceutical compositions containing an ANGPTL3 dsRNA agent and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the ANGPTL3 dsRNA agent can be used in methods of the invention to reduce ANGPTL3 gene expression and ANGPTL3 activity in a cell and is useful to treat an ANGPTL3-associated disease or condition. Such pharmaceutical compositions can be formulated based on the mode of delivery. Non-limiting examples of formulations for modes of delivery are: a composition formulated for subcutaneous delivery, a composition formulated for systemic administration via parenteral delivery, a composition formulated for intravenous (IV) delivery, a composition formulated for intrathecal delivery, a composition formulated for direct delivery into brain, etc. Administration of a pharmaceutic composition of the invention to deliver an ANGPTL3 dsRNA agent into a cell may be done using one or more means such as: topical (e.g., by a transdermal patch) , pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration. An ANGPTL3 dsRNA agent can also be delivered directly to a target tissue, for example directly into the liver, directly into a kidney, etc. It will be understood that “delivering an ANGPTL3 dsRNA agent” into a cell encompasses delivering an ANGPTL3 dsRNA agent, directly as well as expressing an ANGPTL3 dsRNA agent in a cell from an encoding vector that is delivered into a cell, or by any suitable means with which the ANGPTL3 dsRNA becomes present in a cell. Preparation and use of formulations and means for delivering inhibitory RNAs are well known and routinely used in the art.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of an ANGPTL3 dsRNA agent of the invention and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in drug formulations are described further herein below.
Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials that are well-known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Pat. No. 5,211,657 and others are known by those skilled in the art. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
The pharmaceutical compositions comprising an ANGPTL3 RNAi construct suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to includeisotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient (s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The compositions for use in the methods of the present invention generally may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with free amino groups) derived from inorganic acids (e.g., hydrochloric or phosphoric acids) , or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like) . Salts formed with the free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like) . Sodium salts of the ANGPTL3 RNAi constructs are particularly useful for therapeutic administration to human subjects. Thus, in certain preferred embodiments, the ANGPTL3 RNAi constructs are in the form of a sodium salt. In other embodiments, the ANGPTL3 RNAi constructs are in the form of a potassium salt.
For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion or injection, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580) . For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA standards. In some embodiments, a pharmaceutical composition for use in the methods of the invention comprises or consists of a sterile saline solution and an ANGPTL3 RNAi construct described herein. In some embodiments, a pharmaceutical composition for use in the methods of the invention comprises or consists of an ANGPTL3 RNAi construct described herein and sterile water (e.g. water for injection, WFI) . In some embodiments, a pharmaceutical composition for use in the methods of the invention comprises or consists of an ANGPTL3 RNAi construct described herein and phosphate-buffered saline (PBS) .
In certain embodiments, a pharmaceutical composition useful for treating, ameliorating, preventing, or reducing the risk of hyperlipidemia according to the methods of the invention comprises an effective amount of an ANGPTL3 RNAi construct, Ortho-Phosphoric acid, and/or sodium hydroxide. In some embodiments, the pharmaceutical composition comprises about 200 mg/mL of an ANGPTL3 RNAi construct, which is calculated based on 100%pure sodium salt, and water for injection as the diluent, and optionally, sodium hydroxide or 85%ortho-phosphoric acid at 0.1N or 1N solution as the pH adjust mentor when needed. In some embodiments, the pH of any of these pharmaceutical compositions can be in the range of about 5.0 to about 7.0 (e.g., pH of about 6.0, pH of about 6.4, about 6.6, about 6.8 or about 7.0) . A skilled person can add sodium hydroxide or ortho-phosphoric acid as needed to adjust pH to target range.
Delivery of ANGPTL3 dsRNA agents
Certain embodiments of methods of the invention, includes delivery of an ANGPTL3 dsRNA agent into a cell. As used herein the term, “delivery” means facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an ANGPTL3 dsRNA agent can occur through unaided diffusive or active cellular processes, or by use of delivery agents, targeting agents, etc. that may be associated with an ANGPTL3 dsRNA agent of the invention. Delivery means that are suitable for use in methods of the invention include, but are not limited to: in vivo delivery, in which an ANGPTL3 dsRNA agent is in injected into a tissue site or administered systemically. In some embodiments of the invention, an ANGPTL3 dsRNA agent is attached to a delivery agent.
Non-limiting examples of methods that can be used to deliver 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 used successfully in the art to deliver therapeutic RNAi agents for treatment of various diseases and conditions, such as but not limited to: neurodegenerative diseases, liver diseases, acute intermittent porphyria (AIP) , hemophilia, pulmonary fibrosis, etc. Details of various delivery means are found in publications such as: Nikam, R.R. &K.R. Gore (2018) Nucleic Acid Ther, 28 (4) , 209-224 Aug 2018; Springer A.D. &S.F. Dowdy (2018) Nucleic Acid Ther. Jun 1; 28 (3) : 109–118; Lee, K. et al., (2018) Arch Pharm Res, 41 (9) , 867-874; Nair, J.K. et al., (2014) J. Am. Chem. Soc. 136: 16958-16961; Imran Sajid M. et al., (2023) Adv Drug Deliv Rev. 199: 114968, and Padmakumar S. et al., (2022) J Control Release. 352: 121-145; the content each of which is incorporated by reference herein.
Some embodiments of the invention comprise use of lipid nanoparticles (LNPs) to deliver an ANGPTL3 dsRNA agent of the invention to a cell, tissue, and/or subject. LNPs are routinely used for in vivo delivery of ANGPTL3 dsRNA agents, including therapeutic ANGPTL3 dsRNA agents. One benefit of using an LNP or other delivery agent is an increased stability of the ANGPTL3 RNA agent when it is delivered to a subject using the LNP or other delivery agent. In some embodiments of the invention an LNP comprises a cationic LNP that is loaded with one or more ANGPTL3 RNAi molecules of the invention. The LNP comprising the ANGPTL3 RNAi molecule (s) is administered to a subject, the LNPs and their attached ANGPTL3 RNAi molecules are taken up by cells via endocytosis, their presence results in release of RNAi trigger molecules, which mediate RNAi.
Some embodiments of the invention comprise use of functional moieties to deliver an ANGPTL3 dsRNA agent of the invention to a cell, tissue, and/or subject.
A functional moiety is a molecule that confers one or more additional activities to the RNA silencing agent. In certain embodiments, the functional moieties enhance cellular uptake by target cells (e.g., liver cells) . Thus, the disclosure includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 5’ and/or 3' terminus) to another moiety (e.g. a non-nucleic acid moiety such as a peptide) , an organic compound (e.g., a dye) , or the like. The conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al., Drug Deliv. Rev.: 47 (1) , 99-112 (2001) (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles) ; Fattal et al., J. Control Release 53 (1-3) : 137-43 (1998) (describes nucleic acids bound to nanoparticles) ; Schwab et al., Ann. Oncol. 5 Suppl. 4: 55-8 (1994) (describes nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles) ; and Godard et al., Eur. J. Biochem. 232 (2) : 404-10 (1995) (describes nucleic acids linked to nanoparticles) .
