Methods of preventing cardiovascular events by lowering proprotein convertase subtilisin kexin9 (pcsk9) protein

Abstract

The present invention relates to a method of reducing low density lipoprotein cholesterol or preventing a cardiac event in a subject at risk of having an atherosclerotic cardiovascular disease or an atherosclerotic cardiovascular disease, comprising administering to the subject a prophylactically effective amount of an RNAi agent. In addition, the present invention relates to a method of preventing the development of an atherosclerotic cardiovascular disease in a subject, comprising administering to the subject a prophylactically effective amount of an RNAi agent. Furthermore, the present invention relates to a method of treating a subject at risk of having an atherosclerotic cardiovascular disease or equal to an atherosclerotic cardiovascular disease, comprising administering to the subject a therapeutically effective amount of an RNAi agent.

Description

[0001] This application is a divisional application of an invention patent application with an application date of October 18, 2017, application number 201780077941.7, and invention name "Method for preventing cardiovascular events by reducing proprotein convertase subtilisin / kexin 9 (PCSK9) protein".

[0002] Related Applications

[0003] This application claims the benefit of the following U.S. Provisional Patent Applications: U.S. Provisional Patent Application 62 / 409,816 filed on October 18, 2016, U.S. Provisional Patent Application 62 / 422,028 filed on November 14, 2016, U.S. Provisional Patent Application 62 / 472,525 filed on March 16, 2017, and U.S. Provisional Patent Application 62 / 550,426 filed on August 25, 2017, the entire contents of each of these provisional patent applications are hereby incorporated herein by reference.

[0004] This application is also related to U.S. Provisional Patent Application 62 / 209,526 filed on August 25, 2015, PCT Application No. PCT / US2016 / 048666 filed on August 25, 2016, U.S. Provisional Application 61 / 733,518 filed on December 5, 2012, U.S. Provisional Application 61 / 793,530 filed on March 15, 2013, U.S. Provisional Application 61 / 886,916 filed on October 4, 2013, U.S. Provisional Application 61 / 892,188 filed on October 17, 2013, PCT Application No. PCT / US2013 / 073349 filed on December 5, 2013, and U.S. Patent Application No. 14 / 650,128 filed on June 5, 2015, each of which is incorporated herein by reference in its entirety.

[0005] Sequence Listing

[0006] This application contains a sequence listing, which has been submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy created on October 18, 2017 is named A115-2_WO_SL.txt and is 1,800 bytes in size. Background of the Invention

[0008] Atherosclerotic cardiovascular disease (ASCVD) remains a global health challenge. Atherosclerosis is a systemic disease process characterized by the accumulation of fatty deposits, inflammatory cells, and scar tissue within the arterial walls and is the underlying cause of most clinical cardiovascular events. Although various types of treatments and preventive measures have been developed, lowering low-density lipoprotein cholesterol (LDL-C) remains a reliable strategy for combating ASCVD. To this end, statins are a widely used therapy to lower lipid levels, but many subjects at risk still have elevated LDL-C levels.

[0009] Proprotein convertase subtilisin kexin 9 (PCSK9) has recently been identified as a target for lowering LDL-C levels. PCSK9 is a member of the subtilisin family of serine proteases shown to play a role in cholesterol metabolism.

[0010] Monoclonal antibodies that block PCSK9 have been developed and shown to reduce circulating PCSK9 levels and lower LDL-C concentrations. However, monoclonal antibodies to PCSK9 have a short duration of action, which may lead to heavy dosing and economic burden.

[0011] Therefore, there is a need in the art for effective therapeutic or preventive measures aimed at lowering LDL-C levels and ultimately treating or preventing ASCVD. SUMMARY OF THE INVENTION

[0013] The present invention relates to the use of RNA interference (RNAi) agents that inhibit PCSK9 synthesis in a method of prevention or treatment of a subject in need thereof.

[0014] In one aspect, the present invention relates to a method for reducing LDL-C in a subject. The method comprises administering to the subject an effective amount of an RNAi agent, wherein the RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region. The antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 4.

[0015] In another aspect, the present invention relates to a method for preventing cardiovascular events in a subject. The method comprises administering to the subject an effective amount of an RNAi agent, wherein the RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region. The antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 4.

[0016] In another aspect, the present invention relates to a method for reducing cardiovascular mortality and / or morbidity in a subject. The method comprises administering to the subject an effective amount of an RNAi agent, wherein the RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region. The antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 2.

[0017] In certain embodiments, the subject may have ASCVD, ASCVD equivalent risk, elevated cardiovascular disease (CVD) risk, heterozygous familial hypercholesterolemia, or homozygous familial hypercholesterolemia, be in need of lowering of LDL-C, or have a disorder that may benefit from lowering of LDL-C, or a combination of these.

[0018] In another aspect, the present invention relates to a method for preventing the development of ASCVD. The method comprises administering an effective amount of an RNAi agent to a subject, wherein the RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region. The antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 4.

[0019] In another aspect, the present invention relates to a method for treating a subject having ASCVD, ASCVD equivalent risk, elevated CVD risk, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, a subject in need of lowering LDL-C, or a combination thereof. The method comprises administering to the subject an effective amount of an RNAi agent, wherein the RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region. The antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 4.

[0020] In certain embodiments, the methods are used to treat acute coronary syndrome and / or elevated LDL-C levels.

[0021] In some embodiments, the subject may have a baseline LDL-C level above about 70 mg / dl, such as about 100 mg / dl.

[0022] In certain embodiments, more than one dose of the RNAi agent is administered to a subject. For example, multiple doses of the RNAi agent can be administered to a subject. In some embodiments, the RNAi agent is administered to a subject at regular intervals, such as about once a week, about once every two weeks, about once a month, about once every two months, about once every three months, about once every four months, about once every six months, about once a year, etc.

[0023] In certain embodiments, the method includes evaluating the object before applying the RNAi agent. The evaluation may include measuring one or more biochemical parameters of the object, including lipid parameters. The example of the parameter that can be measured before applying the RNAi agent includes LDL-C, high-density lipoprotein cholesterol (HDL-C), PCSK9, total cholesterol, triglycerides, non-HDL-C, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein A1 (Apo-A1), apolipoprotein B (Apo-B), lipoprotein (a) (Lp (a)), C-reactive protein (CRP), glycosylated hemoglobin A1c, alanine aminotransferase, aspartate aminotransferase, alkaline aminotransferase, creatine kinase and total bilirubin. In some embodiments, the evaluation of the object before applying the RNAi agent provides a baseline measurement of biochemical parameters. In certain embodiments, the evaluation of the object before applying the RNAi agent determines and / or affects the application of the RNAi agent, such as the amount of the RNAi agent, the application time of the RNAi agent, etc.

[0024] In certain embodiments, the method includes evaluating the subject between the administration of one or more doses of the RNAi agent. The assessment may include measuring one or more biochemical parameters of the subject, including lipid parameters. Examples of parameters that can be measured between the administration of one or more doses of the RNAi agent include the levels of LDL-C, HDL-C, PCSK9, total cholesterol, triglycerides, non-HDL-C, VLDL-C, Apo-A1, Apo-B, Lp (a), CRP, glycosylated hemoglobin A1c, alanine aminotransferase, aspartate aminotransferase, alkaline aminotransferase, creatine kinase, and total bilirubin. In some embodiments, the assessment result can determine and / or affect the subsequent administration of the RNAi agent, such as the amount of the RNAi agent, the administration time of the RNAi agent, etc.

[0025] In some embodiments, the RNAi agent is used to reduce LDL-C levels by more than about 20% compared to baseline LDL-C levels. In certain embodiments, after the RNAi agent is used, LDL-C levels are reduced by more than about 20% compared to baseline levels and maintained for, at or through 15 days or longer.

[0026] In some embodiments, the level of PCSK9 is reduced by more than about 25% compared to the baseline level of PCSK9 by administering the RNAi agent. In certain embodiments, after administering the RNAi agent, the PCSK9 level is reduced by more than about 25% compared to the baseline level for, for, or until 30 days or longer.

[0027] In some embodiments, the subject may be on background lipid-lowering therapy, such as statin therapy. In certain embodiments, the RNAi agent is administered to the subject while continuing background lipid-lowering therapy. In certain embodiments, the subject may be on maximum tolerated statin therapy. Alternatively, the subject may be on ezetimibe or LDL apheresis.

[0028] In other embodiments, the subject is not on background lipid-lowering therapy.

[0029] In certain embodiments, the subject has no active liver disease. For example, the subject may not exhibit a baseline level of alanine aminotransferase and / or aspartate aminotransferase greater than 2 times the upper limit of normal (ULN). In some embodiments, the subject may not exhibit a baseline level of total bilirubin greater than 1.5 times the ULN.

[0030] In another aspect, the present invention relates to RNAi agents for use in the following methods: (i) reducing LDL-C in a subject; (ii) preventing cardiovascular events in a subject; (iii) reducing cardiovascular mortality and / or morbidity in a subject; (iv) preventing the development of ASCVD in a subject; (v) treating a subject with ASCVD, ASCVD equivalent risk, elevated CVD risk, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, a subject in need of lowering LDL-C, or a combination thereof. The RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region. The antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 4. These methods comprise administering an effective amount of the RNAi agent to a subject.

[0031] The present invention is further illustrated by the following detailed description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 RNAi agents according to embodiments of the present invention are shown. Figure 1 SEQ ID NOs: 4, 3 and 3 are shown in order of appearance, respectively.

[0034] Figure 2 An overview of the dosing regimens used in the studies discussed in the Examples is shown.

[0035] Figure 3 Shown are mean percent changes in LDL-C up to day 360 following single administration of a 200 mg dose, a 300 mg dose, and a 500 mg dose of a RNAi agent on day 1, according to embodiments of the present invention.

[0036] Figure 4 Shown are the average percent changes in LDL-C over time until day 360 following single administration of 200 mg, 300 mg, and 500 mg doses of RNAi agent on day 1 and 100 mg, 200 mg, and 300 mg doses of RNAi agent on days 1 and 90, according to embodiments of the present invention.

[0037] Figure 5 Shown are the average absolute changes in LDL-C over time until day 360 following single administration of 200 mg, 300 mg, and 500 mg doses of RNAi agent on day 1 and 100 mg, 200 mg, and 300 mg doses of RNAi agent on days 1 and 90, according to embodiments of the present invention.

[0038] Figure 6A-6B Figure 2 shows that according to an embodiment of the present invention, after a single administration of a 200 mg dose, a 300 mg dose, and a 500 mg RNAi agent on day 1, the LDL-C of each patient was between baseline and day 270 ( Fig. 6A ) and between baseline and day 360 ( Figure 6B ) percentage change.

[0039] Figure 7 Shown are the mean percent changes in LDL-C up to day 360 following administration of a 100 mg dose, a 200 mg dose, and a 300 mg dose of a RNAi agent on days 1 and 90, according to embodiments of the present invention.

[0040] Figure 8 Shown is the percent change in LDL-C between baseline and Day 180 for each patient following administration of a 300 mg dose of the RNAi agent on Days 1 and 90, according to an embodiment of the invention.

[0041] Figures 9A-9B Figure 2 shows that, according to an embodiment of the present invention, after administration of 100 mg, 200 mg, and 300 mg of the RNAi agent on days 1 and 90, the LDL-C of each patient was between baseline and day 270 ( Fig.9A ) and between baseline and day 360 ( Fig. 9B ) percentage change.

[0042] Figures 10A-10C The change in LDL-C from baseline to Day 180 for each patient randomly assigned to the double-dose placebo group (61 patients) according to an embodiment of the present invention is shown ( Fig. 10A); Change in LDL-C from baseline to Day 180 for each patient randomized to two doses of 300 mg RNAi agent (59 patients) ( Fig. 10B ); and the change in LDL-C from baseline to day 240 in the group randomly assigned to the double-dose 300 mg RNAi agent group (59 patients) ( Fig. 10C The dashed lines represent LDL-C decreases of 39 mg / dL and 78 mg / dL (to convert cholesterol values ​​to mmol / L, multiply by 0.02586).

[0043] Figures 11A-11B According to an embodiment of the present invention, a 300 mg dose of RNAi agent ( Fig.11A ) or 300 mg of RNAi agent at day 1 and day 90 ( Fig. 11B ) after each patient received LDL-C from baseline to Day 90 and Day 270.

[0044] Fig.12 Shown are the average percent changes in PCSK9 levels up to day 270 following single administration of a 200 mg dose, a 300 mg dose, and a 500 mg dose of an RNAi agent on day 1, according to an embodiment of the present invention.

[0045] Fig.13 Shown are the average percent changes in PCSK9 levels up to day 270 following administration of a 100 mg dose, a 200 mg dose, and a 300 mg dose of a RNAi agent on days 1 and 90, according to embodiments of the present invention.

[0046] Fig.14 Shown are modeling results for LDL-C up to 22 months after administration of two or three 300 mg doses of a RNAi agent a year, according to embodiments of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention is based at least in part on the use of small interfering RNA (siRNA) molecules as a means of reducing PCSK9 protein levels, which in turn reduces LDL-C levels. Through RNA interference, siRNA binds to the RNA-induced silencing complex (RISC) in the cell, enabling it to cleave the messenger RNA (mRNA) encoding PCSK9. The cleaved mRNA is degraded and therefore cannot be used for protein translation, resulting in reduced PSCK9 protein levels, thereby reducing LDL-C levels.

[0049] Therefore, the present invention provides a method for reducing LDL-C in a subject, the method comprising administering to the subject an effective amount, such as a preventive effective amount or a therapeutically effective amount of an RNAi agent. The present invention also provides a method for preventing cardiovascular events in a subject, the method comprising administering to the subject an effective amount, such as a preventive effective amount or a therapeutically effective amount of an RNAi agent. In addition, the present invention provides a method for reducing cardiovascular mortality and / or morbidity in a subject, the method comprising administering to the subject an effective amount, such as a preventive effective amount or a therapeutically effective amount of an RNAi agent. For these methods, the subject may have ASCVD, ASCVD equivalent risk, increased CVD risk, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, need to reduce LDL-C or a combination thereof.

[0050] Additionally, the present invention provides a method of preventing the development of ASCVD in a subject, the method comprising administering to the subject an effective amount, eg, a prophylactically effective amount or a therapeutically effective amount, of a RNAi agent.

[0051] In addition, the present invention provides methods for treating subjects with ASCVD, ASCVD equivalent risk, elevated CVD risk, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, need for lowering LDL-C, or a combination thereof. The method may include administering to the subject an effective amount, such as a prophylactically effective amount or a therapeutically effective amount, of an RNAi agent.

[0052] In addition, the present invention relates to RNAi agents for use in the methods described herein, i.e., for lowering LDL-C in a subject, preventing a cardiovascular event in a subject, reducing cardiovascular mortality and / or morbidity in a subject, preventing the development of ASCVD in a subject, and / or treating a subject with ASCVD, ASCVD equivalent risk, increased CVD risk, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, a subject in need of lowering LDL-C, or a combination thereof.

[0053] In some embodiments, the cardiovascular event may be a major adverse cardiovascular event, which includes, but is not limited to, death, non-fatal myocardial infarction, severe recurrent ischemia, stroke, symptomatic pulmonary embolism, and hemorrhage.

[0054] Methods for reducing LDL-C and / or preventing cardiovascular events can be directed to subjects with ASCVD or "ASCVD equivalent risk". In certain embodiments, a subject is an "ASCVD equivalent risk" subject if he or she has one or more of the following conditions: symptomatic atherosclerosis, type 2 diabetes, familial hypercholesterolemia, including subjects with a target LDL-C <100 mg / dl for their 10-year risk of cardiovascular events as assessed by the Framingham risk score (score>20%) or equivalent. In some embodiments, the subject may have heterozygous familial hypercholesterolemia.

[0055] Methods of lowering LDL-C and / or preventing cardiovascular events may also be directed to subjects who present with one or more symptoms or risk factors for ASCVD, who are at "ASCVD equivalent risk," or who are otherwise predisposed to developing heart health problems. These symptoms / risk factors may include having an acute coronary syndrome; having a history of myocardial infarction, stable or unstable angina, coronary or other arterial revascularization, stroke, transient ischemic attack, or peripheral arterial disease from atherosclerosis; being male; having a family history of heart disease, ASCVD, or ASCVD equivalent risk; being a smoker; having limited mobility; having high blood pressure; having high blood cholesterol; having diabetes or prediabetes; being overweight or obese; having a history of preeclampsia during pregnancy; having uncontrolled stress and / or anger; being postmenopausal; having an unhealthy diet, such as a diet high in salt, saturated fat, trans fat, cholesterol, and / or refined sugar; being 55 years of age or older; having sleep apnea; having anemia; or a combination of these. Thus, the method may be directed to, for example, a subject who is young but has a family history of heart disease, or a subject who smokes but does not have hypertension. In certain embodiments, the method may be directed to a subject who has previously experienced a cardiac event.

