Methods for treating atherosclerotic cardiovascular disease with LPA-targeting RNAi constructs

Abstract

The present invention relates to a method for treating or preventing atherosclerotic cardiovascular disease and other conditions associated with elevated levels of lipoprotein(a) (Lp(a)) using an RNAi construct that targets the LPA gene, which encodes apolipoprotein(a), a component of Lp(a) particles. In particular, the present invention relates to a method for lowering serum Lp(a) levels and reducing the risk of cardiovascular events in patients with elevated Lp(a) levels, comprising administering an LPA-targeting RNAi construct according to a specific dosing regimen. Pharmaceutical compositions containing the LPA-targeting RNAi construct for use in the method are also disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 110,309, filed November 5, 2020, which is incorporated herein by reference in its entirety.

[0002] Description of electronically submitted text files

[0001] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of the Sequence Listing in computer readable format, created on November 1, 2021, is titled A-2694-WO-PCT_ST25, and is 3.5 kilobytes in size.

[0003] The present invention relates to pharmaceutical compositions and methods for treating atherosclerotic cardiovascular disease and other conditions associated with elevated lipoprotein(a) (Lp(a)). In particular, the present invention relates to methods for lowering serum levels of Lp(a) and reducing the risk of cardiovascular events, such as cardiovascular death, myocardial infarction, stroke, and coronary revascularization, in patients with elevated Lp(a) by administering an LPA-targeting RNAi construct according to a specific dosing regimen. [Background technology]

[0004] Atherosclerotic cardiovascular disease is highly prevalent and remains the leading cause of mortality worldwide, despite the widespread use of low-density lipoprotein (LDL)-lowering therapy. Although LDL-lowering therapy reduces the risk of major cardiac events, the residual cardiovascular risk faced by some patients with low LDL levels is indicative of other mechanisms of cardiovascular pathology. Over the past few decades, compelling evidence from epidemiological studies and meta-analyses, Mendelian randomization analyses, and genome-wide association studies has demonstrated that increasing serum Lp(a) concentrations are associated with increased risk of coronary artery disease and atherosclerosis-related disorders (Clarke et al., N. Engl. J. Med., Vol. 361:2518-2528, 2009; Kamstrup et al., JAMA, Vol. 301:2331-2339, 2009; Nordestgaard et al., European Heart Journal, Vol. 31:2844-2853, 2010; Helgadottir et al., J. Am. Coll. Cardiol, Vol. 60:722-729, 2012; Thanassoulis et al., JAMA, Vol. 60:722-729, 2012). al.,J.Am.Coll.Cardiol.,Vol.55:2491-2498,2010;Kamstrup et al.,J.Am.Coll. Cardiol.,Vol.63:470-477,2014;Kral et al.,Journal of Cardiology,Vol.118:656-661,2016;Thanassoulis et al. al., J. Lipid Res., Vol. 57:917-924, 2016; Tsimikas et al., J. Am. Coll. Cardiol., Vol. 69: 692-711, 2017). In particular, the relationship between Lp(a) levels and coronary artery disease, myocardial infarction, stroke, peripheral vascular disease, and aortic stenosis has been described in several genetic and observational studies (reviewed in Schmidt et al., J. Lipid Res., Vol. 57:1339-1359, 2016). This risk relationship is known to be continuous, with higher Lp(a) levels associated with proportionally greater effects.This association persisted even after adjusting for other lipid parameters (Emerging Risk Factors Collaboration, JAMA, Vol. 302:412-423, 2009).

[0005] Lp(a) is a low-density lipoprotein (LDL) composed of the glycoprotein apolipoprotein(a) (apo(a)) linked to apolipoprotein B of the LDL particle by a disulfide bond (Schmidt et al., supra). Apo(a) is encoded by the LPA gene and is expressed almost exclusively in primates, including humans. Apo(a) exhibits homology to plasminogen and exists in various isoforms due to intragenic size polymorphism resulting from a variable number of kringle IV type 2 (KIV-2) domain repeats (see Kronenberg and Utermann, J. Intern. Med., Vol. 273:6-30, 2013). An inverse correlation has been observed between the size of apo(a) isoforms and plasma levels of Lp(a) particles (Sandholzer et al., Hum. Genet., Vol. 86;607-614, 1991). Lp(a) contains proinflammatory oxidized phospholipids that contribute to its atherogenic effects (Tsimikas et al., J. Am. Coll. Cardiol., Vol. 63:1724-1734, 2014).