In a certain embodiment, the functional moiety is a hydrophobic moiety. In a certain embodiment, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins. In a certain embodiment, the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA) . In a certain embodiment, the fatty acid selected from the group consisting of Eicosapentaenoic acid (EPA) , Docosahexaenoic acid (DHA) and Docosanoic acid (DCA) . In a certain embodiment, the vitamin selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof. In a certain embodiment, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate.
In certain embodiments, the functional moieties may comprise one or more ligands tethered to an RNA silencing agent to improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell-type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism. Ligands and associated modifications can also increase sequence specificity and consequently decrease off-site targeting. A tethered ligand can include one or more modified bases or sugars that can function as intercalators. These can be located in an internal region, such as in a bulge of RNA silencing agent/target duplex. The intercalator can be an aromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound. A polycyclic intercalator can have stacking capabilities, and can include systems with 2, 3, or 4 fused rings. The universal bases described herein can be included on a ligand. In one embodiment, the ligand can include a cleaving group that contributes to target gene inhibition by cleavage of the target nucleic acid. The cleaving group can be, for example, a bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2) , pyrene, phenanthroline (e.g., O-phenanthroline) , a polyamine, a tripeptide (e.g., lys-tyr-lys tripeptide) , or a metal ion chelating group. The metal ion chelating group can include, e.g., an Lu (III) or EU (III) macrocyclic complex, a Zn (II) 2, 9-dimethylphenanthroline derivative, a Cu (II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the site of the bulge by free metal ions, such as Lu (III) . In some embodiments, a peptide ligand can be tethered to a RNA silencing agent to promote cleavage of the target RNA, e.g., at the bulge region. For example, 1, 8-dimethyl-1, 3, 6, 8, 10, 13-hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (e.g., by an amino acid derivative) to promote target RNA cleavage. A tethered ligand can be an aminoglycoside ligand, which can cause an RNA silencing agent to have improved hybridization properties or improved sequence specificity. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog can increase sequence specificity. For example, neomycin B has a high affinity for RNA as compared to DNA, but low sequence-specificity. An acridine analog, neo-5-acridine, has an increased affinity for the HIV Rev-response element (RRE) . In some embodiments, the guanidine analog (the guanidinoglycoside) of an aminoglycoside ligand is tethered to an RNA silencing agent. In a guanidinoglycoside, the amine group on the amino acid is exchanged for a guanidine group. Attachment of a guanidine analog can enhance cell permeability of an RNA silencing agent. A tethered ligand can be a poly-arginine peptide, peptoid or peptidomimetic, which can enhance the cellular uptake of an oligonucleotide agent.
Exemplary ligands are coupled, either directly or indirectly, via an intervening tether, to a ligand-conjugated carrier. In certain embodiments, the coupling is through a covalent bond. In certain embodiments, the ligand is attached to the carrier via an intervening tether. In certain embodiments, a ligand alters the distribution, targeting or lifetime of an RNA silencing agent into which it is incorporated. In certain embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.
Exemplary ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified RNA silencing agent, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides. Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin, diosgenin) , terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid) , vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal) , carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics. Ligands can include a naturally occurring substance, (e.g., human serum albumin (HSA) , low-density lipoprotein (LDL) , or globulin) ; carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid) ; amino acid, or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL) , poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly (L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N- (2-hydroxypropyl) methacrylamide copolymer (HMPA) , polyethylene glycol (PEG) , polyvinyl alcohol (PVA) , polyurethane, poly (2-ethylacryllic acid) , N-isopropyl acrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL) , spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine (GalNAc) or derivatives thereof, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B 12, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g. acridines and substituted acridines) , crosslinkers (e.g. psoralene, mitomycin C) , porphyrins (TPPC4, texaphyrin, Sapphyrin) , polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrenes) , lys-tyr-lys tripeptide, aminoglycosides, guanidium aminoglycodies, artificial endonucleases (e.g. EDTA) , lipophilic molecules, e.g, cholesterol (and thio analogs thereof) , cholic acid, cholanic acid, lithocholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters, e.g., C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 fatty acids) and ethers thereof, e.g., C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl; e.g., l, 3-bis-O (hexadecyl) glycerol, l, 3-bis-O (octaadecyl) glycerol) , geranyl oxy hexyl group, hexadecylglycerol, borneol, menthol, 1, 3 -propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate) , oleic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (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 facilitators (e.g., aspirin, naproxen, vitamin E, folic acid) , synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles) , dinitrophenyl, HRP or AP. In certain embodiments, the ligand is GalNAc or a derivative thereof.
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose.
In certain embodiments, the functional moiety is linked to the 5’ end and/or 3’ end of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5’ end and/or 3’ end of an antisense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5’ end and/or 3’ end of a sense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 3’ end of a sense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5’ end of a sense strand of the RNA silencing agent of the disclosure.
In certain embodiments, the functional moiety is linked to the RNA silencing agent by a linker. In certain embodiments, the functional moiety is linked to the antisense strand and/or sense strand by a linker. In certain embodiments, the functional moiety is linked to the 3’ end of a sense strand by a linker. In certain embodiments, the linker comprises a divalent or trivalent linker. In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.
Another non-limiting example of a delivery agent that may be used in embodiments of the invention to deliver an ANGPTL3 dsRNA agent of the invention to a cell, tissue and/or subject is an agent comprising GalNAc that is attached to an ANGPTL3 dsRNA agent of the invention and delivers the ANGPTL3 dsRNA agent to a cell, tissue, and/or subject. Examples of certain additional delivery agents comprising GalNAc that can be used in certain embodiments of methods and composition of the invention are disclosed in PCT Application: WO2020191183A1 and WO2023045995 (incorporated herein in its entirety) . A non-limiting example of a GalNAc targeting ligand that can be used in compositions and methods of the invention to deliver an ANGPTL3 dsRNA agent to a cell is a targeting ligand cluster. Examples of targeting ligand clusters that are presented herein are referred to as: GalNAc Ligand with phosphodiester link (GLO) and GalNAc Ligand with phosphorothioate link (GLS) . The term “GLX-n” may be used herein to indicate the attached GalNAc-containing compound is any one of compounds 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 structure of each of which is shown below, with the below with location of attachment of the GalNAc-targeting ligand to an RNAi agent of the invention at far right of each (shown with) . It will be understood that any RNAi and dsRNA molecule of the invention can be attached to the 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, GLO-1 through GLO-16 and GLS-1*through GLS-16*structures are shown below.




In some embodiments of the invention, in vivo delivery can also be by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. In certain embodiments of methods of the invention, an ANGPTL3 dsRNA is delivered without a targeting agent. These RNAs may be delivered as “naked” RNA molecules. As a non-limiting example, an ANGPTL3 dsRNA of the invention may be administered to a subject to treat an ANGPTL3-associated disease or condition in the subject, in a pharmaceutical composition comprising the RNAi agent, but not including a targeting agent such as a GalNAc targeting compound.