[0056] In some embodiments, the method of the present invention may be to reduce LDL-C and / or prevent cardiovascular events in a subject suffering from heterozygous familial hypercholesterolemia, the method comprising administering an effective amount, such as a preventive effective amount or a therapeutically effective amount of an RNAi agent. In some embodiments, the method of the present invention may be to reduce LDL-C and / or prevent cardiovascular events in a subject suffering from ASCVD, the method comprising administering an effective amount, such as a preventive effective amount or a therapeutically effective amount of an RNAi agent. In some embodiments, the method of the present invention may be to reduce LDL-C and / or prevent cardiovascular events in a subject with ASCVD equivalent risk, the method comprising administering an effective amount, such as a preventive effective amount or a therapeutically effective amount of an RNAi agent. In some embodiments, the method of the present invention may be to reduce LDL-C and / or prevent cardiovascular events in a subject suffering from homozygous familial hypercholesterolemia, the method comprising administering an effective amount, such as a preventive effective amount or a therapeutically effective amount of an RNAi agent. In some embodiments, the method of the present invention may be to treat a subject suffering from heterozygous familial hypercholesterolemia, the method comprising administering an effective amount, such as a preventive effective amount or a therapeutically effective amount of an RNAi agent. In some embodiments, the method of the present invention can be a treatment for an object with ASCVD, the method comprising administering an effective amount, such as a preventive effective amount or a therapeutically effective amount of an RNAi agent. In some embodiments, the method of the present invention can be a treatment for an object with an equivalent risk of ASCVD, the method comprising administering an effective amount, such as a preventive effective amount or a therapeutically effective amount of an RNAi agent. In some embodiments, the method of the present invention can be a treatment for an object with homozygous familial hypercholesterolemia, the method comprising administering an effective amount, such as a preventive effective amount or a therapeutically effective amount of an RNAi agent. In these embodiments, the RNAi agent can be a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises a nucleotide sequence of SEQ ID NO: 4. The object can be a diet designed to improve lipid levels, and / or in treatment to reduce LDL, such as statins, ezetimibe or LDL separation. In some embodiments, the object can receive maximum tolerated statin therapy. In addition, the object may need additional reduction in LDL-C.

[0057] In certain embodiments, administration of a RNAi agent can reduce LDL-C levels by greater than about 20%, or greater than about 25%, or greater than about 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50%, or greater than about 55%, or greater than about 60%, compared to baseline LDL-C levels.

[0058] In some embodiments, after administration of the RNAi agent, the reduction in LDL-C levels can be maintained for, maintained for, or maintained for about 15 days or longer, or about 20 days or longer, or about 30 days or longer, or about 40 days or longer, or about 50 days or longer, or about 60 days or longer, or about 70 days or longer, or about 80 days or longer, or about 90 days or longer, or about 100 days or longer, or about 110 days or longer, or about 120 days or longer, or about 130 days or longer, or about 140 days or longer, or about 150 days or longer, or about 160 days or longer, or about 170 days or longer. About 170 days or more, or about 180 days or more, or about 190 days or more, or about 200 days or more, or about 210 days or more, or about 220 days or more, or about 230 days or more, or about 240 days or more, or about 250 days or more, or about 260 days or more, or about 270 days or more, or about 280 days or more, or about 290 days or more, or about 300 days or more, or about 310 days or more, or about 320 days or more, or about 330 days or more, or about 340 days or more, or about 350 days or more, or about 360 days or more.

[0059] Administration of a RNAi agent at a dose of about 100 mg may reduce LDL-C levels by more than about 15% or more than about 20% compared to baseline LDL-C levels on day 15. This reduction in LDL-C may be maintained for, maintained at, or maintained until about 30 days, or about 60 days, or about 90 days or longer. In some embodiments, at day 30, the reduction in LDL-C is more than about 40% and maintained for, maintained at, or maintained until about 60 days, about 90 days or longer compared to baseline LDL-C levels.

[0060] Administration of a RNAi agent at a dose of about 200 mg may reduce LDL-C levels by more than about 15%, or more than about 20%, or more than about 25%, or more than about 30% compared to baseline LDL-C levels on day 15. This reduction in LDL-C may be maintained for, maintained at, or maintained until about 30 days, or about 60 days, or about 90 days, or about 120 days, or about 150 days, or about 180 days, or about 210 days, or about 240 days, or about 270 days, or about 300 days, or about 330 days, or about 360 days, or longer. In some embodiments, at day 30, the reduction in LDL-C is more than about 40% compared to baseline LDL-C levels and maintained for, maintained at, or maintained until about 60 days, about 90 days, or longer. In certain embodiments, at day 15, the reduction in LDL-C is greater than about 30% compared to baseline LDL-C levels and is maintained for, maintained at, or maintained until about 30 days, about 60 days, about 90 days, about 120 days, about 150 days, or longer. In some embodiments, at day 180 or until day 180, LDL-C is reduced by more than about 5% compared to baseline LDL-C levels. In certain embodiments, at day 15, the reduction in LDL-C is greater than about 20% compared to baseline LDL-C levels and is maintained for, maintained at, or maintained until about 30 days, or about 60 days, or about 90 days, or about 120 days, or about 150 days, or about 180 days, or about 210 days, or about 240 days, or about 270 days, or about 300 days, or about 330 days, or about 360 days. In certain embodiments, the reduction in LDL-C may be greater than about 25% at day 180, or greater than about 25% at day 270, or greater than about 30% at day 360, compared to baseline LDL-C levels.

[0061] Administration of a RNAi agent at a dose of about 300 mg may reduce LDL-C levels by more than about 15%, or more than about 20%, or more than about 25%, or more than about 30%, or more than about 40%, on day 15, compared to baseline LDL-C levels. This reduction in LDL-C may be maintained for, maintained at, or maintained until about 30 days, or about 60 days, or about 90 days, or about 120 days, or about 150 days, or about 180 days, or about 210 days, or about 240 days, or about 270 days, or about 300 days, or about 330 days, or about 360 days, or longer. In some embodiments, at day 30, compared to baseline LDL-C levels, the reduction in LDL-C may be more than about 45%, and maintained for, maintained at, or maintained until about 60 days, about 90 days, or longer. In certain embodiments, at day 15, the reduction in LDL-C may exceed about 40% compared to baseline LDL-C levels and be maintained for, maintained at, or maintained until about 30 days, about 60 days, about 90 days, about 120 days, about 150 days, or longer. In some embodiments, at day 15, the reduction in LDL-C may exceed about 35% compared to baseline LDL-C levels and be maintained for, maintained at, or maintained until about 30 days, or about 60 days, or about 90 days, or about 120 days, or about 150 days, or about 180 days, or about 210 days, or longer. In some embodiments, the reduction in LDL-C may be greater than about 30% at day 15, compared to baseline LDL-C levels, and maintained for, at, or until about 30 days, or about 60 days, or about 90 days, or about 120 days, or about 150 days, or about 180 days, or about 210 days, or about 240 days, or about 270 days, or about 300 days, or about 330 days, or about 360 days or longer. In certain embodiments, the reduction in LDL-C may be greater than about 35% at day 180, or greater than about 30% at day 270, or greater than about 30% at day 360, compared to baseline LDL-C levels.

[0062] Administration of a RNAi agent at a dose of about 500 mg may reduce LDL-C levels by more than about 15%, or more than about 20%, or more than about 25%, or more than about 30%, or more than about 40% compared to baseline LDL-C levels on day 15. This reduction in LDL-C may be maintained for, maintained at, or maintained until about 30 days, or about 60 days, or about 90 days, or about 120 days, or about 150 days, or about 180 days, or about 210 days, or about 240 days, or about 270 days, or about 300 days, or about 330 days, or about 360 days, or longer. In some embodiments, at day 30, the reduction in LDL-C may be more than about 50% compared to baseline LDL-C levels, and maintained for, maintained at, or maintained until about 60 days, about 90 days, or longer. In certain embodiments, at day 15, the reduction in LDL-C may exceed about 40% compared to baseline LDL-C levels and be maintained for, maintained at, or maintained until about 30 days, about 60 days, about 90 days, about 120 days, about 150 days, about 180 days, or longer. In some embodiments, at day 15, the reduction in LDL-C may exceed about 35% compared to baseline LDL-C levels and be maintained for, maintained at, or maintained until about 30 days, or about 60 days, or about 90 days, or about 120 days, or about 150 days, or about 180 days, or about 210 days, or about 240 days, or longer. In some embodiments, at day 15, the reduction in LDL-C may be greater than about 30% compared to baseline LDL-C levels, and maintained for, at, or until about 30 days, or about 60 days, or about 90 days, or about 120 days, or about 150 days, or about 180 days, or about 210 days, or about 240 days, or about 270 days, or about 300 days, or longer. In certain embodiments, the reduction in LDL-C may be greater than about 40% at day 180, or greater than about 30% at day 270, or greater than about 30% at day 360 compared to baseline LDL-C levels.

[0063] Administration of a second dose of the RNAi agent on day 90 can further reduce LDL-C levels compared to baseline LDL-C levels. For example, administration of a dose of about 100 mg of the RNAi agent on day 1 can reduce LDL-C levels by more than about 40% on days 104 to 120 and / or 150, more than about 35% on days 104 to 120, 150, and / or 180, more than about 30% on days 104 to 120, 150, 180, and / or 210, and more than about 40% on days 104 to 120, 150, 180, and / or 210, compared to baseline LDL-C levels. In some embodiments, the reduction in LDL-C may be greater than about 35% at day 180, greater than about 25% at day 270, or greater than about 10% at day 360, compared to baseline LDL-C levels.

[0064] Following administration of a dose of about 200 mg of the RNAi agent on day 1, administration of a dose of about 200 mg of the RNAi agent on day 90 can reduce LDL-C levels by more than about 45% on days 104 to 120 and / or 150 compared to baseline LDL-C levels; by more than about 40% on days 104 to 120, 150, 180, 210, and / or 240; by more than about 35% on days 104 to 120, 150, 180, 210, 240, 270, and / or 300; and / or by more than about 30% or less on days 104 to 120, 150, 180, 210, 240, 270, 300, 330, and / or 360. In certain embodiments, the reduction in LDL-C may be greater than about 40% at day 180, or greater than about 35% at day 270, or greater than about 30% at day 360, compared to baseline LDL-C levels.

[0065] After administration of a dose of about 300 mg of the RNAi agent on day 1, administration of a dose of about 300 mg of the RNAi agent on day 90 can reduce LDL-C levels by more than about 50% on days 104 to 120, 150, 180, and / or 210 compared to baseline LDL-C levels; and by more than about 50% on days 104 to 120, 150, 180, 210, 240, 270, and / or 280 compared to baseline LDL-C levels. or more than about 40% at day 300; more than about 35% at day 104 to day 120, day 150, day 180, day 210, day 240, day 270, day 300, and / or day 310; and / or more than about 30% or less at day 104 to day 120, day 150, day 180, day 210, day 240, day 270, day 300, day 330, and / or day 360. In certain embodiments, the reduction in LDL-C may be more than about 50% at day 180, more than about 40% at day 270, or more than about 30% at day 360 compared to baseline LDL-C levels.

[0066] In certain embodiments, administration of a RNAi agent can reduce PCSK9 levels by more than about 25%, or more than about 30%, or more than about 35%, or more than about 40%, or more than about 45%, or more than about 50%, or more than about 55%, or more than about 60%, or more than about 65%, or more than about 70%, or more than about 75%, or more than about 80%, or more than about 85%, or more than about 90%, or more than about 95% compared to baseline PCSK9 levels.

[0067] In some embodiments, after administration of the RNAi agent, the reduction in PCSK9 levels can be maintained for, maintained for, or maintained until about 15 days or more, or about 20 days or more, or about 30 days or more, or about 40 days or more, or about 50 days or more, or about 60 days or more, or about 70 days or more, or about 80 days or more, or about 90 days or more, or about 100 days or more, or about 110 days or more, or about 120 days or more, or about 130 days or more, or about 140 days or more, or about 150 days or more, or about 160 days or more, or about 170 days or more, or about 180 days or more, or about 190 days or more, or about 200 days or more, or about 210 days or more, or about 220 days or more, or about 230 days or more, or about 240 days or more, or about 250 days or more, or about 260 days or more, or about 270 days or more, or about 280 days or more, or about 290 days or more, or about 300 days or more, or about 310 days or more, or about 320 days or more, or about 330 days or more, or about 340 days or more, or about 350 days or more, or about 360 days or more, or about 370 days or more, or about 380 days or more, or about 390 days or more, or about 400 days or more, or about 410 days or more, or about 420 days or more, or about 430 days or more, or about 440 days or more, or about 450 days or more, or about 460 days or more, or about 470 days or more, or about 480 days or more, or about 490 days or more, or or about 170 days or more, or about 180 days or more, or about 190 days or more, or about 200 days or more, or about 210 days or more, or about 220 days or more, or about 230 days or more, or about 240 days or more, or about 250 days or more, or about 260 days or more, or about 270 days or more, or about 280 days or more, or about 290 days or more, or about 300 days or more, or about 310 days or more, or about 320 days or more, or about 330 days or more, or about 340 days or more, or about 350 days or more, or about 360 days or more.

[0068] Administration of a 100 mg dose of the RNAi agent can reduce PCSK9 levels by more than about 20%, or more than about 25%, or more than about 35%, or more than about 40% compared to baseline PCSK9 levels on day 15. This reduction in PCSK9 can be maintained for, during, or until about 30 days, or about 60 days, or about 90 days or longer.

[0069] Administration of a 200 mg dose of RNAi agent may reduce PCSK9 levels by more than about 20%, or more than about 25%, or more than about 30%, or more than about 35%, or more than about 40%, or more than about 45%, or more than about 50% on day 15 compared to baseline PCSK9 levels. This reduction in PCSK9 may be maintained for, maintained at, or maintained until about 30 days, or about 60 days, or about 90 days or longer. In some embodiments, the reduction in PCSK9 may be more than about 60% on day 30 compared to baseline PCSK9 levels, and maintained for, maintained at, or maintained until about 60 days, about 90 days or longer. In certain embodiments, the reduction in PCSK9 may be more than about 30% on day 300 compared to baseline PCSK9 levels.

[0070] Administration of a RNAi agent at a dose of about 300 mg may reduce PCSK9 levels by more than about 20%, or more than about 25%, or more than about 30%, or more than about 35%, or more than about 40%, or more than about 45%, or more than about 50%, or more than about 55%, or more than about 60% on day 15 compared to baseline PCSK9 levels. This reduction in PCSK9 may be maintained for, maintained at, or maintained until about 30 days, or about 60 days, or about 90 days or longer. In some embodiments, the reduction in PCSK9 may exceed about 60% on day 30 compared to baseline PCSK9 levels, and may be maintained for, maintained at, or maintained until about 60 days, about 90 days or longer. In certain embodiments, the reduction in PCSK9 may exceed about 30% on day 300 compared to baseline PCSK9 levels.

[0071] Administration of a RNAi agent at a dose of about 500 mg may reduce PCSK9 levels by more than about 20%, or more than about 25%, or more than about 30%, or more than about 35%, or more than about 40%, or more than about 45%, or more than about 50%, or more than about 55%, or more than about 60% on day 15 compared to baseline PCSK9 levels. This reduction in PCSK9 may be maintained for, maintained at, or maintained until about 30 days, or about 60 days, or about 90 days or longer. In some embodiments, the reduction in PCSK9 may exceed about 70% on day 30 compared to baseline PCSK9 levels, and may be maintained for, maintained at, or maintained until about 60 days, about 90 days or longer. In some embodiments, the reduction in PCSK9 may exceed about 30% on day 300 compared to baseline PCSK9 levels.

[0072] Administration of a dose of about 100 mg of the RNAi agent on day 1, followed by administration of a dose of about 100 mg of the RNAi agent on day 90 can reduce PCSK9 levels by more than about 20%, or more than about 25%, or more than about 30%, or more than about 35%, or more than about 40% compared to baseline PCSK9 levels on day 104. This reduction in PCSK9 can be maintained for about 120 days, maintained at about 120 days, or maintained up to about 120 days. In some embodiments, the reduction in PCSK9 can be more than about 30% on day 104, and maintained for about 15 days, maintained at about 150 days, or maintained up to about 150 days compared to baseline PCSK9 levels.