In addition to certain delivery means described herein, it will be understood that RNAi delivery means, such as but not limited to those described herein and those used in the art, can be used in conjunction with embodiments of ANGPTL3 RNAi agents and treatment methods described herein.
ANGPTL3 dsRNA agents of the invention may be administered to a subject in an amount and manner effective to reduce a level and activity of ANGPTL3 polypeptide in a cell and/or subject. In some embodiments of methods of the invention one or more ANGPTL3 dsRNA agents are administered to a cell and/or subject to treat a disease or condition associated with ANGPTL3 expression and activity. Methods of the invention, in some embodiments, include administering one or more ANGPTL3 dsRNA agents to a subject in need of such treatment to reduce a disease or condition associated with ANGPTL3 expression in the subject. ANGPTL3 dsRNA agents or ANGPTL3 antisense polynucleotide agents of the invention can be administered to reduce ANGPTL3 expression and/or activity in one more of in vitro, ex vivo, and in vivo cells.
Administration methods
A variety of administration routes for an ANGPTL3 dsRNA agent are available for use in methods of the invention. The particular delivery mode selected will depend at least in part, upon the particular condition being treated and the dosage required for therapeutic efficacy. Methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of treatment of an ANGPTL3-associated disease or condition without causing clinically unacceptable adverse effects. In some embodiments of the invention, an ANGPTL3 dsRNA agent may be administered via an oral, enteral, mucosal, subcutaneous, and/or parenteral route. The term “parenteral” includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal, and intrasternal injection, or infusion techniques. Other routes include but are not limited to nasal (e.g., via a gastro-nasal tube) , dermal, vaginal, rectal, sublingual, and inhalation. Delivery routes of the invention may include intrathecal, intraventricular, or intracranial. In some embodiments of the invention, an ANGPTL3 dsRNA agent may be placed within a slow-release matrix and administered by placement of the matrix in the subject. In some aspects of the invention, an ANGPTL3 dsRNA agent may be delivered to a subject cell using nanoparticles coated with a delivery agent that targets a specific cell or organelle. Various delivery means, methods, agents are known in the art. Non-limiting examples of delivery methods and delivery agents are additionally provided elsewhere herein. In some aspects of the invention, the term “delivering” in reference to an ANGPTL3 dsRNA agent may mean administration to a cell or subject of one or more “naked” ANGPTL3 dsRNA agent sequences and in certain aspects of the invention “delivering” means administration to a cell or subject via transfection means, delivering a cell comprising an ANGPTL3 dsRNA agent to a subject, delivering a vector encoding an ANGPTL3 dsRNA agent into a cell and/or subject, etc. Delivery of an ANGPTL3 dsRNA agent using a transfection means may include administration of a vector to a cell and/or subject.
In some methods of the invention one or more ANGPTL3 dsRNA agents may be administered in formulations, which may be administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. In some embodiments of the invention an ANGPTL3 dsRNA agent may be formulated with another therapeutic agent for simultaneous administration. According to methods of the invention, an ANGPTL3 dsRNA agent may be administered in a pharmaceutical composition. In general, a pharmaceutical composition comprises an ANGPTL3 dsRNA agent and optionally, a pharmaceutically-acceptable carrier. Pharmaceutically-acceptable carriers are well-known to those of ordinary skill in the art. As used herein, a pharmaceutically-acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients, e.g., the ability of the ANGPTL3 dsRNA agent to inhibit ANGPTL3 gene expression in a cell or subject. Numerous methods to administer and deliver dsRNA agents s for therapeutic use are known in the art and may be utilized in methods of the invention.
Some embodiments of methods of the invention include administering one or more ANGPTL3 dsRNA agents directly to a tissue. In some embodiments, the tissue to which the compound is administered is a tissue in which the ANGPTL3-associated disease or condition is present or is likely to arise, non-limiting examples of which are the liver or kidney. Direct tissue administration may be achieved by direct injection or other means. Many orally delivered compounds naturally travel to and through the liver and kidneys and some embodiments of treatment methods of the invention include oral administration of one or more ANGPTL3 dsRNA agents to a subject. ANGPTL3 dsRNA agents, either alone or in conjunction with other therapeutic agents, may be administered once, or alternatively they may be administered in a plurality of administrations. If administered multiple times, the ANGPTL3 dsRNA agent may be administered via different routes. For example, though not intended to be limiting, a first (or first several) administrations may be made via subcutaneous means and one or more additional administrations may be oral and/or systemic administrations.
For embodiments of the invention in which it is desirable to administer an ANGPTL3 dsRNA agent systemically, the ANGPTL3 dsRNA agent may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with or without an added preservative. ANGPTL3 dsRNA agent formulations (also referred to as pharmaceutical compositions) may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. 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, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's , or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose) , and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day may be used as needed to achieve appropriate systemic or local levels of one or more ANGPTL3 dsRNA agents and to achieve appropriate reduction in ANGPTL3 protein activity.
In yet other embodiments, methods of the invention include use of a delivery vehicle such as biocompatible microparticle, nanoparticle, or implant suitable for implantation into a recipient, e.g., a subject. Exemplary bioerodible implants that may be useful in accordance with this method are described in PCT Publication No. WO 95/24929 (incorporated by reference herein) , which describes a biocompatible, biodegradable polymeric matrix for containing a biological macromolecule.
Both non-biodegradable and biodegradable polymeric matrices can be used in methods of the invention to deliver one or more ANGPTL3 dsRNA agents s to a subject. In some embodiments, a matrix may be biodegradable. Matrix polymers may be natural or synthetic polymers. A polymer can be selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months can be used. The polymer optionally is in the form of a hydrogel that can absorb up to about 90%of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers.
In general, ANGPTL3 dsRNA agents may be delivered in some embodiments of the invention using the bioerodible implant by way of diffusion, or by degradation of the polymeric matrix. Exemplary synthetic polymers for such use are well known in the art. Biodegradable polymers and non-biodegradable polymers can be used for delivery of ANGPTL3 dsRNA agents s using art-known methods. Bioadhesive polymers such as bioerodible hydrogels (see H.S. Sawhney, C.P. Pathak and J.A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated by reference herein) may also be used to deliver ANGPTL3 dsRNA agents for treatment of an ANGPTL3-associated disease or condition. Additional suitable delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of an ANGPTL3 dsRNA agent, increasing convenience to the subject and the medical care professional. Many types of release delivery systems are available and known to those of ordinary skill in the art. (See for example: U.S. Pat. Nos. 5,075,109; 4,452,775; 4,675,189; 5,736,152; 3,854,480; 5,133,974; and 5,407,686 (the teaching of each of which is incorporated herein by reference) . In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
Use of a long-term sustained release implant may be suitable for prophylactic treatment of subjects and for subjects at risk of developing a recurrent ANGPTL3-associated disease or condition. Long-term release, as used herein, means that the implant is constructed and arranged to deliver a therapeutic level of an ANGPTL3 dsRNA agent for at least up to 10 days, 20 days, 30 days, 60 days, 90 days, six months, a year, or longer. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
Therapeutic formulations of ANGPTL3 dsRNA agents may be prepared for storage by mixing the molecule or compound having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers [Remington's Pharmaceutical Sciences 21st edition, (2006) ] , in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; 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, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes) ; and/or non-ionic surfactants such asor polyethylene glycol (PEG) .