[0073] Administration of a dose of about 200 mg of the RNAi agent on day 1, followed by administration of a dose of about 200 mg of the RNAi agent on day 90 can reduce PCSK9 levels by more than about 20%, or more than about 25%, or more than about 30%, or more than about 35%, or more than about 40%, or more than 45%, or more than 50% compared to baseline PCSK9 levels on day 104. This reduction in PCSK9 can be maintained for about 120 days, maintained at about 120 days, or maintained up to about 120 days. In some embodiments, the reduction in PCSK9 can be more than about 40% on day 104, and maintained for about 1250 days, maintained at about 150 days, or maintained up to about 150 days compared to baseline PCSK9 levels.

[0074] Administration of a dose of about 300 mg of the RNAi agent on day 1 followed by administration of a dose of about 300 mg of the RNAi agent on day 90 can reduce PCSK9 levels by more than about 20%, or more than about 25%, or more than about 30%, or more than about 35%, or more than about 40%, or more than 45%, or more than 50% compared to baseline PCSK9 levels on day 104. This reduction in PCSK9 can be maintained for about 120 days, maintained at about 120 days, or maintained up to about 120 days, or up to 150 days. In some embodiments, the reduction in PCSK9 can be more than about 50% or more than about 55% on day 120, and maintained for about 150 days, maintained at about 150 days, or maintained up to about 150 days, compared to baseline PCSK9 levels.

[0075] Administration of a dose of about 100 mg of the RNAi agent can reduce total cholesterol levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20% on day 90, compared to baseline total cholesterol levels; can reduce non-HDL-C levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than about 30% on day 90, compared to baseline non-HDL-C levels; can reduce Apo-B levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25% on day 90, compared to baseline Apo-B levels; and / or can reduce Lp(a) levels by more than about 5%, or more than about 10%, or more than about 15% on day 90, compared to baseline Lp(a) levels.

[0076] Administration of a dose of about 200 mg of the RNAi agent can reduce total cholesterol levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25% on day 90, compared to baseline total cholesterol levels; can reduce non-HDL-C levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than about 30%, or more than about 35% on day 90, compared to baseline non-HDL-C levels; can reduce Apo-B levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than about 30% on day 90, compared to baseline Apo-B levels; and / or can reduce Lp(a) levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20% on day 90, compared to baseline Lp(a) levels. In some embodiments, at day 180, total cholesterol is reduced by more than about 15%, non-HDL-C is reduced by more than about 25%, and Apo-B is reduced by more than about 20%.

[0077] Administration of a dose of about 300 mg of the RNAi agent can reduce total cholesterol levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25% at day 90, compared to baseline total cholesterol levels; can reduce non-HDL-C levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than about 30%, or more than about 35%, or more than about 40% at day 90, compared to baseline non-HDL-C levels; can reduce Apo-B levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than about 30%, or more than about 35% at day 90, compared to baseline Apo-B levels; and / or can reduce Lp(a) levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20% at day 90, compared to baseline Lp(a) levels.

[0078] Administration of a dose of about 500 mg of the RNAi agent can reduce total cholesterol levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than about 30% at day 90, compared to baseline total cholesterol levels; can reduce non-HDL-C levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than about 30%, or more than about 35%, or more than about 40% at day 90, compared to baseline non-HDL-C levels; can reduce Apo-B levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than about 30%, or more than about 35%, or more than about 40% at day 90, compared to baseline Apo-B levels; and / or can reduce Lp(a) levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20% at day 90, compared to baseline Lp(a) levels. In some embodiments, at day 180, the reduction in total cholesterol is greater than about 25%, and the reduction in Apo-B is greater than about 30%.

[0079] Administration of a dose of about 100 mg of the RNAi agent on day 1 followed by administration of a dose of about 100 mg of the RNAi agent on day 90 can reduce total cholesterol levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20% on day 180 compared to baseline total cholesterol levels; can reduce non-HDL-C levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than about 30% on day 180 compared to baseline non-HDL-C levels; can reduce Apo-B levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25% on day 180 compared to baseline Apo-B levels; and / or can reduce Lp(a) levels by more than about 5% or more than about 10% on day 180 compared to baseline Lp(a) levels.

[0080] Administration of a dose of about 200 mg of the RNAi agent on day 1 followed by administration of a dose of about 200 mg of the RNAi agent on day 90 may reduce total cholesterol levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25% on day 180, compared to baseline total cholesterol levels; may reduce non-HDL-C levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than about 30%, or more than 35% on day 180, compared to baseline non-HDL-C levels; may reduce Apo-B levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than 30%, or more than 35% on day 180, compared to baseline Apo-B levels; and / or may reduce Lp(a) levels by more than about 5%, or more than about 10%, or more than 15% on day 180, compared to baseline Lp(a) levels.

[0081] Administration of a dose of about 300 mg of the RNAi agent on day 1 and then administration of a dose of about 300 mg of the RNAi agent on day 90 can reduce total cholesterol levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than 30% compared to baseline total cholesterol levels on day 180; and can reduce non-HDL-C levels by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25% compared to baseline non-HDL-C levels on day 180. or more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than 30%, or more than 35%, or more than 40%; at day 180, Apo-B levels may be reduced by more than about 5%, or more than about 10%, or more than about 15%, or more than about 20%, or more than about 25%, or more than 30%, or more than 35%, or more than 40% compared to baseline Apo-B levels; and / or Lp(a) levels may be reduced by more than about 5%, or more than about 10%, or more than 15%, or more than 20%, or more than 25% at day 180 compared to baseline Lp(a) levels.

[0082] In certain embodiments, the method may further include evaluating the subject before administering the RNAi agent. The evaluation may be performed on the same day but before administering the RNAi agent, or may be performed one day before, or two days before, or three days before, or five days before, or six days before, or seven days before, or eight days before, or nine days before, or ten days before, or 11 days before, or 12 days before, or 13 days before, or 14 days before. In some embodiments, the evaluation may be performed more than one day before administering the RNAi agent.

[0083] In some embodiments, the assessment can include measuring one or more physiological parameters or characteristics of the subject, including but not limited to age, height, weight, body mass index, race, gender, whether the subject is undergoing any other treatment (e.g., lipid-lowering therapy such as statin therapy), diagnosis of cardiovascular disease, diagnosis of diabetes, heart rate, blood pressure, electrocardiogram parameters, etc.

[0084] In certain embodiments, assessment may include one or more biochemical parameters of the measurement object. In some embodiments, the measurement performed before the RNAi agent is applied can be considered as a "baseline" measurement. Examples of biochemical parameters including lipid parameters may be, but are not limited to, LDL-C, HDL-C, PCSK9, total cholesterol, triglycerides, non-HDL-C, VLDL-C, Apo-A1, Apo-B, Lp (a), CRP, glycosylated hemoglobin A1c, alanine aminotransferase, aspartate aminotransferase, alkaline aminotransferase, creatine kinase and total bilirubin levels. In some embodiments, the assessment of the object before the RNAi agent is applied can provide a baseline measurement of biochemical parameters. In certain embodiments, the assessment of the object before the RNAi agent is applied can determine and / or affect the application of the RNAi agent, such as the amount of the RNAi agent, the application time of the RNAi agent, etc.

[0085] In certain embodiments, the method may include evaluating the subject between one or more administrations of the RNAi agent. The evaluation may include measuring one or more biochemical parameters between administrations, including lipid parameters, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 21 days, 28 days, 30 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 56 days, 60 days, 63 days, 70 days, 77 days, 80 days, 84 days and / or 90 days after administration of the RNAi agent. Biochemical measurements may include, but are not limited to, levels of LDL-C, HDL-C, PCSK9, total cholesterol, triglycerides, non-HDL-C, VLDL-C, Apo-A1, apolipoprotein B, Lp(a), CRP, glycated hemoglobin A1c, alanine aminotransferase, aspartate aminotransferase, alkaline aminotransferase, creatine kinase, and total bilirubin.

[0086] In some embodiments, assessments between one or more administrations of the RNAi agent can measure physiological parameters or characteristics of the subject, including but not limited to body weight, body mass index, heart rate, blood pressure, electrocardiogram parameters, and the like.

[0087] In some embodiments, the evaluation results can determine and / or affect the subsequent administration of the RNAi agent, such as the amount of the RNAi agent, the time of administration of the RNAi agent, etc. For example, one or more results obtained from the measurement can increase or decrease the subsequent dosage of the RNAi agent by about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or more.

[0088] A variety of biochemical parameters can determine / affect the dosage of the RNAi agent to be administered. For example, measuring alanine aminotransferase or aspartate aminotransferase exceeding 2 times of ULN, and / or total bilirubin exceeding 1.5 times of ULN, can result in a reduction in the dosage of the RNAi agent to be subsequently administered. As another example, measuring glycated hemoglobin A1c exceeding 10% may result in a reduction in the dosage of the RNAi agent to be subsequently administered. Measuring LDL-C to be less than about 1% or less than about 5% or less than about 10% or less than about 15% of the baseline measurement may result in an increase in the dosage of the RNAi agent to be subsequently administered. The dosage may be reduced or increased, for example, by about 1%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or more.

[0089] In certain embodiments, after evaluating the subject before administering the RNAi agent as described above, lipid-lowering therapy can be administered to the subject. Lipid-lowering therapy can be a treatment regimen in which lipid-lowering therapy is administered to the subject at regular intervals. In some embodiments, the subject has not previously undergone lipid-lowering therapy. In certain embodiments, after administering the lipid-lowering treatment regimen, the subject can be additionally evaluated to measure one or more biochemical parameters of the subject, including lipid parameters, as described above. RNAi agents as described herein can then be administered to the subject.

[0090] Administration of RNAi agents

[0091] The methods of the invention comprise administering to a subject an effective amount, such as a prophylactically effective amount or a therapeutically effective amount, of a RNAi agent.

[0092] A "prophylactically effective amount" may include an amount of an RNAi agent sufficient to prevent or improve the disorder or one or more symptoms of the disorder when administered to a subject who has not yet experienced or shown symptoms of the disorder but is predisposed to the disorder. Improving a disorder includes slowing the progression of the disorder or reducing the severity of a subsequent disorder. A "prophylactically effective amount" may vary depending on the RNAi agent, how the RNAi agent is administered, the degree of risk of the disorder, medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0093] A "therapeutically effective amount" may include an amount of a RNAi agent that is sufficient to achieve treatment of a condition (e.g., reduce, improve, or maintain an existing condition or one or more symptoms of a condition) when administered to a subject. A "therapeutically effective amount" may vary depending on the RNAi agent, how the RNAi agent is administered, the condition and its severity, as well as medical history, age, weight, family history, genetic makeup, stage of pathological progression, type of previous or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0094] "Baseline" refers to the untreated condition, such as the condition prior to administration of treatment.

[0095] RNAi agent can be administered to the subject at a fixed dose. "Fixed dose" (e.g., dosage in mg) is a dosage for all subjects, regardless of any specific subject-related factors such as body weight. Alternatively, RNAi agent can be administered to the subject as a dosage based on body weight (e.g., dosage in mg / kg), which is the dosage of the RNAi agent changed according to the body weight of the subject. In embodiments where the subject receives multiple doses, RNAi agent can be administered in a combination of a fixed dose and a dosage based on body weight.

[0096] In certain embodiments, the RNAi agent is administered at about 50 mg to about 800 mg, about 100 mg to about 800 mg, about 150 mg to about 800 mg, about 200 mg to about 800 mg, about 250 mg to about 800 mg, about 300 mg to about 800 mg, about 350 mg to about 800 mg, about 400 mg to about 800 mg, about 450 mg to about 800 mg, about 500 mg to about 800 mg, about 550 mg to about 800 mg, about 600 mg to about 800 mg, about 650 mg to about 800 mg, about 700 mg to about 800 mg, about 750 mg to about 800 mg. about 800 mg, about 50 mg to about 750 mg, about 100 mg to about 750 mg, about 150 mg to about 750 mg, about 200 mg to about 750 mg, about 250 mg to about 750 mg, about 300 mg to about 750 mg, about 350 mg to about 750 mg, about 400 mg to about 750 mg, about 450 mg to about 750 mg, about 500 mg to about 750 mg, about 550 mg to about 750 mg, about 600 mg to about 750 mg, about 650 mg to about 750 mg, about 700 mg to about 750 mg, about 50 mg to about 700 mg, about 100 mg to about 1000 mg, about 150 mg to about 10 ... about 100 mg to about 700 mg, about 150 mg to about 700 mg, about 200 mg to about 700 mg, about 250 mg to about 700 mg, about 300 mg to about 700 mg, about 350 mg to about 700 mg, about 400 mg to about 700 mg, about 450 mg to about 700 mg, about 500 mg to about 700 mg, about 550 mg to about 700 mg, about 600 mg to about 700 mg, about 650 mg to about 700 mg, about 50 mg to about 650 mg, about 100 mg to about 650 mg, about 150 mg to about 650 mg, about 200 mg to about 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 50 mg to about 550 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 50 mg to about 500 mg, about 100 mg to about 500mg, about 150mg to about 500mg, about 200mg to about 500mg, about 250mg to about 500mg, about 300mg to about 500mg, about 350mg to about 500mg, about 400mg to about 500mg, about 450mg to about 500mg, about 50mg to about 450mg, about 100mg to about 450mg, about 150mg to about 450mg, about 200mg to about 450mg, about 250mg to about 450mg, about 300mg to about 450mg, about 350mg to about 450mg, about 400mg to about 450mg, about 50 mg to about 400 mg, about 100 mg to about 400 mg, about 150 mg to about 400 mg, about 200 mg to about 400 mg, about 250 mg to about 400 mg, about 300 mg to about 400 mg, about 350 mg to about 400 mg, about 50 mg to about 350 mg, about 100 mg to about 350 mg, about 150 mg to about 350 mg, about 200 mg to about 350 mg, about 250 mg to about 350 mg, about 300 mg to about 350 mg, about 50 mg to about 300 mg, about 100 mg to about 300 mg, about 150 mg to about 300 mg , about 200 mg to about 300 mg, or about 250 mg to about 300 mg fixed dose, for example about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 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,A fixed dose of about 700 mg, about 725 mg, about 750 mg, about 775 mg or about 800 mg is administered to a subject. Values ​​and ranges between the aforementioned values ​​are also part of the present invention.

[0097] The RNAi agent can be administered in multiple doses, such as repeated at regular intervals. For example, the RNAi agent can be administered to a subject at intervals of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 1 year, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months or longer, such as long-term administration. In certain embodiments, the fixed dose can be administered to a subject once or more times per year, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more times. In some embodiments, the fixed dose can be administered to a subject once every about 4 weeks, every about 5 weeks, every about 6 weeks, every about 7 weeks, every about 8 weeks, every about 9 weeks, every about 10 weeks, every about 11 weeks, every about 12 weeks, every about 13 weeks, every about 14 weeks, every about 15 weeks, every about 16 weeks, every about 17 weeks, every about 18 weeks, every about 19 weeks, every about 20 weeks, every about 22 weeks, every about 24 weeks, every about 26 weeks, every about 28 weeks, every about 30 weeks, every about 32 weeks, every about 34 weeks, every about 36 weeks, every about 38 weeks, every about 40 weeks, every about 42 weeks, every about 44 weeks, every about 46 weeks, every about 48 weeks, every about 50 weeks, every about 52 weeks, or more.In certain embodiments, the fixed dose can be administered once a day, once every about 2 days, once every about 3 days, once every about 4 days, once every about 5 days, once every about 6 days, once every about 7 days, once every about 8 days, once every about 9 days, once every about 10 days, once every about 11 days, once every 12 days, once every about 13 days, once every about 14 days, once every about 15 days, once every about 16 days, once every about 17 days, once every about 18 days, once every about 19 days, once every about 20 days, once every about 30 days, once every about 40 days, once every about 50 days, once every about 60 days, once every about 70 days, once every about 80 days, once every about 90 days, once every about 100 days, once every about 110 days, once every about 120 days, once every about 130 days, once every about 140 days, once every about 150 days, once every about 160 days, once every about 170 days, once every about 180 days, once every about 190 days, once every about 200 days, once every about 210 days, once every about 220 days, once every about 230 days, once every about 240 days, once every about 250 days, once every about 260 days, once every about 270 days, once every about 280 days, once every about 290 days, once every about 300 days, once every about 310 days, once every about 321 days, once every about 330 days, once every about 340 days The subject may be administered once every about 120 days, once every about 130 days, once every about 140 days, once every about 150 days, once every about 160 days, once every about 170 days, once every about 180 days, once every about 190 days, once every about 200 days, once every about 210 days, once every about 220 days, once every about 230 days, once every about 240 days, once every about 250 days, once every about 260 days, once every about 270 days, once every about 280 days, once every about 290 days, once every about 300 days, once every about 310 days, once every about 320 days, once every about 330 days, once every about 340 days, once every about 350 days, once every about 360 days, once every about 365 days, or more frequently.

[0098] In certain embodiments, the RNAi agent can be administered in a dosing regimen that includes a "loading phase" administered at shorter intervals, followed by a "maintenance phase" in which the RNAi agent is administered at longer intervals. For example, after a month of weekly or biweekly administration, administration can be repeated monthly for six months or a year or longer, such as long-term administration.

[0099] In some embodiments, the loading phase can include administering the RNAi agent during the first week, first two weeks, first three weeks, first month, etc.

[0100] In certain embodiments, one or more doses can be administered during the loading phase. The loading phase can include a first administration of the RNAi agent on day 1, followed by administration of the RNAi agent on about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, about day 7, about day 8, about day 9, about day 10, about day 11, about day 12, about day 13, about day 14, about day 15, about day 16, about day 17, about day 18, about day 19, about day 20, about day 30, about day 40, about day 50, about day 60, about day 70, about day 80, about day 90, about day 100, about day 110, about day 121, about day 132, about day 143, about day 154, about day 165, about day 176, about day 187, about day 198, about day 209, about day 210, about day 211, about day 212, about day 213, about day 214, about day 215, about day 216, about day 217, about day 218, about day 219, about day 220, about day 221 The one or more RNAi agents are administered after about 0 days, about 130 days, about 140 days, about 150 days, about 160 days, about 170 days, about 180 days, about 190 days, about 200 days, about 210 days, about 220 days, about 230 days, about 240 days, about 250 days, about 260 days, about 270 days, about 280 days, about 290 days, about 300 days, about 310 days, about 320 days, about 330 days, about 340 days, about 350 days, about 360 days, about 365 days or more. In some embodiments, the loading phase may include administering the RNAi agent on day 1, and then administering the RNAi agent a second time after about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 13 weeks, about 15 weeks, about 20 weeks, about 25 weeks, about 26 weeks, about 30 weeks, about 35 weeks, about 40 weeks, about 45 weeks, about 50 weeks, about 52 weeks, or more. In certain embodiments, the loading phase may include administering the RNAi agent on day 1, and then administering the RNAi agent a second time after about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, or more. In some embodiments, the loading phase may include additional doses of the RNAi agent (i.e., more than two doses), and the interval between each administration may be different.

[0101] In certain embodiments, the RNA agent can be administered on day 1 and again on about day 90.

[0102] In certain embodiments, the RNAi agent is administered to a subject during the loading phase at a fixed dose of about 50 mg to about 800 mg, about 100 mg to about 800 mg, about 150 mg to about 800 mg, about 200 mg to about 800 mg, about 250 mg to about 800 mg, about 300 mg to about 800 mg, about 350 mg to about 800 mg, about 400 mg to about 800 mg, about 450 mg to about 800 mg, about 500 mg to about 800 mg, about 550 mg to about 800 mg, about 600 mg to about 800 mg, about 650 mg to about 800 mg, about 700 mg to about 800 mg. About 750 mg to about 800 mg, about 750 mg to about 800 mg, about 50 mg to about 750 mg, about 100 mg to about 750 mg, about 150 mg to about 750 mg, about 200 mg to about 750 mg, about 250 mg to about 750 mg, about 300 mg to about 750 mg, about 350 mg to about 750 mg, about 400 mg to about 750 mg, about 450 mg to about 750 mg, about 500 mg to about 750 mg, about 550 mg to about 750 mg, about 600 mg to about 750 mg, about 650 mg to about 750 mg, about 700 mg to about 750 mg g, about 50 mg to about 700 mg, about 100 mg to about 700 mg, about 150 mg to about 700 mg, about 200 mg to about 700 mg, about 250 mg to about 700 mg, about 300 mg to about 700 mg, about 350 mg to about 700 mg, about 400 mg to about 700 mg, about 450 mg to about 700 mg, about 500 mg to about 700 mg, about 550 mg to about 700 mg, about 600 mg to about 700 mg, about 650 mg to about 700 mg, about 50 mg to about 650 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 50 mg to about 550 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 50 mg to about 500mg, about 100mg to about 500mg, about 150mg to about 500mg, about 200mg to about 500mg, about 250mg to about 500mg, about 300mg to about 500mg, about 350mg to about 500mg, about 400mg to about 500mg, about 450mg to about 500mg, about 50mg to about 450mg, about 100mg to about 450mg, about 150mg to about 450mg, about 200mg to about 450mg, about 250mg to about 450mg, about 300mg to about 450mg, about 350mg to about 450mg, about 40 0 mg to about 450 mg, about 50 mg to about 400 mg, about 100 mg to about 400 mg, about 150 mg to about 400 mg, about 200 mg to about 400 mg, about 250 mg to about 400 mg, about 300 mg to about 400 mg, about 350 mg to about 400 mg, about 50 mg to about 350 mg, about 100 mg to about 350 mg, about 150 mg to about 350 mg, about 200 mg to about 350 mg, about 250 mg to about 350 mg, about 300 mg to about 350 mg, about 50 mg to about 300 mg, about 100 mg to about 300 mg, about 150 mg to about 300 mg, about 200 mg to about 300 mg, or about 250 mg to about 300 mg, for example, a fixed dose of about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 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, about 700 mg, about 725 mg, about 750 mg, about 775 mg or about 800 mg. Values ​​and ranges between the aforementioned values ​​are also part of the present invention.

[0103] In some embodiments, each dose of the RNAi agent administered during the loading phase is the same dose, or may be different.

[0104] In certain embodiments, the RNAi agent can be administered at a dose of about 300 mg on day 1 and at a dose of about 300 mg on or at about day 90.

[0105] In certain embodiments, the maintenance phase may include administering one or more doses of the RNAi agent to the subject. The administration may be once every 1 month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, once every 12 months, once every 13 months, once every 14 months, once every 15 months, once every 16 months, once every 17 months, once every 18 months, once every 19 months, once every 20 months, once every 21 months, once every 22 months, once every 23 months, once every 24 months, once every 25 months. In one embodiment, the maintenance dose is administered to a subject once every 3 months, once every 6 months, once every 9 months, once every 30 months, once every 31 months, once every 32 months, once every 33 months, once every 34 months, once every 35 months, once every 36 months, once every 37 months, once every 38 months, once every 39 months, once every 40 months, once every 41 months, once every 42 months, once every 43 months, once every 44 months, once every 45 months, once every 46 months, once every 47 months, once every 48 months or more. In a specific embodiment, the maintenance dose is administered to a subject once every 3 months, or once every 6 months, or once every 9 months, or once a year.

[0106] In embodiments of the invention, the maintenance phase can include administering the RNAi agent in multiple doses, such as repeated at regular intervals. For example, the RNAi agent can be administered to a subject at intervals of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 1 year, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months or longer, such as long-term administration. In certain embodiments, the fixed dose can be administered to a subject one or more times per year, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more times. In some embodiments, the fixed dose can be administered to a subject once every about 4 weeks, every about 5 weeks, every about 6 weeks, every about 7 weeks, every about 8 weeks, every about 9 weeks, every about 10 weeks, every about 11 weeks, every about 12 weeks, every about 13 weeks, every about 14 weeks, every about 15 weeks, every about 16 weeks, every about 17 weeks, every about 18 weeks, every about 19 weeks, every about 20 weeks, every about 22 weeks, every about 24 weeks, every about 26 weeks, every about 28 weeks, every about 30 weeks, every about 32 weeks, every about 34 weeks, every about 36 weeks, every about 38 weeks, every about 40 weeks, every about 42 weeks, every about 44 weeks, every about 46 weeks, every about 48 weeks, every about 50 weeks, every about 52 weeks, or more.In certain embodiments, the fixed dose can be once a day, once every about 2 days, once every about 3 days, once every about 4 days, once every about 5 days, once every about 6 days, once every about 7 days, once every about 8 days, once every about 9 days, once every about 10 days, once every about 11 days, once every about 12 days, once every about 13 days, once every about 14 days, once every about 15 days, once every about 16 days, once every about 17 days, once every about 18 days, once every about 19 days, once every about 20 days, once every about 30 days, once every about 40 days, once every about 50 days, once every about 60 days, once every about 70 days, once every about 80 days, once every about 90 days, once every about 100 days, once every about 110 days, once every about 120 days, once every about 130 days, once every about 140 days, once every about 150 days, once every about 160 days, once every about 170 days, once every about 180 days, once every about 190 days, once every about 200 days, once every about 210 days, once every about 211 days, once every about 212 days, once every about 213 days, once every about 214 days, once every about 215 days, once every about 216 days, once every about 217 days, once every about 218 days, once every about 219 days, once every about 220 days, once every about 221 days, once every about 222 days The subject may be administered once every about 120 days, once every about 130 days, once every about 140 days, once every about 150 days, once every about 160 days, once every about 170 days, once every about 180 days, once every about 190 days, once every about 200 days, once every about 210 days, once every about 220 days, once every about 230 days, once every about 240 days, once every about 250 days, once every about 260 days, once every about 270 days, once every about 280 days, once every about 290 days, once every about 300 days, once every about 310 days, once every about 320 days, once every about 330 days, once every about 340 days, once every about 350 days, once every about 360 days, once every about 365 days or more.

[0107] In certain embodiments, the maintenance phase can include administering a dose of the RNAi agent to the subject every about 3 months, every about 4 months, every about 6 months, every about 9 months, or about every year.

[0108] One or more maintenance doses may be the same or different from one or more loading doses. For example, a maintenance dose may be about 25 mg to about 800 mg administered to a subject, such as about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, 225 mg, about 250 mg, about 275 mg, about 300 mg, 325 mg, about 350 mg, about 375 mg, about 400 mg, 425 mg, about 450 mg, about 475 mg, about 500 mg, 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, 725 mg, about 750 mg, about 775 mg, or about 800 mg. Values ​​and ranges between the aforementioned values ​​are also part of the present invention.

[0109] In certain embodiments, the maintenance phase can include administering a 300 mg dose of the RNAi agent to the subject every 3 months, every 4 months, every 6 months, every 9 months, or yearly.

[0110] In certain embodiments, the RNAi agent can be administered in a dosing regimen comprising (1) a loading phase, in which the RNAi agent is administered at a dose of 300 mg on day 1 and approximately day 90; and (2) a maintenance phase, in which 300 mg of the RNAi agent is administered every approximately 6 months after administration on approximately day 90.

[0111] The RNAi agent can be administered to a subject using any administration method known in the art, including but not limited to subcutaneously, intravenously, intramuscularly, intraocularly, intrabronchially, intrapleurally, intraperitoneally, intraarterially, lymphatically, cerebrospinally, and any combination thereof. In a preferred embodiment, the RNAi agent is administered subcutaneously.

[0112] In some embodiments, the depot injection is administered by a depot injection. The depot injection may include a subcutaneous injection or an intramuscular injection. In certain embodiments, the depot injection is a subcutaneous injection.

[0113] In some embodiments, administration is by a pump. The pump can be an external pump or a surgically implanted pump. In certain embodiments, the pump is an osmotic pump implanted subcutaneously. In other embodiments, the pump is an infusion pump. An infusion pump can be used for intravenous, subcutaneous, arterial or epidural infusion. In certain 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.

[0114] Other modes of administration include epidural, intracerebral, intraventricular, intranasal, intraarterial, intracardiac, intraosseous infusion, intrathecal and intravitreal, pulmonary, oral, topical, intratracheal, epidermal or transdermal. The mode of administration can be selected based on whether local or systemic treatment is desired and based on the area to be treated. The route and site of administration can be selected to enhance targeting.

[0115] RNAi agent can be used by intravenous infusion during the following time, for example, at 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, 35, 40, 45, 50, 55 or 60 minutes or longer. For example, it is possible to repeat administration regularly, for example, once a week, once in 2 weeks (i.e., every two weeks) for a total of 1 month, 2 months, 3 months, 4 months or longer. After the initial treatment regimen, treatment can be applied less frequently. For example, once a week or once in 2 weeks after three months, it is possible to repeat administration once a month, for 6 months or one year or longer.

[0116] RNAi agent can be used in combination with one or more other preventive or therapeutic agents. RNAi agent can be in the same preparation with one or more other preventive or therapeutic agents, or RNAi agent can be in different preparations with one or more other preventive or therapeutic agents. RNAi agent and one or more other preventive or therapeutic agents can be used simultaneously, or each other in a short time, for example, in about 1 minute, or about 5 minutes, or about 15 minutes, or about 30 minutes, or about 60 minutes, or about 2 hours, or about 3 hours, or about 4 hours, or about 6 hours, or about 9 hours, or about 12 hours, or about 15 hours, or about 18 hours, or about 24 hours.

[0117] The one or more other preventive or therapeutic agents include those known to treat lipid disorders, such as hypercholesterolemia, atherosclerosis or dyslipidemia. For example, the one or more other preventive or therapeutic agents can be HMG-CoA reductase inhibitors, fibrates, bile acid sequestrants, nicotinic acid, antiplatelet agents, angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists, acyl CoA cholesterol acetyltransferase (ACAT) inhibitors, cholesterol absorption inhibitors, cholesterol ester transfer protein (CETP) inhibitors, microsomal triglyceride transfer protein (MTTP) inhibitors, cholesterol regulators, bile acid regulators, peroxisome proliferator-activated receptor (PPAR) agonists, gene-based therapy, composite vascular protectors (such as AGI-1067 from Atherogenics), glycoprotein IIb / IIIa inhibitors, aspirin or aspirin-like compounds, IBAT inhibitors, squalene synthase inhibitors or monocyte chemoattractant protein (MCP)-I inhibitors. As a further example, the one or more additional prophylactic or therapeutic agents can be an anti-PCSK9 antibody, such as alirocumab (Praluent), evolocumab (Repatha), bococizumab, lodelcizumab, ralpancizumab, RG7652, LY3015014, LPD1462, AX1, ALD306, or Ig1-PA4.

[0118] Subjects to be administered RNAi agents

[0119] The subject of administering the RNAi agent can be a human or non-human animal, in certain embodiments a vertebrate, in a specific embodiment a mammal. In a preferred embodiment, the subject is a human. In certain embodiments, the subject is an adult. In some embodiments, the subject is a patient.

[0120] The subject's baseline LDL-C level is about 70 mg / dl or higher. In certain embodiments, the baseline LDL-C level is about 80 mg / dl or higher, or about 90 mg / dl or higher, or about 100 mg / dl or higher, or about 110 mg / dl or higher, or about 120 mg / dl or higher, or about 130 mg / dl or higher, or about 140 mg / dl or higher, or about 150 mg / dl or higher, or about 160 mg / dl or higher, or about 170 mg / dl or higher, or about 180 mg / dl or higher , or about 190 mg / dl or more, or about 200 mg / dl or more, or about 210 mg / dl or more, or about 220 mg / dl or more, or about 230 mg / dl or more, or about 240 mg / dl or more, or about 250 mg / dl or more, or about 260 mg / dl or more, or about 270 mg / dl or more, or about 280 mg / dl or more, or about 290 mg / dl or more, or about 300 mg / dl or more.

[0121] In some embodiments, the subject is in need of lowering of LDL-C. A subject in need of lowering of LDL-C has an LDL-C level of about 50 mg / dl or more, or about 60 mg / dl or more, or about 70 mg / dl or more, or about 80 mg / dl or more, or about 90 mg / dl or more, or about 100 mg / dl or more, or about 110 mg / dl or more, or about 120 mg / dl or more, or about 130 mg / dl or more, or about 140 mg / dl or more, or about 150 mg / dl or more, or about 160 mg / dl or more, or about 170 dl or more, or about 180 mg / dl or more, or about 190 mg / dl or more, or about 200 mg / dl or more, or about 210 mg / dl or more, or about 220 mg / dl or more, or about 230 mg / dl or more, or about 240 mg / dl or more, or about 250 mg / dl or more, or about 260 mg / dl or more, or about 270 mg / dl or more, or about 280 mg / dl or more, or about 290 mg / dl or more, or about 300 mg / dl or more.