Treatment
ANGPTL3-associated diseases and conditions in which a decrease in a level and/or activity of ANGPTL3 polypeptide is effective to treat the disease or condition, can be treated using methods and ANGPTL3 dsRNA agents of the invention to inhibit ANGPTL3 expression. Examples of diseases and conditions that may be treated with an ANGPTL3 dsRNA agent of the invention and a treatment method of the invention, include, but are not limited to: hyperlipidemia, hypertriglyceridemia, severe hypertriglyceridemia (SHTG) , familial chylomicronemia syndrome, mixed dyslipidemia, hypercholesterolemia, homozygous familial hypercholesterolemia (HoFH) , heterozygous familial hypercholesterolemia (HeFH) , abnormal lipid and/or cholesterol metabolism, statin resistant hypercholesterolemia, cardiometabolic disease, obesity, atherosclerosis, type II diabetes mellitus, cardiovascular disease, coronary artery disease, cerebrovascular disease, peripheral artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis caused by hypertriglyceridemia. Such diseases and conditions may be referred to herein as “ANGPTL3-associated diseases and conditions” and “diseases and conditions caused and/or modulated by ANGPTL3. ”
In certain aspects of the invention, a subject may be administered an ANGPTL3 dsRNA agent of the invention at a time that is one or more of before or after diagnosis of an ANGPTL3-associated disease or condition. In some aspects of the invention, a subject is at risk of having or developing an ANGPTL3-associated disease or condition. A subject at risk of developing an ANGPTL3-associated disease or condition is one who has an increased probability of developing the ANGPTL3-associated disease or condition, compared to a control risk of developing the ANGPTL3-associated disease or condition. In some embodiments of the invention, a level of risk may be statistically significant compared to a control level of risk. A subject at risk may include, for instance, a subject who is, or will be, a subject who has a preexisting disease and/or a genetic abnormality that makes the subject more susceptible to an ANGPTL3-associated disease or condition than a control subject without the preexisting disease or genetic abnormality; a subject having a family and/or personal medical history of the ANGPTL3-associated disease or condition; and a subject who has previously been treated for an ANGPTL3-associated disease or condition. It will be understood that a preexisting disease and/or a genetic abnormality that makes the subject more susceptible to an ANGPTL3-associated disease or condition, may be a disease or genetic abnormality that when present has been previously identified as having a correlative relation to a higher likelihood of developing an ANGPTL3-associated disease or condition.
It will be understood that an ANGPTL3 dsRNA agent may be administered to a subject based on a medical status of the individual subject. For example, a health-care provided for a subject may assess a lipid level measured in a sample obtained from a subject and determine it is desirable to reduce the subject’s lipid level, by administration of an ANGPTL3 dsRNA agent of the invention. In this example, the lipid level may be considered to be a physiological characteristic of an ANGPTL3-associated condition, even if the subject is not diagnosed as having an ANGPTL3-assoicated disease such as one disclosed herein. A healthcare provider may monitor changes in the subject’s lipid level, as a measure of efficacy of the administered ANGPTL3 dsRNA agent of the invention. In a non-limiting example, a biological sample, such as a blood or serum sample may be obtained from a subject and a lipid level for the subject determined in the sample. An ANGPTL3 dsRNA agent is administered to the subject and a blood or serum sample is obtained from the subject following the administration and the lipid level determined using the sample and the results compared to the results determined in the subject’s pre-administration (prior) sample. A reduction in the subject’s lipid level in the later sample compared to the pre-administration level indicates the administered ANGPTL3 dsRNA agent efficacy in reducing the lipid level in the subject.
Certain embodiments of methods of the invention include adjusting a treatment that includes administering an ANGPTL3 dsRNA agent of the invention to a subject based at least in part on assessment of a change in one or more of the subject’s physiological characteristics of an ANGPTL3-associated disease or condition resulting from the treatment. For example, in some embodiments of the invention, an effect of an administered dsRNA agent of the invention may be determined for a subject and used to assist in adjusting an amount of a dsRNA agent of the invention subsequently administered to the subject. In a non-limiting example, a subject is administered a dsRNA agent of the invention, the subject’s lipid level is determined after the administration, and based at least in part on the determined level, a greater amount of the dsRNA agent is determined to be desirable in order to increase the physiological effect of the administered agent, for example to reduce or further reduce the subject’s lipid level. In another non-limiting example, a subject is administered a dsRNA agent of the invention, the subject’s lipid level is determined after the administration and based at least in part on the determined level, a lower amount of the dsRNA agent is desirable to administer to the subject.
Thus, some embodiments of the invention include assessing a change in one or more physiological characteristics of resulting from a subject’s previous treatment to adjust an amount of a dsRNA agent of the invention subsequently administered to the subject. Some embodiments of methods of the invention include 1, 2, 3, 4, 5, 6, or more determinations of a physiological characteristic of an ANGPTL3-associated disease or condition to assess and/or monitor the efficacy of an administered ANGPTL3 dsRNA agent of the invention, and optionally using the determinations to adjust one or more of: a dose, administration regimen, and or administration frequency of a dsRNA agent of the invention to treat an ANGPTL3-associated disease or condition in a subject. In some embodiments of methods of the invention, a desired result of administering an effective amount of a dsRNA agent of the invention to a subject is a reduction of the subject’s lipid level, serum lipid level, LDL level, LDL : HDL ratio, triglyceride level, TC level, fat present in a subject’s liver, etc., as compared to a prior level determined for the subject, or to a control level.
As used herein, the terms “treat” , “treated” , or “treating” when used with respect to an ANGPTL3-associated disease or condition may refer to a prophylactic treatment that decreases the likelihood of a subject developing the ANGPTL3-associated disease or condition, and also may refer to a treatment after the subject has developed an ANGPTL3-associated disease or condition in order to eliminate or reduce the level of the ANGPTL3-associated disease or condition, prevent the ANGPTL3-associated disease or condition from becoming more advanced (e.g., more severe) , and/or slow the progression of the ANGPTL3-associated disease or condition in a subject compared to the subject in the absence of the therapy to reduce activity in the subject of ANGPTL3 polypeptide. The term "patient" as used herein, refers to a mammal, including humans, and can be used interchangeably with the term "subject" . In preferred embodiments, the patient is a human patient.