[0122] In some embodiments, the subject has no active liver disease. Active liver disease can be determined by measuring one or more biochemical parameters of alanine aminotransferase, aspartate aminotransferase, and total bilirubin. The biochemical parameters can be measured at baseline. In certain embodiments, the subject's alanine aminotransferase level may be no higher than two times (2×) the ULN. In some embodiments, the subject's alanine aminotransferase level may be about 1.5 times the ULN, or about the same as or lower than the ULN.

[0123] In some embodiments, the subject's aspartate aminotransferase level may be no greater than two times (2×) the ULN. In certain embodiments, the subject's aspartate aminotransferase level may be about 1.5 times the ULN, or about the same as or below the ULN.

[0124] In some embodiments, the subject's total bilirubin level may be no greater than 1.5 times the ULN. In certain embodiments, the subject's total bilirubin level may be about the same as or lower than the ULN.

[0125] Alternatively, the subject may have active liver disease. In certain embodiments, the subject's alanine aminotransferase level may be greater than two times (2×) ULN, such as about 2.5 times ULN, or about 3 times ULN, or about 3.5 times ULN, or about 4 times ULN, or more. In some embodiments, the subject's aspartate aminotransferase level may be greater than two times (2×) ULN, such as about 2.5 times ULN, or about 3 times ULN, or about 3.5 times ULN, or about 4 times ULN, or more. In certain embodiments, the subject's total bilirubin level may be greater than 1.5 times ULN, such as about 2 times ULN, or about 2.5 times ULN, or about 3 times ULN, or about 3.5 times ULN, or about 4 times ULN, or more.

[0126] Those skilled in the art are aware of the ULNs for alanine aminotransferase, aspartate aminotransferase, and total bilirubin.

[0127] In some embodiments, the subject may be on background lipid-lowering therapy. The subject may continue to receive background lipid-lowering therapy while administering the RNAi agent, or the subject may stop background lipid-lowering therapy. In certain embodiments, background lipid-lowering therapy may be statin therapy, for example including but not limited to atorvastatin, pravastatin, simvastatin, lovastatin, fluvastatin, cerivastatin, rosuvastatin and pitavastatin. The subject may receive maximum tolerated statin therapy, and the maximum dose of statins is known in the art.

[0128] In some embodiments, the subject may be on another type of lipid-lowering therapy, such as ezetimibe, LDL apheresis, bile acid sequestrants, niacin, and fibric acid therapy.

[0129] In other embodiments, the subject is not on background lipid-lowering therapy.

[0130] In certain embodiments, the subject may be in a condition where their diet is designed, for example, to improve lipid levels. Such diets are known in the art. For example, a diet designed to improve lipid levels may include eating lean cuts of meat, removing fat from meat, not eating fried foods or high-fat sauces, not eating egg yolks, using low-fat dairy products such as skim milk or 1% milk, low-fat frozen yogurt, low-fat ice cream, and low-fat cheese, and eating fiber sources such as fruits and vegetables.

[0131] The subject's triglyceride level may be no higher than about 400 mg / dl. For example, the subject's baseline triglyceride level may be about 380 mg / dl, or about 360 mg / dl, or about 340 mg / dl, or about 320 mg / dl, or about 300 mg / dl, or about 280 mg / dl, or about 260 mg / dl, or about 240 mg / dl, or about 220 mg / dl, or about 200 mg / dl, or about 180 mg / dl, or about 160 mg / dl, or about 140 mg / dl, or about 120 mg / dl, or about 100 mg / dl, or about 80 mg / dl, or about 60 mg / dl.

[0132] In other embodiments, the subject's triglyceride level may be above about 400 mg / dl. For example, the subject's baseline triglyceride level may be about 420 mg / dl, or about 440 mg / dl, or about 460 mg / dl, or about 480 mg / dl, or about 500 mg / dl, or about 520 mg / dl, or about 540 mg / dl, or about 560 mg / dl, or about 580 mg / dl, or about 600 mg / dl.

[0133] In some embodiments, triglyceride levels are measured at baseline.

[0134] The estimated glomerular filtration rate (eGFR) of the subject may be at least about 30 ml / min. For example, the eGFR may be about 30 ml / min, or about 35 ml / min, or about 40 ml / min, or about 45 ml / min, or about 50 ml / min, or higher.

[0135] In some embodiments, the subject suffers from hyperlipidemia, such as hypercholesterolemia. In certain embodiments, the subject suffers from heterozygous familial hypercholesterolemia. In other embodiments, the subject suffers from homozygous familial hypercholesterolemia.

[0136] Alternatively, the subject may have an eGFR of less than about 30 ml / min, such as an eGFR of about 25 ml / min, or about 20 ml / min, or about 15 ml / min.

[0137] In some embodiments, eGFR is measured at baseline.

[0138] The subject may not have poorly controlled type 2 diabetes, e.g., type 2 diabetes that is not properly treated or resolved, or not at all. Poorly controlled diabetes can be identified by a glycated hemoglobin A1c level of at least about 10%. Thus, the subject may have a glycated hemoglobin A1c level of less than about 10%, e.g., about 9%, or about 8%, or about 7%, or about 6%, or about 5% or less.

[0139] In other embodiments, the subject may have poorly controlled type 2 diabetes as evidenced by a glycated hemoglobin A1c level of at least about 10%, such as about 15%, or about 20%, or about 25%, or about 30% or more.

[0140] In some embodiments, glycated hemoglobin A1c levels are measured at baseline.

[0141] The subject may not have heart failure characterized by New York Heart Association (NYHA) class II, III or IV. Alternatively, the subject may have heart failure characterized by New York Heart Association (NYHA) class II, III or IV.

[0142] The subject may have a ventricular ejection fraction of 30% or more, such as about 35% or about 40%, or about 45%, or about 50% or more. In other embodiments, the subject may have a ventricular ejection fraction of less than 30%, such as about 25%, or about 20%, or about 15% or less. The ventricular ejection fraction may be measured as a baseline measurement, or may be the most recently known ventricular ejection fraction that was previously measured.

[0143] The object may not have experienced major adverse cardiac events within six months of applying the RNAi agent. Major adverse cardiac events include, but are not limited to, death, non-fatal myocardial infarction, severe recurrent ischemia, stroke, symptomatic pulmonary embolism, and hemorrhage. In some embodiments, major adverse cardiac events do not occur within about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, about 24 months, about 30 months, about 36 months or longer of applying the RNAi agent, including no major cardiac events. In another embodiment, the object may experience major cardiac events within six months of applying the RNAi agent, including about 5 months, or about 4 months, or about 3 months, or about 2 months, or about 1 month, or about 4 weeks, or about 3 weeks, or about 2 weeks, or about 1 week, or earlier.

[0144] The object may not suffer from severe hypertension. In some embodiments, severe hypertension can be determined by a systolic pressure exceeding about 180mmHg and / or a diastolic pressure exceeding about 110mmHg, such as a systolic pressure of about 190mmHg, or about 200mmHg, or about 220mmHg, or about 240mmHg, or about 260mmHg, or about 280mmHg, or about 300mmHg, or higher; such as a diastolic pressure of about 120mmHg, or about 140mmHg, or about 160mmHg, or about 180mmHg, or about 200mmHg, or about 220mmHg, or about 240mmHg or higher. In certain embodiments, the object may not have uncontrolled severe hypertension. Uncontrolled severe hypertension can be identified by a systolic pressure exceeding about 180mmHg and / or a diastolic pressure exceeding about 110mmHg despite antihypertensive treatment. Alternatively, the subject may suffer from severe hypertension and / or uncontrolled severe hypertension.

[0145] In some embodiments, blood pressure is measured at baseline.

[0146] The subject may not have any history of hemorrhagic stroke. Alternatively, the subject may have experienced a hemorrhagic stroke.

[0147] The subject may be free of arrhythmia within 3 months after administration of the RNAi agent, including within about 4 months, or within about 5 months, or within about 6 months, or within about 7 months, or within about 8 months, or within about 9 months, or within about 10 months, or within about 11 months, or within about 12 months, or longer. In other embodiments, the subject suffers from arrhythmia within 3 months of administering the RNAi agent, but the arrhythmia is controlled, for example, by drug therapy or by ablation. Alternatively, the subject may suffer from arrhythmia, for example, that is not controlled by drug therapy or by ablation within three months.

[0148] In certain embodiments, the subject has one or more of the following: a history of myocardial infarction, stable or unstable angina, coronary or other arterial revascularization, stroke, transient ischemic attack, or peripheral arterial disease of atherosclerotic origin; is male; has a family history of heart disease, ASCVD, or ASCVD risk equivalent; is a smoker; is physically inactive; has high blood pressure; has high blood cholesterol; has diabetes and prediabetes; is overweight or obese; has a history of preeclampsia during pregnancy; has uncontrolled stress and / or anger; is postmenopausal; has an unhealthy diet, such as a diet high in salt, saturated fat, trans fat, cholesterol, and / or refined sugar; is 55 years of age or older; has sleep apnea; has anemia; or a combination of these.

[0149] In some embodiments, the subject may have a cognitive impairment, such as Alzheimer's disease, dementia, memory loss, etc. In other embodiments, the subject does not have any cognitive impairment.

[0150] RNAi Agents

[0151] The RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand, forming a double-stranded region. The antisense strand comprises the nucleotide sequence of SEQ ID NO: 1, i.e., 5'-ACAAAAGCAAAACAGGUCUAGAA-3'. The sense strand comprises the nucleotide sequence of SEQ ID NO: 2, i.e., 5'-CUAGACCUGUTUUGCUUUUGU-3'.

[0152] In embodiments of the invention, at least one nucleotide on the antisense strand, at least one nucleotide on the sense strand, or at least one nucleotide on both the antisense strand and the sense strand is a modified nucleotide. In some embodiments, substantially all nucleotides of the antisense strand, substantially all nucleotides of the sense strand, or substantially all nucleotides of both the antisense strand and the sense strand are modified nucleotides. In certain embodiments, all nucleotides of the antisense strand, all nucleotides of the sense strand, or all nucleotides of both the antisense strand and the sense strand are modified nucleotides.

[0153] The nucleotides may be modified using techniques and methods known in the art. The modifications may include those described in PCT Application PCT / US2016 / 048666 filed on August 25, 2016 and U.S. Patent Application Serial No. 14 / 650,128 filed on June 5, 2015, which are incorporated herein by reference.

[0154] In some embodiments, one or more nucleotides of the antisense strand or sense strand may be 2'-O-methyl (2'-OMe) or 2'-fluoro (2'-F) modified. In certain embodiments, one or more nucleotides of the antisense strand or sense strand may be linked by a 3'-5' phosphodiester bond.

[0155] In some embodiments, the double-stranded RNA comprises a ligand. In certain embodiments, the ligand is conjugated to the 3' end of the sense strand of the double-stranded RNA. Examples of ligands are described in PCT application PCT / US2016 / 048666 filed on August 25, 2016 and U.S. application serial number 14 / 650,128 filed on June 5, 2015, which are incorporated herein by reference. In certain embodiments, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0156] In certain embodiments, the RNAi agent is a double-stranded ribonucleic acid comprising an antisense strand of the nucleotide sequence shown in 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 3) and a sense strand of the nucleotide sequence shown in 5'-csusagacCfuGfudTuugcuuuugu-3' (SEQ ID NO: 4), wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C or U; Af, Gf, Cf or Uf are 2'-fluoro A, G, C or U; dT is 2'-deoxythymidine; s is a phosphorothioate bond. The double-stranded ribonucleic acid has a covalently attached triantennary GalNAc ligand.

[0157] In some embodiments, the RNAi agent is Figure 1 shown.

[0158] The RNAi agent can be in a "naked" form, or in a "free RNA". The "naked" form refers to the absence of a pharmaceutical composition. For example, the naked RNAi agent can be in a suitable buffer solution, which may include, for example, acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. In some embodiments, the buffer solution is phosphate buffered saline (PBS). The pH and osmotic pressure of the buffer solution containing the RNAi agent can be adjusted to make it suitable for administration to a subject. In certain embodiments, the RNAi agent can be in water for injection.

[0159] Alternatively, the RNAi agent can be formulated into a pharmaceutical composition comprising the RNAi agent and one or more pharmaceutically acceptable carriers. The pharmaceutical composition can be formulated based on the mode of delivery. For example, the composition can be formulated for systemic administration by parenteral delivery, such as by intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or the composition can be formulated for direct delivery to the brain parenchyma, such as by infusion into the brain such as by continuous pump infusion; or the composition can be formulated. Alternatively, the composition can be formulated for oral administration; topical (e.g., by transdermal patch); pulmonary, such as by inhalation or insufflation of powder or aerosol, including by nebulizer; intratracheal; intranasal; epidermal or transdermal administration.

[0160] Compositions and preparations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include, but are not limited to, sterile aqueous solutions, which may also contain buffers, diluents and other suitable additives such as penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. Compositions and preparations for oral administration may include, but are not limited to, powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or mini-tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or adhesives may also be used. Pharmaceutical compositions and preparations for topical administration may include, but are not limited to, transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Pharmaceutical compositions of the present invention may also include, but are not limited to, solutions, emulsions and liposome-containing preparations. Compositions may be formulated into various forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories and enemas.

[0161] Examples of pharmaceutically acceptable carriers and specific compositions are described in PCT Application No. PCT / US2016 / 048666, filed on August 25, 2016, and U.S. Application Serial No. 14 / 650,128, filed on June 5, 2015, which are incorporated herein by reference.

[0162] In certain embodiments, the RNAi agent can be formulated at a suitable concentration so that administration delivers a fixed dose of about 50 mg to about 800 mg, about 100 mg to about 800 mg, about 150 mg to about 800 mg, about 200 mg to about 800 mg, about 250 mg to about 800 mg, about 300 mg to about 800 mg, about 350 mg to about 800 mg, about 400 mg to about 800 mg, about 450 mg to about 800 mg, about 500 mg to about 800 mg, about 550 mg to about 800 mg, about 600 mg to about 800 mg, about 650 mg to about 800 mg, about 70 .... 0 mg to about 800 mg, about 750 mg to about 800 mg, about 50 mg to about 750 mg, about 100 mg to about 750 mg, about 150 mg to about 750 mg, about 200 mg to about 750 mg, about 250 mg to about 750 mg, about 300 mg to about 750 mg, about 350 mg to about 750 mg, about 400 mg to about 750 mg, about 450 mg to about 750 mg, about 500 mg to about 750 mg, about 550 mg to about 750 mg, about 600 mg to about 750 mg, about 650 mg to about 750 mg, about 700 mg to about 750 mg, about 50 mg to about 700 mg, about 100 mg to about 700 mg, about 150 mg to about 700 mg, about 200 mg to about 700 mg, about 250 mg to about 700 mg, about 300 mg to about 700 mg, about 350 mg to about 700 mg, about 400 mg to about 700 mg, about 450 mg to about 700 mg, about 500 mg to about 700 mg, about 550 mg to about 700 mg, about 600 mg to about 700 mg, about 650 mg to about 700 mg, about 50 mg to about 650 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 50 mg to about 550 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 50 mg to about 500mg, about 100mg to about 500mg, about 150mg to about 500mg, about 200mg to about 500mg, about 250mg to about 500mg, about 300mg to about 500mg, about 350mg to about 500mg, about 400mg to about 500mg, about 450mg to about 500mg, about 50mg to about 450mg, about 100mg to about 450mg, about 150mg to about 450mg, about 200mg to about 450mg, about 250mg to about 450mg, about 300mg to about 450mg, about 350mg to about 450mg, about 40 0 mg to about 450 mg, about 50 mg to about 400 mg, about 100 mg to about 400 mg, about 150 mg to about 400 mg, about 200 mg to about 400 mg, about 250 mg to about 400 mg, about 300 mg to about 400 mg, about 350 mg to about 400 mg, about 50 mg to about 350 mg, about 100 mg to about 350 mg, about 150 mg to about 350 mg, about 200 mg to about 350 mg, about 250 mg to about 350 mg, about 300 mg to about 350 mg, about 50 mg to about 300 mg, about 100 mg to about 300 mg, about 150 mg to about 300 mg, about 200 mg to about 300 mg, or about 250 mg to about 300 mg, for example, a fixed dose of about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 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, about 700 mg, about 725 mg, about 750 mg, about 775 mg or about 800 mg. Values ​​and ranges between the aforementioned values ​​are also part of the present invention.