As used herein, the term “hyperlipidemia” , which can be used interchangeably with “dyslipidemia” , as used herein, means a metabolic disorder characterized by abnormally high or low amounts of any or all lipids (e.g. fats, triglycerides, cholesterol, phospholipids) or lipoproteins in the blood. Dyslipidemia is a risk factor for the development of atherosclerotic cardiovascular diseases (ASCVD) which include coronary artery disease, cerebrovascular disease, and peripheral artery disease.
Certain embodiments of agents, compositions, and methods of the invention can be used to inhibit ANGPTL3 gene expression. As used herein in reference to expression of an ANGPTL3 gene, the terms “inhibit, ” “silence, ” “reduce, ” “down-regulate, ” and “knockdown” mean the expression of the ANGPTL3 gene, as measured by one or more of: a level of RNA transcribed from the gene, a level of activity of ANGPTL3 expressed, and a level of ANGPTL3 polypeptide, protein or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the ANGPTL3 gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is contacted with (e.g., treated with) an ANGPTL3 dsRNA agent of the invention, compared to a control level of RNA transcribed from the ANGPTL3 gene, a level of activity of expressed ANGPTL3, or a level of ANGPTL3 translated from the mRNA, respectively. In some embodiments, a control level is a level in a cell, tissue, organ or subject that has not been contacted with (e.g. treated with) the ANGPTL3 dsRNA agent.
In certain embodiments of the invention, an ANGPTL3 dsRNA agent is administered to a subject to treat an ANGPTL3-associated disease or condition in conjunction with one or more additional therapeutic regimens for treating the ANGPTL3-associate disease or condition. Non-limiting examples of additional therapeutic regimens are: administering a non-ANGPTL3 dsRNA therapeutic agent, and a behavioral modification. An additional therapeutic regimen may be administered at a time that is one or more of: prior to, simultaneous with, and following administration of an ANGPTL3 dsRNA agent of the invention. It will be understood that simultaneous with as used herein, within five minutes of time zero, within 10 minutes of time zero, within 30 minutes of time zero, within 45 minutes of time zero, and within 60 minutes of time zero, with “time zero” the time of administration of the ANGPTL3 dsRNA agent of the invention to the subject. Non-limiting examples of behavioral modifications are: a dietary regimen, counseling, and an exercise regimen. These and other therapeutic agents and behavior modifications are known in the art and used to treat an ANGPTL3-associated disease or condition in a subject and may be administered to a subject in combination with the administration of one or more ANGPTL3 dsRNA agents of the invention to treat the ANGPTL3-associated disease or condition. An ANGPTL3 dsRNA agent of the invention administered to a subject to treat an ANGPTL3-associated disease or condition may act in a synergistic manner with one or more other therapeutic agents or activities and increase the effectiveness of the one or more therapeutic agents or activities and/or to increase the effectiveness of the ANGPTL3 dsRNA agent at treating the ANGPTL3-associated disease or condition.
As used herein, the terms “subsequent dose” and “additional dose” may be used interchangeably, meaning the dose after the second dose, and may be administered for once or at regular intervals.
Treatment methods of the invention that include administration of an ANGPTL3 dsRNA agent can be used prior to the onset of an ANGPTL3-associated disease or condition and/or when an ANGPTL3-associated disease or condition is present, including at an early stage, mid-stage, and late stage of the disease or condition and all times before and after any of these stages. Methods of the invention may also be to treat subjects who have previously been treated for an ANGPTL3-associated disease or condition with one or more other therapeutic agents and/or therapeutic activities that were not successful, were minimally successful, and/or are no longer successful at treating the ANGPTL3-associated disease or condition in the subject.
Kits
Also within the scope of the invention are kits that comprise one or more ANGPTL3 dsRNA agents and/or ANGPTL3 antisense polynucleotide agents and instructions for its use in methods of the invention. Kits of the invention may include one or more of an ANGPTL3 dsRNA agent, ANGPTL3 sense polynucleotide, and ANGPTL3 antisense polynucleotide agent that may be used to treat an ANGPTL3-associated disease or condition. Kits containing one or more ANGPTL3 dsRNA agents, ANGPTL3 sense polynucleotides, and ANGPTL3 antisense polynucleotide agents can be prepared for use in treatment methods of the invention. Components of kits of the invention may be packaged either in aqueous medium or in lyophilized form. A kit of the invention may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means or series of container means such as test tubes, vials, flasks, bottles, syringes, or the like. A first container means or series of container means may contain one or more compounds such as an ANGPTL3 dsRNA agent and/or ANGPTL3 sense or antisense polynucleotide agent. A second container means or series of container means may contain a targeting agent, a labelling agent, a delivery agent, etc. that may be included as a portion of an ANGPTL3 dsRNA agent to be administered in an embodiment of a treatment method of the invention.
A kit of the invention may also include instructions. Instructions typically will be in written form and will provide guidance for carrying-out a treatment embodied by the kit and for making a determination based upon that treatment.
The following examples are provided to illustrate specific instances of the practice of the present invention and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods.
Examples
Example 1.
Synthesis of ANGPTL3 RNAi Agents.
A series of siRNA agent duplexes targeting the human ANGPTL3 gene (SEQ ID NO: 1) were designed and synthesized according to prior arts. These same sequences were also synthesized with various nucleotide modifications and conjugated with a GalNAc. Further description related to the synthesis of ANGPTL3 RNAi agents (including the targeting group) may be found, for example, in WO2023045994 and WO2023045995, each of which is incorporated by reference herein in its entirety. ANGPTL3 RNAi agents can then be formulated by dissolving in suitable pharmaceutically acceptable excipients. The sense and antisense strand sequences of ANGPTL3 RNAi duplexes are shown in Table 1.



Example 2 A Phase I, Randomized, Double-Blind, Placebo-Controlled, Single Ascending Dose Study to Evaluate the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Subcutaneously Administered AD00112-2 in Adult Subjects
This was a multi-site, randomized, double-blind, placebo-controlled, single ascending dose study evaluating the safety, tolerability, PK, and PD of SC doses of AD00112-2 in subjects with elevated LDL-C and TG who were not receiving lipid-lowering therapy.
Subjects are enrolled in the study if they meet all the following key inclusion criteria:
1. Must have given written informed consent and be able to comply with all study requirements.
2. Males or females aged 18 to 65 aged years, inclusive, at the time of informed consent.
3. All subjects are required to have 100 mg/dL [2.69 mmol/L] ≤ LDL-C < 190 mg/dL [4.91 mmol/L] and 100 mg/dL [1.13 mmol/L] ≤ TG <500 mg/dL [5.65 mmol/L] in a fasting state and have not been treated with lipid-lowering medicines in the past 3 months (Fasting is defined as no food or caloric beverages for at least 8 hours) .
4. BMI ≥18 and ≤35 kg/m2 with body weight >50 kg.
5. For smokers, subjects aged > 50 need to have stopped smoking more than 12 months prior to screening; subjects aged ≤ 50 need to have stopped smoking 30 days prior to screening.