[0163] In some embodiments, the RNAi agent can be formulated into a suitable concentration, thereby administering a suitable volume of the composition to the subject, such as about 1.0 ml, about 1.1 ml, about 1.2 ml, about 1.3 ml, about 1.4 ml, about 1.5 ml, about 1.6 ml, about 1.7 ml, about 1.8 ml, about 1.9 ml, or about 2.0 ml of the pharmaceutical composition. For example, in one embodiment, the RNAi agent is formulated in a suitable pharmaceutical formulation at about 200 mg / ml, thereby administering about 1.5 ml of the formulation to the subject to provide a 300 mg fixed dose of the RNAi agent.

[0164] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein, as well as sequence tables and drawings are incorporated by reference in their entirety. If a conflict occurs, the present specification including definitions will prevail. In addition, the materials, methods and embodiments are illustrative only and are not intended to be restrictive. Example

[0165] A placebo-controlled, double-blind, randomized trial was conducted to compare the effects of single or multiple subcutaneous injections of different doses of an RNAi agent in patients with high cardiovascular risk and high LDL-C.

[0166] method

[0167] The RNAi agent is a double-stranded ribonucleic acid comprising an antisense strand of a 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 3) nucleotide sequence and a sense strand of a 5'-csusagacCfuGfudTuugcuuuugu-3' nucleotide sequence (SEQ ID NO: 4), wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, or U; Af, Gf, Cf, or Uf are 2'-fluoro A, G, C, or U; dT is 2'-deoxythymidine; s is a phosphorothioate bond. The double-stranded ribonucleic acid has a covalently linked triantennary GalNAc ligand.

[0168] The primary objective of this trial is to evaluate the effect of RNAi treatment on LDL-C levels at day 180. Secondary objectives are to evaluate the effect of RNAi on: (i) LDL-C levels at day 90; (ii) LDL-C levels at other time points; (iii) changes in PCSK9 levels over time; (iv) other lipids, lipoproteins, apolipoproteins; (v) the proportion of patients meeting pre-specified global lipid guidelines; (vi) individual responsiveness to different doses; (vii) the duration of lipid-lowering effect of different doses; and (viii) the safety and tolerability of the RNAi. This study also collects / evaluates the effect of RNAi on: (a) cardiovascular events, such as cardiovascular death, non-fatal myocardial infarction, resuscitated cardiac arrest, and non-fatal stroke (ischemic and hemorrhagic); and (b) anti-drug antibodies to the RNAi agent.

[0169] This study included 501 patients with ASCVD or ASCVD-equivalent risk (e.g., diabetes and familial hypercholesterolemia) and elevated LDL-C. Patients were included if they met all of the following inclusion criteria prior to randomization:

[0170] (a) Male or female patients aged ≥18 years;

[0171] (b) history of ASCVD or ASCVD equivalent risk (symptomatic atherosclerosis, type 2 diabetes, familial hypercholesterolemia, including patients whose target LDL-C is <100 mg / dl as assessed by the Framingham Risk Score* or equivalent for 10-year risk of cardiovascular events;

[0172] (c) serum LDL-C ≥1.8 mmol / l (≥70 mg / dl) in ASCVD patients or ≥2.6 mmol / l (≥100 mg / dl) in ASCVD patients with equivalent risk at screening;

[0173] (d) fasting triglyceride <4.52 mmol / l (<400 mg / dl) at screening;

[0174] (e) glomerular filtration rate >30 ml / min calculated by estimated glomerular filtration rate (eGFR) using standardized local clinical methods;

[0175] (f) Patients taking statins should receive the maximum tolerated dose (at the discretion of the investigator);

[0176] (g) Patients receiving lipid-lowering therapy (e.g., statins and / or ezetimibe) should be on stable doses for ≥30 days and then screened with no planned medication or dose changes during study participation; and

[0177] (h) Be willing and able to give informed consent before any research-related procedures are initiated and be willing to comply with all required research procedures.

[0178] *Framingham risk score > 20%

[0179] Patients will be excluded from the study if they have any of the following exclusion criteria immediately prior to randomization:

[0180] (a) any uncontrolled or serious illness, or any medical or surgical condition that may interfere with participation in a clinical investigation and / or that places the patient at significant risk (in the judgment of the Investigator [or his or her delegate]) if the patient participates in a clinical investigation;

[0181] (b) any known underlying disease or surgical, physical or medical condition that the Investigator (or his or her representative) believes may interfere with the interpretation of the clinical study results;

[0182] (c) New York Heart Association (NYHA) class II, III, or IV heart failure or a recently known left ventricular ejection fraction of <30%;

[0183] (d) arrhythmia not controlled by medication or ablation within 3 months before randomization;

[0184] (e) any history of hemorrhagic stroke;

[0185] (f) major adverse cardiac events within 6 months before randomization;

[0186] (g) uncontrolled severe hypertension: systolic blood pressure >180 mmHg or diastolic blood pressure >110 mmHg before randomization despite antihypertensive treatment;

[0187] (h) poorly controlled type 2 diabetes, i.e., glycosylated hemoglobin A1c (HbA1c) > 10.0% before randomization;

[0188] (i) Active liver disease confirmed by repeated measurements at least one week apart at screening, defined as any known current infection, neoplasm, or metabolic lesion of the liver or unexplained elevation of alanine aminotransferase, aspartate aminotransferase greater than 2 times the ULN, or total bilirubin greater than 1.5 times the ULN;

[0189] (j) severe comorbidities where the patient's life expectancy is shorter than the duration of the trial (e.g., acute systemic infection, cancer, or other serious illness); this includes all cancers other than treated basal cell carcinoma occurring more than 5 years before screening;

[0190] (k) women who are pregnant or breastfeeding, or women of childbearing potential who do not wish to use at least two methods of contraception (oral contraceptives, barrier methods, approved contraceptive implants, long-term injectable contraceptives, intrauterine contraceptive devices, or tubal ligation)**; women who are more than 2 years postmenopausal, defined as ≥1 year since the last menstrual period, and women who are younger than 55 years of age and have a negative pregnancy within 24 hours of randomization or are surgically infertile are exempt from this exclusion;

[0191] (l) men who were unwilling to use an acceptable method of birth control (i.e., condoms containing spermicide) throughout the study period;

[0192] (m) known history of alcohol and / or drug abuse;

[0193] (n) Treatment with other investigational medicinal products or devices within 30 days or 5 half-lives, whichever is longer;

[0194] (o) Use of other investigational medicinal products or devices during the study;

[0195] (p) Any conditions that the researcher believes may interfere with the research, such as but not limited to:

[0196] (i) Unsuitable for this study, including patients who are unable to communicate or cooperate with the researchers;

[0197] (ii) Inability to understand protocol requirements, instructions, and study-related limitations, the nature, scope, and possible consequences of the study (including patients suspected of cooperation due to drug abuse or alcohol dependence);

[0198] (iii) unlikely to comply with protocol requirements, instructions, and study-related limitations (e.g., uncooperative attitude, inability to return for follow-up, and unlikely to complete the study);

[0199] (iv) have any medical or surgical condition that the investigator believes would increase the risk for patients participating in the study;

[0200] (v) related to the person directly conducting the research or his / her relatives;

[0201] (vi) any known cognitive impairment (e.g. Alzheimer's disease); and

[0202] (q) Previous or current treatment with monoclonal antibodies against PCSK9 (within 90 days of screening).

[0203] **Over the entire study period.

[0204] Patients were screened and randomized to 6 RNAi agent groups and a placebo group. Treatment assignment was stratified by country and current use of statins or other lipid-repairing therapies. Each patient received: (i) one or two injections of RNAi agent or placebo only on day 1, or (ii) a single injection on day 1 and a second injection of RNAi agent or placebo on day 90.

[0205] The study design is summarized in Figure 2 Shown in. On day 1, all eligible patients were randomized and received the first subcutaneous administration of RNAi agent or placebo. RNAi agent was administered as a single subcutaneous injection (dose: 200 mg, 300 mg) or two injections (dose: 500 mg). Patients who were randomized to receive the second dose received a second injection of RNAi agent or placebo on day 90.

[0206] Placebo was administered by one or two subcutaneous injections of saline solution. The placebo volume was matched to the RNAi agent volume within each dose and injection regimen, but not between injection regimens. For example, the 200 mg placebo group received 1.0 ml of placebo, while the 300 mg placebo group received 1.5 mL of placebo.

[0207] The duration of patient participation in the study was approximately 224 days, including screening, study drug administration, single or multiple injections, and a follow-up period to day 210. If additional follow-up is required, the maximum duration of participation is 374 days. The end-of-study assessment was performed on day 210, except for those patients whose LDL-C levels had not returned to within 20% of the starting level; these patients were assessed on day 210 and followed up every 30 days thereafter until day 360, or LDL-C returned to within 20% of the starting level (whichever occurred first).

[0208] For the single-dose group (one or two injections on Day 1), patients followed the following schedule:

[0209] (a) Screening: Day -14 to Day -1,

[0210] (b) Randomization, start of study medication: Day 1

[0211] (c) Treatment phase: Day 1

[0212] (d) Follow-up:

[0213] (i) Follow-up: Day 2 to Day 210; EOS on Day 210

[0214] (ii) Additional follow-up (for patients whose LDL-C levels have not recovered to more than 80% of baseline values; patients will return for monthly follow-up until this level is reached or until Day 360): Days 240, 270, 300, 330, and 360.

[0215] For the two-dose group (one injection each on Day 1 and Day 90), patients followed the following schedule:

[0216] (a) Screening: Day -14 to Day -1,

[0217] (b) Randomization, start of study medication: Day 1

[0218] (c) Treatment phase: Day 1 to Day 90

[0219] (d) Follow-up:

[0220] (i) Follow-up: Day 91 to Day 210; EOS on Day 210

[0221] (ii) Additional follow-up (for patients whose LDL-C levels have not recovered to more than 80% of baseline values; patients will return for monthly follow-up until this level is reached or until Day 360): Days 240, 270, 300, 330, and 360.

[0222] The primary endpoint was assessed by determining the percent change from baseline in LDL-C to day 180.

[0223] Secondary endpoints were assessed by determining: (i) percent change from baseline in LDL-C at day 90; (ii) percent change from baseline in LDL-C at days 14, 30, 60, 120, 150, and 210; (iii) proportion of patients in each group with LDL-C greater than 80% of baseline value at days 180 and 210; (iv) duration of treatment in which patients’ LDL-C or PCSK9 protein recovered to 80% or greater of baseline levels; (v) individual responders defined as achieving a therapeutic LDL-C level of <25 mg / dl, <5 mg / dl, or <10 mg / dl at days 90, 120, and 180. The results of the present study showed that the number of patients with LDL-C levels in each group decreased by 0 mg / dl, <70 mg / dl, and <100 mg / dl at each visit; (vi) the proportion of patients in each group with a reduction of greater than or equal to 50% from baseline in LDL-C levels at days 90, 120, and 180; (vii) the percent change from baseline in PCSK9 levels at days 14, 30, 60, 90, 104, 120, 150, 180, and 210; (viii) the percent change from baseline in other lipids, lipoproteins, and apolipoproteins at each visit through day 210; and (ix) the proportion of patients in each group whose ASCVD risk level reached the global lipid restoration goal.

[0224] Efficacy assessments included measuring the effects of RNAi agents on LDL-C lipid and lipoprotein levels, including total cholesterol, triglycerides, HDL-C, non-HDL-C, VLDL-C, Apo-A1, Apo-B, Lp(a), CRP, and PCSK9.

[0225] Adverse events, serious adverse events, vital signs, clinical laboratory values ​​(hematology, coagulation tests, chemistry analysis, and urinalysis), and electrocardiograms (ECGs) were collected at designated follow-up visits through the EOS visit (day 210). Adverse events, serious adverse events, and clinical laboratory values ​​continued to be assessed during additional monthly follow-up (for patients whose LDL-C levels had not recovered to >80% of baseline values). Cardiovascular events were reported as adverse events with pooled cardiovascular event information, such as cardiovascular death, nonfatal myocardial infarction, major coronary events (CHD death, resuscitated cardiac arrest, nonfatal myocardial infarction), ischemic stroke, and hemorrhagic stroke.

[0226] In addition, anti-drug antibodies to RNAi agents were evaluated. The formation of anti-drug antibodies was evaluated on day 1 (before and 4 hours after injection) and days 30, 60, 90, 120, 150, 180 (days 150 and 180 only in patients who received the second dose of RNAi agent) and day 210 or until any anti-drug antibody response became negative during the study duration. For patients whose LDL-C levels did not return to >80% of baseline values, the formation of anti-drug antibodies was evaluated when LDL-C returned to normal limits or at 1-year follow-up.

[0227] An independent Data Monitoring Committee (DMC) reviewed safety data starting after the first 40 patients received their first injection of RNAi or placebo and completing the 14-day follow-up. Thereafter, the DMC reviewed safety data every two months until the end of the trial.

[0228] The primary endpoint was analyzed by the least mean square mean percentage change from baseline to the 180th day. This was calculated by a repeated measurement linear effect model, including the study group, baseline value, scheduled follow-up, and the interaction between the study group and the planned follow-up. The PROC MIXED program in the SAS software with an autoregressive variance structure was used for analysis, with treatment inclusion at each visit as a fixed effect and patients as random effects. For the primary and secondary endpoints, multiple comparisons were performed using the Dunnett test in the six RNAi groups and the placebo control group to adjust the P value. Each dosing regimen (i.e., single dose and two doses) was analyzed separately. For a two-sided test, the type I error significance level was 0.05.

[0229] All patients who received at least one dose of RNAi agent or placebo were included in the safety analysis (safety population). The scheduled modified intention-to-treat population was defined as all randomly assigned patients who received at least one dose of study drug and had baseline and 180-day follow-up LDL-C level measurements available. Imputation was used for patients with missing data for intention-to-treat analysis.

[0230] Time course data are presented as means with 95% confidence intervals. Variations in responses between patients are depicted graphically using waterfall plots. Analyses were performed using SAS software, version 9.2 and higher (SAS Institute).

[0231] result

[0232] Baseline demographics and history are shown in Tables 1 and 2, indicating that the patients were well balanced. In the study, 73% of the patients were treated with statins and 31% with ezetimibe.

[0233] A single administration of all RNAi doses on day 1 resulted in mean reductions in LDL-C ranging from 44.5% to 51.5% below baseline on day 30 (see Figure 3 and 4 ), with a nadir at approximately day 60. A single 200 mg dose of the RNAi agent reduced LDL-C levels by more than 40% from baseline at days 30, 60, and 90, and by 25% at day 270. Figure 3 A single administration of the 300 mg and 500 mg doses of the RNAi agent reduced LDL-C by more than 40% below baseline from day 15 to day 150 and by less than 30% below baseline by day 270 ( Figure 3 For each RNAi dose strength, LDL-C remained more than 20% below baseline by day 360 ( Figure 3 ). The least mean square reduction after a single dose of the RNAi agent (reduction of 27.9%-41.9%) was significantly greater than that of the placebo group (increase of 2.1%) (P<0.001 for all doses) (Table 4). Administration of the 200 mg, 300 mg, and 500 mg doses resulted in a mean reduction in LDL-C from baseline of 26%-34.3% on day 270 and 30.2%-32.2% on day 360. In addition, the percentage change in LDL-C over time between baseline and day 360 (i.e., the average of the percentage change in LDL-C at each time point when LDL-C was measured between baseline and day 360) was -31.6%, -38.1%, and -39.8%, respectively, for the 200 mg, 300 mg, and 500 mg doses (see Figure 4The absolute change in LDL-C over time between baseline and day 360 (i.e., the mean of the absolute change in LDL-C at each time point measured between baseline and day 360) was -39.1 mg / dl, -43.6 mg / dl, and -53.0 mg / dl for the 200 mg, 300 mg, and 500 mg doses, respectively (see Figure 5 ). Translated to individual patient responses, the coefficients of variation for changes in LDL-C in individual patients given 200 mg, 300 mg, and 500 mg were 45.2%, 60.8%, and 50.7% from baseline to Day 270, and 45.1%, 56.8%, and 50.2% from baseline to Day 360, respectively (see Figure 6A-6B ).

[0234] Administration of a second dose of RNAi agent on day 90 resulted in further reductions in LDL-C compared to baseline. Across all doses, mean reductions in LDL-C ranged from 34.2% to 44.1% on day 90 and from 41.1% to 54.6% on day 120 (see Figure 7 The difference between the two-dose regimen and placebo was significant: at day 180, the least squares reduction in LDL-C levels from baseline for patients who received two doses of the RNAi agent ranged from 35.5% to 52.6%, while the placebo group increased from baseline by 1.8% (P<0.001 for all comparisons with placebo) (see Table 4). The second administration of the 100 mg and 200 mg doses of the RNAi agent maintained LDL-C levels below baseline by more than 30% and 40% at day 210 (see Table 4). Figure 7 The second 300 mg dose maintained LDL-C levels at more than 50% below baseline from day 120 to day 210 ( Figure 7 ). Administration of the 100 mg, 200 mg, and 300 mg doses resulted in mean reductions in LDL-C levels from baseline ranging from 25.6% to 43.4% at Day 270 and from 13.3% (100 mg dose) to 33.3% (300 mg dose) at Day 360. In addition, the percent change in LDL-C over time between baseline and Day 360 was -31.0%, -41.2%, and -46.8% for the 200 mg, 300 mg, and 500 mg doses, respectively (see Figure 4 The absolute changes over time in LDL-C between baseline and Day 360 were -39.6 mg / dl, -39.1 mg / dl, and -57.7 mg / dl for the 200 mg, 300 mg, and 500 mg doses, respectively (see Figure 5 ). Individual patient responses Figure 8 At day 180, the mean reduction in LDL-C levels from baseline was 52.6%, with a maximum of 81% (see Figure 8 The coefficients of variation for changes in LDL-C in patients given 100 mg, 200 mg, and 300 mg were 45.7%, 82.3%, and 73.7% from baseline to Day 270, and 46.0%, 77.5%, and 74.2% from baseline to Day 360, respectively (see Figures 9A-9B ).

[0235] In patients who received placebo against a background of maximal possible statin therapy, there was a substantial change from baseline in LDL cholesterol levels at day 180 (mean [±SD] absolute difference, -0.7±25.6 mg / dL [-0.02±0.66 mmol / L] (see Fig. 10A In contrast, all patients who received two 300-mg doses of the RNAi agent had decreased LDL-C levels at day 180 (mean absolute change in LDL cholesterol level, -64.2 ± 20.7 mg / dl [-1.66 ± 0.54 mmol / L]) (see Fig. 10B ), 54% of patients had a 50% or greater reduction. In this RNAi dose group, 5%, 48%, and 66% of patients had LDL-C levels below 25 mg / dl (0.65 mmol / L), below 50 mg / dl (1.3 mmol / L), and below 70 mg / dl (1.8 mmol / L), respectively, at day 180. At day 240, in the same patient group, individual cholesterol levels remained below baseline (see Fig. 10C ).

[0236] Comparison of individual patient data from administration of a single 300 mg dose and a double 300 mg dose suggests that administration of 300 mg of the RNAi agent on day 1, day 90, day 270, and every 6 months thereafter should reduce intra-patient variability and provide a sustained reduction in LDL-C of approximately 50% (see Figures 11A-11B ).

[0237] Levels of non-HDL-C and Apo-B were also significantly reduced in patients randomized to receive the RNAi agent, with no significant changes in high-sensitivity C-reactive protein levels. The percent changes from baseline in other lipid measurements are shown in Tables 3-5.

[0238] like Fig.12 and 13As shown, after the RNAi agent was administered on day 1, PCSK9 levels were reduced by 59.6% and 68.7% from baseline levels within the RNAi agent dosage range of 100 mg-500 mg. On day 30, PCSK9 levels were further reduced to between 66.2%-74.0% below baseline levels, and similar reductions occurred on days 60 and 90. In patients receiving a single administration of RNAi agents, the average reduction in PCSK9 levels on day 180 ranged from 47.9%-59.3% (P<0.001 for each dose compared to placebo) (see Table 4). For patients receiving a single dose of 300 mg or 500 mg, PCSK9 levels were still more than 60% below baseline by day 150. In contrast, in patients receiving two doses of RNAi agents, further reductions in PCSK9 levels were observed after the second dose was administered. At day 90, these patients had PCSK9 levels reduced by 47.0%-62.8%, and at day 120, PCSK9 levels reduced by 60.4%-74.5%. At day 180, the mean reduction in PCSK9 levels from baseline in patients who received two doses of the RNAi agent ranged from 53.2%-69.1% (P<0.001 for each dose compared to placebo) (see Table 4). For patients who received two doses of 200 mg or 300 mg, PCSK9 levels were still more than 60% below baseline by day 240. In association with the single-dose and two-dose RNAi agent dosing regimens, the reduction in PCSK9 levels at day 270 was still greater than about 40% (see Table 4). Fig.12 and 13 ).

[0239] A model was developed and validated to project LDL-C levels over time when 300 mg of the RNAi agent was administered twice or three times per year. The model was validated based on the minimum difference between the observed and expected data at day 210. The results at day 270 were correctly predicted by the pharmacodynamic model using data from patient follow-up to day 210. Fig.14 Modeled LDL-C levels through 22 months are shown.

[0240] At day 210, adverse events were reported in 76% of patients receiving RNAi agents and 76% of patients receiving placebo (Table 7; see also Table 6). Most of these events (95%) were mild or moderate in severity (Grade 1 or 2). The incidence of serious adverse events was 11% in patients receiving RNAi agents and 8% in patients receiving placebo. Two patients discontinued participation in the trial due to adverse events: one patient because of herpes zoster infection (placebo group) and the other because of influenza or nasopharyngitis (two doses of 100 mg RNAi group). The most common adverse events (occurring in >2% of patients) were myalgia, headache, fatigue, nasopharyngitis, back pain, hypertension, diarrhea, and dizziness, and the incidence of these events was not significantly different between the groups receiving RNAi agents and the groups receiving placebo.

[0241] Injection site reactions occurred in 4% of patients who received a single dose of the RNAi agent and in 7% of patients who received two doses (one or two doses) of the RNAi agent (combined incidence of 5%); no patients who received placebo experienced injection site reactions (Table 7; see also Tables 8 and 9).

[0242] Two patients had elevated hepatic aspartate aminotransferase levels (>3 times the upper limit of the normal range), one in the single-dose placebo group and one in the single-dose 300 mg RNAi group; the patient in the 300 mg RNAi group also had elevated hepatic alanine aminotransferase levels. Two additional patients (one in the two-dose 100 mg RNAi group and one in the two-dose 300 mg RNAi group) also had elevated alanine aminotransferase levels. All elevations in aminotransferase levels were transient. In patients with normal bilirubin levels at baseline, there were no increases in bilirubin levels related to the RNAi agent or placebo, and no cases met the definition of Hy's rule, which states that patients are at high risk for fatal drug-induced liver injury if the drug is administered to cause icteric hepatocellular injury (non-cholestatic injury). One patient was positive for antidrug antibodies before the first injection; no other cases of antidrug antibodies were reported (see Table 10).

[0243] There were no additional safety findings during follow-up from Day 210 to Day 360 (see Table 11).

[0244] Two deaths occurred late in the trial. The first occurred in a patient randomized to a single dose of 500 mg RNAi agent who had long-standing vasculopathy and frequent angina. He suffered a cardiac arrest on site and died on day 104. The second death occurred in a male patient in the two doses of 200 mg RNAi agent group who underwent percutaneous repair of a thoracic aortic aneurysm after entering the study, followed by fistula and sepsis, and died on day 198 of entering the trial.

[0245] in conclusion

[0246] These results show that RNAi agents significantly reduce patients' LDL-C over multiple months. Administration of RNAi agents reduced Lp(a) and total cholesterol among other lipid parameters, and increased HDL-C. RNAi agents showed dose-response effects on PCSK9, LDL-C, non-HDL-C, and Apo-B. LDL-C variability within individual patients was virtually eliminated, and there was a sustained effect between infrequent administrations.

[0247] In addition, the RNAi agent was well tolerated by patients without major safety concerns. TEAEs were rare, mild to moderate events, and injection burden was minimal.

[0248] Overall, RNAi agents offer an opportunity to improve patient compliance.

[0249] 1. A method for reducing low-density lipoprotein cholesterol in a subject, the method comprising administering to the subject a therapeutically effective amount of an interfering ribonucleic acid (RNAi) agent,

[0250] The RNAi is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprising the nucleotide sequence of SEQ ID NO: 4.

[0251] 2. A method for preventing a cardiac event in a subject, the method comprising administering to the subject a therapeutically effective amount of an interfering ribonucleic acid (RNAi) agent,

[0252] The RNAi is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprising the nucleotide sequence of SEQ ID NO: 4.

[0253] 3. A method of reducing cardiovascular mortality and morbidity in a subject, the method comprising administering to the subject a therapeutically effective amount of an interfering RNA (RNAi) agent,

[0254] The RNAi is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprising the nucleotide sequence of SEQ ID NO: 4.

[0255] 4. The method of any one of schemes 1-3, wherein the subject has atherosclerotic cardiovascular disease (ASCVD), ASCVD equivalent risk, increased cardiovascular disease risk, heterozygous familial hypercholesterolemia or homozygous familial hypercholesterolemia, or is in need of lowering LDL-C, or a combination thereof.

[0256] 5. The method of any one of schemes 1-4, wherein the subject has a baseline low-density lipoprotein (LDL-C) level of about 70 mg / dl or higher.

[0257] 6. The method of any one of schemes 1-5, wherein the subject does not have active liver disease.

[0258] 7. The method of any one of schemes 1-6, wherein active liver disease is identified by one or more of the following characteristics: alanine aminotransferase (ALT) greater than two times the upper limit of normal (ULN), aspartate aminotransferase (AST) greater than two times the ULN, and total bilirubin greater than 1.5 times the ULN.

[0259] 8. The method of any one of schemes 1-7, wherein the subject has active liver disease.

[0260] 9. The method of any of claim 8, wherein active liver disease is identified by one or more of the following features: alanine aminotransferase (ALT) greater than two times the upper limit of normal (ULN), aspartate aminotransferase (AST) greater than two times the ULN, and total bilirubin greater than 1.5 times the ULN.

[0261] 10. The method of any one of schemes 1-9, wherein the subject is on background lipid-lowering therapy.

[0262] 11. The method of claim 10, wherein the background lipid-lowering therapy is selected from statins, ezetimibe, and LDL apheresis.

[0263] 12. The method of scheme 10 or 11, wherein the background lipid-lowering therapy is a statin.

[0264] 13. The method of any of schemes 10-12, wherein the subject is on maximally tolerated statin therapy.

[0265] 14. The method of any one of schemes 10-13, wherein the background lipid-lowering therapy is maintained while the subject is administered the RNAi agent.

[0266] 15. The method of any one of schemes 1-9, wherein the subject is not on background lipid-lowering therapy.

[0267] 16. The method of claim 1-15, wherein the subject has a baseline triglyceride level of no greater than about 400 mg / dl.

[0268] 17. The method of any one of claims 1-16, wherein the subject has a baseline estimated glomerular filtration rate (eGFR) of at least about 30 ml / min.

[0269] 18. The method of any one of schemes 1-17, wherein the subject does not have poorly controlled type 2 diabetes.

[0270] 19. The method of claim 18, wherein poorly controlled type 2 diabetes is identified by a baseline glycated hemoglobin A1c level of at least about 10%.

[0271] 20. The method of any one of schemes 1-19, wherein the subject does not suffer from New York Heart Association (NYHA) class II, III or IV heart failure.

[0272] 21. The method of any one of claims 1-20, wherein the subject has a last known ventricular ejection fraction of 30% or greater.

[0273] 22. The method of any one of schemes 1-21, wherein the subject has not experienced a major adverse cardiac event within six months of administration of the RNAi agent.

[0274] 23. The method of any one of schemes 1-22, wherein the subject has not experienced a hemorrhagic stroke.

[0275] 24. The method of any one of schemes 1-23, wherein the subject is free of arrhythmia within three months of administration of the RNAi agent.

[0276] 25. The method of any one of schemes 1-24, wherein the subject does not suffer from arrhythmia that is not controlled by medication or by ablation within three months of administration of the RNAi agent.

[0277] 26. The method of any one of schemes 1-25, wherein the subject does not have arrhythmia.

[0278] 27. The method of any one of schemes 1-26, wherein the subject is an adult.

[0279] 28. The method of any one of schemes 1-27, wherein the subject has heterozygous familial hypercholesterolemia or homozygous familial hypercholesterolemia.

[0280] 29. The method of any one of schemes 1-28, wherein the subject is in need of lowering LDL-C.

[0281] 30. The method of any one of claims 1-29, wherein administration of the RNAi agent reduces LDL-C levels by greater than about 20% compared to baseline LDL-C levels.

[0282] 31. The method of any one of schemes 1-30, wherein after administration of the RNAi agent, the reduction in LDL-C level of greater than about 20% compared to the baseline level is maintained for 15 days or longer.

[0283] 32. The method of any one of schemes 1-31, wherein administration of the RNAi agent reduces the level of PCSK9 by greater than about 25% compared to a PCSK9 baseline level, wherein the PCSK9 baseline level is measured before administration of the RNAi agent.

[0284] 33. The method of any one of schemes 1-32, wherein after administration of the RNAi agent, the reduction of PCSK9 level by greater than about 25% compared to baseline level is maintained for 30 days or longer.

[0285] 34. The method of any one of schemes 2 or 4-33, wherein the cardiac event is selected from death, non-fatal myocardial infarction, severe recurrent ischemia, stroke, symptomatic pulmonary embolism, and hemorrhage.

[0286] 35. The method of any one of schemes 1-34, wherein administration of the RNAi agent comprises a loading phase and a maintenance phase.

[0287] 36. The method of claim 35, wherein the loading phase comprises administering the RNAi agent in one or more doses.

[0288] 37. The method of claim 36, wherein the loading phase comprises administering the RNAi agent in at least two doses.

[0289] 38. The method of claim 37, wherein the at least two doses are separated by a time interval.

[0290] 39. The method of claim 38, wherein the time interval is from about 1 day to about 180 days.

[0291] 40. The method of claim 38 or 39, wherein the time interval is about 60 to about 120 days.

[0292] 41. The method of any one of schemes 38-40, wherein the at least two doses are separated by a time interval of about 90 days.

[0293] 42. The method of any one of schemes 36-41, wherein the dose comprises about 50 mg to about 800 mg of the RNAi agent.

[0294] 43. The method of claim 42, wherein the dose comprises about 100 mg to about 500 mg of the RNAi agent.

[0295] 44. The method of claim 42 or 43, wherein the dose comprises approximately 300 mg of the RNAi agent.

[0296] 45. The method of any one of schemes 35-44, wherein the maintenance phase comprises administering the RNAi agent in one or more doses.

[0297] 46. ​​The method of claim 45, wherein the loading phase comprises administering the RNAi agent in at least two doses.

[0298] 47. The method of scheme 46, wherein the at least two doses are separated by a time interval.

[0299] 48. The method of claim 47, wherein the at least two doses are separated by regular time intervals.

[0300] 49. The method of claim 48, wherein the regular time intervals are from about 1 month to about 12 months.

[0301] 50. The method of claim 48 or 49, wherein the regular time intervals are from about 3 months to about 9 months.

[0302] 51. The method of any one of claims 48-50, wherein the regular time intervals are about 6 months.

[0303] 52. The method of any one of schemes 45-51, wherein the dose comprises about 50 mg to about 800 mg of the RNAi agent.

[0304] 53. The method of claim 52, wherein the dose comprises about 100 mg to about 500 mg of the RNAi agent.

[0305] 54. The method of scheme 52 or 53, wherein the dose comprises approximately 300 mg of the RNAi agent.

[0306] 55. A method of preventing the development of atherosclerotic cardiovascular disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an interfering RNA (RNAi) agent,

[0307] The RNAi is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprising the nucleotide sequence of SEQ ID NO: 4.

[0308] 56. A method of treating a subject having ASCVD, ASCVD equivalent risk, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, in need of lowering LDL-C, or a combination thereof, the method comprising administering to the subject a therapeutically effective amount of an interfering ribonucleic acid (RNAi) agent,

[0309] The RNAi is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprising the nucleotide sequence of SEQ ID NO: 4.

[0310] 57. The method of claim 55 or 56, wherein the subject has a baseline LDL-C level of about 70 mg / dl or greater.

[0311] 58. The method of any one of schemes 55-57, wherein the subject has active liver disease.

[0312] 59. The method of claim 58, wherein active liver disease is identified by one or more of the following features: alanine aminotransferase (ALT) greater than two times the upper limit of normal (ULN), aspartate aminotransferase (AST) greater than two times the ULN, and total bilirubin greater than 1.5 times the ULN.

[0313] 60. The method of any one of schemes 55-57, wherein the subject has active liver disease.

[0314] 61. The method of claim 60, wherein active liver disease is identified by one or more of the following features: alanine aminotransferase (ALT) greater than two times the upper limit of normal (ULN), aspartate aminotransferase (AST) greater than two times the ULN, and total bilirubin greater than 1.5 times the ULN.