6. Negative cotinine test at screening and on Day -1.
7. Triplicate 12-lead electrocardiogram (ECG) after ≥5 minutes resting in a supine or semisupine position without clinically significant findings at Screening and on Day -1.
8. Females must be non-pregnant and non-lactating, and either surgically sterile or postmenopausal. Women of child-bearing potential, defined as all women physiologically capable of becoming pregnant, can participate if they are using highly effective methods of contraception from 28 days prior to screening until 24 weeks following administration of the study drug. The dual contraception is required, which is defined as use of a highly effective method of contraception by the female subject combined with use of a condom by the male partner.
Note: Only female subjects who engage in intercourse that may result in pregnancy are required to use contraception.
9. Male subjects agreeing to use acceptable methods of contraception if the male subject’s partner could become pregnant from the time of the administration of study medication from screening until 24 weeks following administration of the study drug. A condom is not required for a male if in same-sex relationship. The dual contraception is required, which is defined as use of a condom by the male subject combined with a highly effective method of contraception by the female partner.
Highly effective methods of contraception are listed below:
a) Hormonal methods of contraception including oral contraceptives containing combined estrogen and progesterone, a vaginal ring, injectable and implantable hormonal contraceptives, intrauterine hormone-releasing system (e.g. Mirena) and progestogen-only hormonal contraception associated with inhibition of ovulation
b) Nonhormonal intrauterine device (IUD)
c) Bilateral tubal occlusion
d) Vasectomised subject/partner with documented azoospermia 90 days after procedure if that partner is the sole sexual partner.
e) True abstinence: when this is in line with the preferred and usual lifestyle of the subject. Periodic abstinence (eg, calendar, ovulation, symptothermal, postovulation methods) and withdrawal are not acceptable methods of contraception.
For female subjects and female partners of subjects, hormonal contraceptives should begin at least 1 month prior to screening to ensure contraceptive is in full effect. Subjects will not be allowed to donate ova or sperm during the study.
10. On a usual diet for at least 4 weeks prior to screening with no plans to significantly alter diet or weight over course of study.
Eligible and enrolled subjects are planned for the 4 cohorts as follows:
Cohort 1: 50 mg AD00112-2 or placebo
Cohort 2: 150 mg AD00112-2 or placebo
Cohort 3: 300 mg AD00112-2 or placebo
Cohort 4: 600 mg AD00112-2 or placebo
Single dose administration of AD00112-2 was evaluated in Cohorts 1 to 4 at dose levels of 50, 150, 300, and 600 mg, respectively. AD00112-2 was provided as a solution for subcutaneous (SC) injection (200 mg/mL [as sodium salt form] , presented as 1 mL extractable volume per vial) . Placebo was provided as sodium chloride injection, 0.9%w/v administered via SC injection. Properties of ANGPTL3 dsRNA agent (AD00112-2) are described in Table 3. A schematic representation of ANGPTL3 dsRNA agent (AD00112-2) is shown in FIG. 8. A sentinel dosing approach was applied in each cohort. Two sentinel subjects (1 received AD00112-2 and 1 received placebo) were dosed first. If deemed safe and tolerated by the Investigator, the remaining subjects in the same cohort were dosed after at least 24 hours of dosing of the 2nd sentinel subject.
The decision to proceed to the next sequential cohort was taken by the safety review committee (SRC) and was based on the cumulative review of the blinded safety, tolerability, and available PK/PD data.
For each study cohort, screening for eligibility was scheduled to take place within 28 days before study drug administration (Day -28 to Day -2) . Eligible subjects were admitted to a clinical study site on Day -1 (the day before dosing of study drug) , dosed on Day 1, and discharged on Day 2 after completing the 24-hour post-dose follow-up assessments. Subjects returned to the clinical study site on an outpatient basis for safety, tolerability, PK, and PD monitoring at specified time points through Day 169.
Serial PK and PD samples (ANGPTL3 and lipid panel) were collected at selected time points through Day 169. All lipid results from the baseline measurements on Day 1 onward were blinded to the Investigators in all sites and Sponsor personnel involved in the study. Final safety assessments included adverse events and the results from physical examinations, ECGs, clinical laboratory tests (hematology, serum chemistry, coagulation, and urinalysis) , and injection site reactions.
Serum ANGPTL3 levels were determined using an ELISA assay and the levels of LDL-C (low-density lipoprotein cholesterol) , triglyceride (TG) , ApoB (apolipoprotein B) and TC (total cholesterol) were determined using enzymatic colorimetric assay (Roche Cobas kit) , and VLDL-C (very low-density lipoprotein cholesterol) and non-HDL-C was determined by calculation.
The cohort demographics and the baseline characteristics of the subjects in the study are provided in Table 2.
AD00112-2 was generally well tolerated when administered subcutaneously as a single dose from 50 mg to 600 mg. There was no TEAEs leading to death or study discontinuation in this study. There was only one SAE cellulitis caused by cat scratch unrelated to study drug in one subject with placebo and no SAEs in subjects with AD00112-2.
The reduction of ANGPTL3 serum levels, shown as mean percentage reductions from baseline, is shown in FIG. 1, and the lowering of LDL-C, TG and non-HDL-C levels in the study, shown as a percent mean LDL-C, TG and non-HDL-C lowering relative to baseline, is shown in FIG. 2, FIG. 3 and FIG. 4. In the study, the effect on ANGPTL3 and LDL-C, TG and non-HDL-C levels remained significantly reduced for at least 169 days post-treatment.
The data demonstrate that 50 mg, 150 mg, 300 mg, and 600 mg groups serum ANGPTL3 concentrations started reducing at Day 2, reaching maximal mean reductions of 60%, 77%, 83%and 90%between Day 15 and Day 29 after the single dose, and has a sustained efficacy with a mean percentage reduction from baseline at Day 169 of 31%, 38%, 51%and 81%respectively. Change from baseline in ANGPTL3 levels in subjects receiving AD00112-2 50-600 mg (n=6-7 per dose group) , was significantly greater than in placebo-treated subjects through day 169 treatment.