[0315] 62. The method of any one of schemes 55-61, wherein the subject is on background lipid-lowering therapy.

[0316] 63. The method of scheme 62, wherein the background lipid-lowering therapy is selected from statins, ezetimibe and LDL apheresis.

[0317] 64. The method of scheme 62 or 63, wherein the background lipid-lowering therapy is maintained while the RNAi agent is administered to the subject.

[0318] 65. The method of any one of schemes 55-61, wherein the subject is not on background lipid-lowering therapy.

[0319] 66. The method of any of claims 55-65, wherein the subject has a baseline triglyceride level greater than about 400 mg / dl.

[0320] 67. The method of any one of schemes 55-66, wherein the subject has a baseline estimated glomerular filtration rate (eGFR) of less than about 30 ml / min.

[0321] 68. The method of any one of schemes 55-67, wherein the subject has poorly controlled type 2 diabetes.

[0322] 69. The method of claim 68, wherein poorly controlled type 2 diabetes is identified by a baseline glycated hemoglobin A1c level of at least about 10%.

[0323] 70. The method of any one of schemes 55-69, wherein the subject suffers from New York Heart Association (NYHA) class II, III or IV heart failure.

[0324] 71. The method of any one of claims 55-70, wherein the subject has a ventricular ejection fraction of less than 30%.

[0325] 72. The method of any one of schemes 55-71, wherein the subject experiences a major adverse cardiac event within six months of administration of the RNAi agent.

[0326] 73. The method of any one of schemes 55-72, wherein the subject has experienced at least one hemorrhagic stroke.

[0327] 74. The method of any one of schemes 55-73, wherein the subject suffers from arrhythmia within three months of administration of the RNAi agent.

[0328] 75. The method of any one of schemes 55-74, wherein the subject does not have arrhythmia that is not controlled by medication or by ablation within three months of administration of the RNAi agent.

[0329] 76. The method of any one of schemes 55-75, wherein the subject does not have arrhythmia.

[0330] 77. The method of any one of schemes 55-76, wherein the subject is an adult.

[0331] 78. The method of any one of schemes 55-77, wherein the subject has heterozygous familial hypercholesterolemia or homozygous familial hypercholesterolemia.

[0332] 79. The method of any one of schemes 55-78, wherein the subject is in need of lowering LDL-C.

[0333] 80. The method of any one of schemes 55-79, wherein after administration of the RNAi agent, the reduction of low-density lipoprotein by greater than about 20% compared to baseline levels is maintained for 15 days or longer.

[0334] 81. The method of any one of schemes 55-80, wherein administration of the RNAi agent reduces PCSK9 levels by greater than about 25% compared to PCSK9 baseline levels, wherein the PCSK9 baseline levels are measured prior to administration of the RNAi agent.

[0335] 82. The method of claim 81, wherein after administration of the RNAi agent, a reduction of greater than about 25% in PCSK9 levels compared to baseline levels is maintained for 30 days or longer.

[0336] 83. The method of any one of schemes 55-82, wherein administration of the RNAi agent comprises a loading phase and a maintenance phase.

[0337] 84. The method of scheme 83, wherein the loading phase comprises administering the RNAi agent in one or more doses.

[0338] 85. The method of scheme 84, wherein the loading phase comprises administering the RNAi agent in at least two doses.

[0339] 86. The method of scheme 85, wherein the at least two doses are separated by a time interval.

[0340] 87. The method of claim 86, wherein the time interval is from about 1 day to about 180 days.

[0341] 88. The method of claim 86 or 87, wherein the time interval is about 60 to about 120 days.

[0342] 89. The method of any one of schemes 86-88, wherein the time interval is about 90 days.

[0343] 90. The method of any one of schemes 83-89, wherein the dose comprises about 50 mg to about 800 mg of the RNAi agent.

[0344] 91. The method of claim 90, wherein the dose comprises about 100 mg to about 500 mg of the RNAi agent.

[0345] 92. The method of claim 90 or 91, wherein the dose comprises approximately 300 mg of the RNAi agent.

[0346] 93. The method of any one of schemes 83-92, wherein the maintenance phase comprises administering the RNAi agent in one or more doses.

[0347] 94. The method of claim 93, wherein the loading phase comprises administering the RNAi agent in at least two doses.

[0348] 95. The method of scheme 94, wherein the at least two doses are separated by a time interval.

[0349] 96. The method of scheme 95, wherein the at least two doses are separated by regular time intervals.

[0350] 97. The method of claim 96, wherein the regular time intervals are from about 1 month to about 12 months.

[0351] 98. The method of claim 96 or 97, wherein the regular time intervals are from about 3 months to about 9 months.

[0352] 99. The method of any one of schemes 96-98, wherein the regular time interval is about 6 months.

[0353] 100. The method of any one of schemes 93-99, wherein the dose comprises about 50 mg to about 800 mg of the RNAi agent.

[0354] 101. The method of scheme 100, wherein the dose comprises about 100 mg to about 500 mg of the RNAi agent.

[0355] 102. The method of scheme 100 or 101, wherein the dose comprises approximately 300 mg of the RNAi agent.

[0356] 103. A method of preventing a cardiac event in a subject who is on a diet designed to improve blood lipid levels and who is on maximally tolerated statin therapy, wherein the subject has heterozygous familial hypercholesterolemia and is in need of additional lowering of low-density lipoprotein cholesterol,

[0357] The method comprises administering a prophylactically effective amount of a RNAi agent,

[0358] The RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 4.

[0359] 104. A method of preventing a cardiac event in a subject who is on a diet designed to improve lipid levels and who is on maximally tolerated statin therapy, wherein the subject has atherosclerotic cardiovascular disease and is in need of additional lowering of low-density lipoprotein cholesterol,

[0360] The method comprises administering a prophylactically effective amount of a RNAi agent,

[0361] The RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 4.

[0362] 105. A method of preventing a cardiac event in a subject who is on a diet designed to improve lipid levels and who is on a low-density lipoprotein cholesterol lowering therapy, wherein the subject has homozygous familial hypercholesterolemia and is in need of additional low-density lipoprotein cholesterol lowering,

[0363] The method comprises administering a prophylactically effective amount of a RNAi agent,

[0364] The RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 4.

[0365] 106. A method of treating a subject who is on a diet designed to improve lipid levels and who is on maximally tolerated statin therapy, wherein the subject has heterozygous familial hypercholesterolemia and is in need of additional lowering of low-density lipoprotein cholesterol,

[0366] The method comprises administering a therapeutically effective amount of a RNAi agent,

[0367] The RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 4.

[0368] 107. A method of treating a subject who is on a diet designed to improve lipid levels and who is on maximally tolerated statin therapy, wherein the subject suffers from atherosclerotic cardiovascular disease and is in need of additional lowering of low-density lipoprotein cholesterol,

[0369] The method comprises administering a therapeutically effective amount of a RNAi agent,

[0370] The RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 4.

[0371] 108. A method of treating a subject who is on a diet designed to improve lipid levels and who is on a low-density lipoprotein cholesterol lowering therapy, wherein the subject has homozygous familial hypercholesterolemia and is in need of additional low-density lipoprotein cholesterol lowering,

[0372] The method comprises administering a therapeutically effective amount of a RNAi agent,

[0373] The RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 4.

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388] Having thus described embodiments of the present invention in detail, it should be understood that the invention defined by the preceding paragraphs is not limited to the specific details set forth in the above description, but that many obvious variations thereof are possible without departing from the spirit or scope of the present invention.

[0389] Sequence Listing

[0390] SEQ ID NO: 1

[0391] 5'-ACAAAAGCAAAACAGGUCUAGAA-3'

[0392] SEQ ID NO: 2

[0393] 5'-CUAGACCUGUTUUGCUUUUGU-3'

[0394] SEQ ID NO: 3

[0395] 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3', wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C or U; Af, Gf, Cf or Uf is 2'-fluoro A, G, C or U; dT is 2'-deoxythymidine; and s is a phosphorothioate bond.

[0396] SEQ ID NO: 4

[0397] 5'-csusagacCfuGfudTuugcuuuugu-3', wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C or U; Af, Gf, Cf or Uf is 2'-fluoro A, G, C or U; dT is 2'-deoxythymidine and s is a phosphorothioate bond.

Claims

1. A method for reducing low-density lipoprotein cholesterol in a subject, the method comprising administering to the subject a therapeutically effective amount of an interfering ribonucleic acid (RNAi) agent, The RNAi is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprising the nucleotide sequence of SEQ ID NO:

4.

2. A method for preventing a cardiovascular event in a subject, the method comprising administering to the subject a therapeutically effective amount of an interfering ribonucleic acid (RNAi) agent, The RNAi is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprising the nucleotide sequence of SEQ ID NO:

4.

3. A method of reducing cardiovascular mortality and morbidity in a subject, the method comprising administering to the subject a therapeutically effective amount of an interfering RNA (RNAi) agent, The RNAi is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprising the nucleotide sequence of SEQ ID NO:

4.

4. A method for preventing the development of atherosclerotic cardiovascular disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an interfering RNA (RNAi) agent, The RNAi is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprising the nucleotide sequence of SEQ ID NO:

4.

5. The method of any one of claims 1-4, wherein the subject has atherosclerotic cardiovascular disease (ASCVD), ASCVD equivalent risk, elevated cardiovascular disease risk, heterozygous familial hypercholesterolemia, or homozygous familial hypercholesterolemia, or is in need of lowering LDL-C, or a combination thereof.

6. The method of any one of claims 1-5, wherein the subject has a baseline low-density lipoprotein (LDL-C) level of about 70 mg / dl or more.

7. The method of any one of claims 1-6, wherein the subject does not have active liver disease.

8. The method of any one of claims 1-7, wherein the subject has active liver disease.

9. The method of any one of claims 7-8, wherein active liver disease is identified by one or more of the following features: alanine aminotransferase (ALT) greater than two times the upper limit of normal (ULN), aspartate aminotransferase (AST) greater than two times the ULN, and total bilirubin greater than 1.5 times the ULN.

10. The method of any one of claims 1-9, wherein the subject is on background lipid-lowering therapy; wherein the background lipid-lowering therapy is selected from the group consisting of statins, ezetimibe, and LDL apheresis.

11. The method of claim 10, wherein the background lipid-lowering therapy is a statin.

12. The method of any one of claims 10-11, wherein the subject is on maximally tolerated statin therapy.

13. The method of any one of claims 1-12, wherein the subject has one or more of the following characteristics: a) the subject has a baseline triglyceride level of no greater than about 400 mg / dl, b) the subject has a baseline estimated glomerular filtration rate (eGFR) of at least about 30 ml / min, c) the subject does not have poorly controlled type 2 diabetes, d) the subject does not suffer from New York Heart Association (NYHA) class II, III or IV heart failure, e) the subject's last known ventricular ejection fraction is 30% or greater, f) the subject has not experienced a major adverse cardiovascular event within six months of administration of the RNAi agent, g) the subject has not experienced a hemorrhagic stroke, h) the subject is free of cardiac arrhythmia within three months of administration of the RNAi agent, i) the subject does not suffer from arrhythmia that is not controlled by medication or by ablation within three months of administration of the RNAi agent, j) the subject does not have arrhythmia, k) the subject is an adult, l) the subject suffers from heterozygous familial hypercholesterolemia or homozygous familial hypercholesterolemia, m) the subject needs to lower LDL-C, n) the subject has a baseline low-density lipoprotein (LDL-C) level of about 70 mg / dl or higher.

14. The method of claim 13, wherein poorly controlled type 2 diabetes is identified by a baseline glycated hemoglobin A1c level of at least about 10%.

15. The method of any one of claims 1-14, wherein the reduction in LDL-C levels of greater than about 20% or 25% compared to baseline levels is maintained for 15 days or longer following administration of the RNAi agent.

16. The method of any one of claims 1-15, wherein after administration of the RNAi agent, a reduction of greater than about 25% in PCSK9 levels compared to baseline levels is maintained for 30 days or longer, wherein the PCSK9 baseline levels are measured prior to administration of the RNAi agent.

17. The method of any one of claims 2 or 5-16, wherein the cardiovascular event is selected from cardiovascular death, non-fatal myocardial infarction, severe recurrent ischemia, stroke, symptomatic pulmonary embolism, and hemorrhage.

18. The method of any one of claims 1-17, wherein administering the RNAi agent comprises a loading phase and a maintenance phase.

19. The method of claim 18, wherein the loading phase comprises administering the RNAi agent in one or more doses.

20. The method of claim 19, wherein the loading phase comprises administering the RNAi agent in at least two doses.

21. The method of claim 20, wherein the at least two doses are separated by a time interval.

22. The method of claim 21, wherein the time interval is from about 60 to about 120 days.

23. The method of any one of claims 21-22, wherein the at least two doses are separated by a time interval of about 90 days.

24. The method of claims 19-23, wherein the dose comprises about 50 mg to about 800 mg of the RNAi agent; or comprises about 100 mg to about 500 mg of the RNAi agent.

25. The method of claim 24, wherein the dose comprises approximately 300 mg of the RNAi agent.

26. The method of any one of claims 18-25, wherein the maintenance phase comprises administering the RNAi agent in one or more doses.

27. The method of claim 26, wherein the loading phase comprises administering the RNAi agent in at least two doses.

28. The method of claim 27, wherein the at least two doses are separated by a time interval.

29. The method of claim 28, wherein the at least two doses are separated by regular time intervals.

30. The method of claim 29, wherein the regular time intervals are from about 3 months to about 9 months.

31. The method of any one of claims 29-30, wherein the regular time intervals are about 6 months.

32. The method of claims 26-31, wherein the dose comprises about 50 mg to about 800 mg of the RNAi agent; or comprises about 100 mg to about 500 mg of the RNAi agent.

33. The method of claim 32, wherein the dose comprises approximately 300 mg of the RNAi agent.

34. The method of any one of claims 1-4, wherein the subject is on a diet designed to improve blood lipid profiles and is on maximally tolerated statin therapy, and the subject has heterozygous familial hypercholesterolemia and is in need of additional lowering of low-density lipoprotein cholesterol.

35. The method of any one of claims 1-4, wherein the subject is on a diet designed to improve lipid levels and is on maximally tolerated statin therapy, and the subject has atherosclerotic cardiovascular disease and is in need of additional lowering of low-density lipoprotein cholesterol.

36. The method of any one of claims 1-4, wherein the subject is on a diet designed to improve lipid levels and is on treatment to lower low-density lipoprotein cholesterol, and the subject has homozygous familial hypercholesterolemia and is in need of additional lowering of low-density lipoprotein cholesterol.

37. A method of treating a subject who is on a diet designed to improve lipid levels and who is on maximally tolerated statin therapy, wherein the subject has heterozygous familial hypercholesterolemia and is in need of additional lowering of low-density lipoprotein cholesterol, The method comprises administering a therapeutically effective amount of a RNAi agent, The RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO:

4.

38. A method of treating a subject who is on a diet designed to improve lipid levels and who is on maximally tolerated statin therapy, wherein the subject has atherosclerotic cardiovascular disease and is in need of additional lowering of low-density lipoprotein cholesterol, The method comprises administering a therapeutically effective amount of a RNAi agent, The RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO:

4.

39. A method of treating a subject who is on a diet designed to improve lipid levels and who is on a low-density lipoprotein cholesterol lowering therapy, wherein the subject has homozygous familial hypercholesterolemia and is in need of additional low-density lipoprotein cholesterol lowering, The method comprises administering a therapeutically effective amount of a RNAi agent, The RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3, and the sense strand comprises the nucleotide sequence of SEQ ID NO:

4.

40. The method of any one of claims 1-39, wherein the subject has one or more risk factors selected from the group consisting of: having an acute coronary syndrome; having a history of myocardial infarction, stable or unstable angina, coronary or other arterial revascularization, stroke, transient ischemic attack, or peripheral arterial disease of atherosclerotic origin; being male; having a family history of heart disease, ASCVD, or ASCVD risk equivalents; being a smoker; being physically inactive; having high blood pressure; having high blood cholesterol; having diabetes or prediabetes; being overweight or obese; having a history of preeclampsia during pregnancy; having uncontrolled stress and / or anger; being postmenopausal; having an unhealthy diet; being 55 years of age or older; having sleep apnea; and having anemia.

41. The method of any one of claims 1-40, wherein the RNAi agent is as shown in Figure 1.

42. The method of any one of claims 1-41, wherein the RNAi agent is formulated in a suitable pharmaceutical formulation at about 200 mg / ml, such that administration of about 1.5 ml of the formulation to a subject provides the fixed dose of the RNAi agent.

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