The data further demonstrate that 50 mg, 150 mg, 300 mg, and 600 mg groups reductions in fasting LDL-C concentrations were observed reaching maximal mean reductions of 16%, 36%, 24%and 42%between Day 15 and Day 57 after the single dose, and has a sustained efficacy with mean percentage reductions from baseline to Day 169 of 4%, 22%, 6%, and 28%respectively. In AD00112-2 50 mg, 150 mg, 300 mg, and 600 mg groups reductions in fasting TG concentrations were observed reaching maximal mean reductions of 45%, 62%, 61%and 64%between Day 22 and Day 43 after the single dose, and has a sustained efficacy with mean percentage reduction from baseline to Day 169 of 19%, 50%, 34%, and 48%respectively. In AD00112-2 50 mg, 150 mg, 300 mg, and 600 mg groups reductions in fasting non-HDL-C concentrations were observed reaching maximal mean reductions of 21%, 40%, 32%and 46%between Day 29 and Day 85 after the single dose, and has a sustained efficacy with mean percentage reduction from baseline to Day 169 of 10%, 28%, 15%, and 30%respectively. In AD00112-2 50 mg, 150 mg, 300 mg, and 600 mg groups reductions in fasting ApoB concentrations were observed reaching maximal mean reductions between Day 15 and Day 57 after the single dose, from 17%in 50 mg to 31%in 600 mg, with mean percentage reduction from baseline to Day 169 of 11%, 21%, 12%, and 20%respectively. In AD00112-2 50 mg, 150 mg, 300 mg, and 600 mg groups reductions in fasting TC concentrations were observed reaching maximal mean reductions between Day 43 and Day 85 after the single dose, from 18%in 50 mg to 41%in 600 mg, with mean percentage reduction from baseline to Day 169 of 6%, 22%, 10%, and 27%respectively. In AD00112-2 50 mg, 150 mg, 300 mg, and 600 mg groups reductions in fasting VLDL-C concentrations were observed reaching maximal mean reductions between Day 22 and Day 127 after the single dose, from 45%in 50 mg to 88%in 150 mg, with mean percentage reduction from baseline to Day 169 of 20%, 75%, 56%, and 32%respectively.
Table 2 Demographics and Baseline Characteristics


BMI: Body Mass Index. SD: Standard Deviation. ANGPTL3: Angiopoietin-like protein 3. LDL-C: Low density lipoprotein cholesterol. HDL-C: 
High density lipoprotein cholesterol.
Table 3 Properties of ANGPTL3 dsRNA agent (AD00112-2)
Equivalents
Although several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. 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 invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto; the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present invention.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
The indefinite articles “a” and “an, ” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one. ”
The term “or” as used herein means “and/or, ” and is used interchangeably with the latter, unless clearly excluded from context. The phrase “and/or, ” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. If there are more than two elements and are separated by commas, the commas before “and/or” have the same meaning as “and/or” , correspondingly representing “and” or “or” . Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified, unless clearly indicated to the contrary.
The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means+10%. In certain embodiments, about means+5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.
The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least” , and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 18 nucleotides of a 21 nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.
As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.
All references, patents and patent applications and publications that are cited or referred to in this application are incorporated herein in their entirety herein by reference.

Claims (66)

  1. A method of inhibiting the expression of an angiopoietin-like protein 3 (ANGPTL3) gene in a subject, comprising administering to the subject a fixed dose of 50 mg to 600 mg of a double stranded ribonucleic acid (dsRNA) agent, or salt thereof, wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 2, and optionally, wherein the sense strand is conjugated to a ligand attached at the 5′-terminus.
  2. A method for treating, reducing, or preventing a disease or a disorder that would benefit from reduction in ANGPTL3 expression in a patient in need thereof comprising administering to the patient a fixed dose of 50 mg to 600 mg of a double stranded ribonucleic acid (dsRNA) agent, or salt thereof, wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 2, and optionally, wherein the sense strand is conjugated to a ligand attached at the 5′-terminus.
  3. The method of any one of claims 1-2, wherein the antisense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 0, 1, 2, 3 nucleotides from the complement of nucleotides 1350-1418 of the nucleotide sequence of SEQ ID NO: 1.
  4. The method of any one of claims 1-2, wherein the antisense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 0, 1, 2, 3 nucleotides from the complement of nucleotides 1358-1378 of the nucleotide sequence of SEQ ID NO: 1.
  5. The method of any one of claims 1-4, wherein the antisense strand of dsRNA comprising a nucleotide sequence II: 5’ -z1UAGAGUAUAACCUUCCz2-3’ , wherein z1 is selected from C, G, A or U, z2 is a nucleotide sequence IV, preferably, the nucleotide sequence IV is selected from A, AU, AA, AC, AG, AUU, AUA, AUC, AUG, AUUG, AUUU, AUUA, AUUC, AUUUU, AUUUUG, AUUCUU, AUUCGA, AUUUUGA, AUUUUGAG, AUUUUGAGA or AUUUUGAGACUUCCA.
  6. The method of any one of claims 1-5, wherein the sense strand of dsRNA comprising a nucleotide sequence III: 5’ -z3GGAAGGUUAUACUCUAz4-3’ , wherein z3 is a nucleotide sequence V, z4 is selected from C, G, A or U, preferably, the nucleotide sequence V is selected from U, AU, UU, GU, CU, AAU, UAU, GAU, CAU, GAAU, CAAU, AAAU, UAAU, AAAAU, CAAAAU, UCAAAAU, CUCAAAAU, UCUCAAAAU or UGGAAGUCUCAAAAU.
  7. The method of any one of claims 1-6, wherein the ligand attached to the sense strand is any one selected from GLO-1 through GLO-16 and GLS-1*through GLS-16*, preferably, the ligand is GLS-15*



  8. The method of any one of claims 1-7, wherein the sense strand of the ANGPTL3 dsRNA agent comprises or consists of the sequences according to any one of SEQ ID NO: 5-39 and the antisense strand of the ANGPTL3 dsRNA agent comprises or consists of the sequences according to any one of SEQ ID NO: 40-74, preferably, the ANGPTL3 dsRNA agent comprises or consists of any one of sequences set forth as a duplex sequence in Table 1.
  9. The method of any one of claims 1-8, wherein the ANGPTL3 dsRNA agent is in a salt form, preferably, the salt form is sodium salt form.
  10. The method of any one of claims 8-9, wherein the sense strand of the ANGPTL3 dsRNA agent comprises or consists of the nucleotide sequence of 5’ - (GLS-15*) (invab*) gaauggaaGfgUfuAfuacucuaa* (invab) -3’ (SEQ ID NO: 39) and the antisense strand of the ANGPTL3 RNAi agent comprises or consists of the nucleotide sequence of 5’ -u*Uf*agagUfauaaCfcUfuCfcau*u*c-3’ (SEQ ID NO: 74) , and preferably, the ANGPTL3 dsRNA agent is in sodium salt form.
  11. The method of any one of claims 1-10, wherein the disease or disorder that would benefit from reduction in ANGPTL3 expression is dyslipidemia, preferably, the disease or disorder is one or more of:hypertriglyceridemia, severe hypertriglyceridemia, familial chylomicronemia syndrome, mixed dyslipidemia, hypercholesterolemia, homozygous and heterozygous familial hypercholesterolemia, abnormal lipid and/or cholesterol metabolism, statin resistant hypercholesterolemia, cardiometabolic disease, obesity, atherosclerosis, type II diabetes mellitus, cardiovascular disease, coronary artery disease, cerebrovascular disease, peripheral artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis caused by hypertriglyceridemia.
  12. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 50 mg.
  13. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 150 mg.
  14. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 200 mg.
  15. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 300 mg.
  16. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 600 mg.
  17. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 50 mg once a quarter.
  18. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 150 mg once a quarter.
  19. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 200 mg once a quarter.
  20. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 300 mg once a quarter.
  21. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 600 mg once a quarter.
  22. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 900 mg once a quarter.
  23. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 50 mg biannually.
  24. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 150 mg biannually.
  25. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 200 mg biannually.
  26. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 300 mg biannually.
  27. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 600 mg biannually.
  28. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 900 mg biannually.
  29. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 50 mg every nine months.
  30. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 150 mg every nine months.
  31. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 200 mg every nine months.
  32. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 300 mg every nine months.
  33. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 600 mg every nine months.
  34. The method of any one of claims 1-11, wherein the subject is administered a fixed dose of 900 mg every nine months.
  35. The method of any one of claims 1-11, wherein the disease or disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 50 mg once a quarter.
  36. The method of any one of claims 1-11, wherein the disease or disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 150 mg once a quarter.
  37. The method of any one of claims 1-11, wherein the disease or disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 200 mg once a quarter.
  38. The method of any one of claims 1-11, wherein the disease or disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 300 mg once a quarter.
  39. The method of any one of claims 1-11, wherein the disease or disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 600 mg once a quarter.
  40. The method of any one of claims 1-11, wherein the disease or disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 150 mg biannually.
  41. The method of any one of claims 1-11, wherein the disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 200 mg biannually.
  42. The method of any one of claims 1-11, wherein the disease or disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 300 mg biannually.
  43. The method of any one of claims 1-11, wherein the disease or disorder is severe hypertriglyceridemia and the subject is administered a fixed dose of about 600 mg biannually.
  44. The method of any one of claims 1-11, wherein the disease or disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 50 mg once a quarter.
  45. The method of any one of claims 1-11, wherein the disease or disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 150 mg once a quarter.
  46. The method of any one of claims 1-11, wherein the disease or disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 200 mg once a quarter.
  47. The method of any one of claims 1-11, wherein the disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 300 mg once a quarter.
  48. The method of any one of claims 1-11, wherein the disease or disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 600 mg once a quarter.
  49. The method of any one of claims 1-11, wherein the disease or disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 900 mg once a quarter.
  50. The method of any one of claims 1-11, wherein the disease or disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 50 mg biannually.
  51. The method of any one of claims 1-11, wherein the disease or disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 150 mg biannually.
  52. The method of any one of claims 1-11, wherein the disease or disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 200 mg biannually.
  53. The method of any one of claims 1-11, wherein the disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 300 mg biannually.
  54. The method of any one of claims 1-11, wherein the disease or disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 600 mg biannually.
  55. The method of any one of claims 1-11, wherein the disease or disorder is mixed dyslipidemia and the subject is administered a fixed dose of about 900 mg biannually.
  56. The method of any one of claims 1-11, wherein the method further comprises administering a second dose of the ANGPTL3 dsRNA agent about one month or three months after the initial dose, wherein the second dose is equal to the initial dose.
  57. The method of claim 56, further comprises administering additional doses after the second dose, wherein the additional doses are administered about six months apart.
  58. The method of claims 56, further comprises administering additional doses after the second dose, wherein the additional doses are administered about nine months apart.
  59. The method of claim 57, wherein the additional doses are administered about six months apart for once or at a regular interval.
  60. The method of claim 58, wherein the additional doses are administered about nine months apart for once or at a regular interval.
  61. The method of any one of claims 1-60, wherein the subject is a human.
  62. The method of any one of claims 1-61, wherein the ANGPTL3 dsRNA agent is administered to the subject subcutaneously, preferably, the double stranded RNAi agent is administered to the subject by subcutaneous injection.
  63. The method of any one of claims 1-36, wherein the method further comprising administering an additional therapeutic agent to the subject, wherein the additional therapeutic agent is selected from statins, Niacin based lipid-lowering drugs, Omega-3 fatty acids, fibrates, and therapeutic agent targeting PCSK9.
  64. A unit dosage form comprising the ANGPTL3 dsRNA agent of any one of claims 1-11 at an amount of between about 50mg to about 900mg of the ANGPTL3 dsRNA agent.
  65. A pharmaceutical composition for use in inhibiting the expression of an angiopoietin-like protein 3 (ANGPTL3) gene in a subject in need thereof, wherein the use comprises the administration of the pharmaceutical composition according to a dosing regimen of any one of claims 12-63.
  66. A pharmaceutical composition for use in treating, reducing, or preventing a disease or a disorder that would benefit from reduction in ANGPTL3 expression in a patient in need thereof, wherein the use comprises the administration of the pharmaceutical composition according to a dosing regimen of any one of claims 12-63.
PCT/CN2025/084552 2024-03-25 2025-03-25 Method for treating dyslipidemia with angiopoietin-like 3 (angptl3) -targeted rnai agents Pending WO2025201277A1 (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140179768A1 (en) * 2011-06-21 2014-06-26 Alnylam Pharmaceuticals, Inc. ANGIOPOIETIN-LIKE 3 (ANGPTL3) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
US20180087054A1 (en) * 2015-04-13 2018-03-29 Alnylam Pharmaceuticals, Inc. ANGIOPOIETIN-LIKE 3 (ANGPTL3) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
US20220290153A1 (en) * 2021-03-04 2022-09-15 Alnylam Pharmaceuticals, Inc. Angiopoietin-like 3 (angptl3) irna compositions and methods of use thereof
WO2023045994A1 (en) * 2021-09-23 2023-03-30 Shanghai Argo Biopharmaceutical Co., Ltd. Compositions and methods for inhibiting expression of angiopoietin-like 3 (angptl3) protein
US20230159930A1 (en) * 2021-11-19 2023-05-25 Sanegene Bio Usa Inc. Double stranded rna targeting angiopoietin-like 3 (angptl-3) and methods of use thereof
US20230257750A1 (en) * 2020-09-30 2023-08-17 Nanopeptide (Qingdao) Biotechnology Ltd. Sirna of angptl3 and use thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140179768A1 (en) * 2011-06-21 2014-06-26 Alnylam Pharmaceuticals, Inc. ANGIOPOIETIN-LIKE 3 (ANGPTL3) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
US20180087054A1 (en) * 2015-04-13 2018-03-29 Alnylam Pharmaceuticals, Inc. ANGIOPOIETIN-LIKE 3 (ANGPTL3) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
US20230257750A1 (en) * 2020-09-30 2023-08-17 Nanopeptide (Qingdao) Biotechnology Ltd. Sirna of angptl3 and use thereof
US20220290153A1 (en) * 2021-03-04 2022-09-15 Alnylam Pharmaceuticals, Inc. Angiopoietin-like 3 (angptl3) irna compositions and methods of use thereof
WO2023045994A1 (en) * 2021-09-23 2023-03-30 Shanghai Argo Biopharmaceutical Co., Ltd. Compositions and methods for inhibiting expression of angiopoietin-like 3 (angptl3) protein
US20230159930A1 (en) * 2021-11-19 2023-05-25 Sanegene Bio Usa Inc. Double stranded rna targeting angiopoietin-like 3 (angptl-3) and methods of use thereof

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