[0006] High plasma Lp(a) concentrations are genetically defined, remain at stable levels, cannot be regulated by habitual changes (diet, exercise, or other environmental factors), and are not effectively controlled by any of the currently available lipid-lowering drugs. Currently, there are no approved therapies shown to reduce the risk of cardiovascular events by lowering Lp(a). Moderate reductions in Lp(a) (approximately 20–30%) have been observed with proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, niacin, or mipomersen (Santos et al., Arterioscler. Thromb. Vasc. Biol., Vol. 35:689–699, 2015; Yeang et al., Curr. Opin. Lipidol., Vol. 26:169–178, 2015; and Landray et al., N. Engl. J. Med., Vol. 371:203–212, 2014). Apheresis is effective in lowering Lp(a), but is currently available only in a few countries with limited access (Julius, J. Cardiovasc. Dev. Dis., Vol. 5:27–37, 2018). Additionally, apheresis is an invasive, extremely expensive procedure requiring frequent office visits, making it unavailable as a long-term treatment for subjects requiring lifelong therapy (Khan et al., Eur. Heart J., Vol. 38:1561-1569, 2017; Roeseler et al., Arterioscler. Thromb. Vasc. Biol., Vol. 36:2019-2027, 2016; Leebmann et al., Circulation, Vol. 128:2567-2576, 2013; Safarova et al., Atheroscler. Suppl., Vol. 14:93-99, 2013).

[0007] An antisense oligonucleotide targeting the apo(a) messenger RNA transcript (AKCEA-APO(a)-LRx; also known as ISIS 681257 and TQJ230) has been developed and is currently undergoing clinical investigation (reviewed in Graham et al., J Lipid Res., Vol. 57:340-351, 2016). When administered at doses of 10 mg, 20 mg, or 40 mg on days 1, 3, 5, 8, 15, and 22 to healthy subjects with baseline Lp(a) levels of 75 nmol / L or higher, this molecule was reported to reduce Lp(a) concentrations by an average of 66%, 80%, and 92%, respectively, at day 36 and by an average of 39%, 53%, and 58%, respectively, at day 113 (Viney et al., Lancet, Vol. 388:2239-2253, 2016). In a subsequent phase 2 study, AKCEA-APO(a)-LRx reduced Lp(a) levels by an average of 35%, 56%, and 72% at week 25 in patients with established cardiovascular disease and baseline Lp(a) levels of 150 nmol / L or higher when administered once every 4 weeks at doses of 20 mg, 40 mg, or 60 mg, respectively (Tsimikas et al., New England Journal of Medicine, Vol. 382:244-255, 2020). A phase 3 cardiovascular outcomes trial in which the molecule is administered monthly at an 80 mg dose is ongoing (ClinicalTrials.gov identifier NCT04023552). However, there remains a need in the art for new therapeutic agents that potently lower Lp(a) levels for long periods of time, allowing for low-dose, less frequent dosing regimens for the treatment and prevention of atherosclerotic cardiovascular disease. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Clarke et al.,N.Engl.J.Med.,Vol.361:2518-2528,2009

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Summary of the Invention

[0009] The present invention is based, in part, on the identification of a therapeutic regimen for an LPA-targeting RNAi construct, particularly olpaciran, for effectively lowering circulating Lp(a) levels for the treatment of atherosclerotic cardiovascular disease. Accordingly, in some embodiments, the present invention provides a method for lowering serum or plasma Lp(a) levels in a patient in need thereof, comprising administering to the patient an LPA RNAi construct described herein at a dose of about 9 mg to about 675 mg with an administration interval of at least 8 weeks. In some such embodiments, the patient to whom the LPA RNAi construct is administered has been diagnosed with or is at risk of developing cardiovascular disease, such as coronary artery disease, carotid artery disease, peripheral artery disease, myocardial infarction, cerebrovascular disease, stroke, aortic stenosis, stable or unstable angina, atrial fibrillation, heart failure, hyperlipidemia, heterozygous familial hypercholesterolemia, or homozygous familial hypercholesterolemia. The patient may have a history or family history of myocardial infarction and / or may be diagnosed with acute coronary syndrome. In other embodiments, the patient to whom the LPA RNAi construct is administered is diagnosed with chronic kidney disease.

[0010] In certain embodiments, the present invention provides methods for treating, attenuating, or preventing atherosclerosis in a patient in need thereof, or for treating, attenuating, or preventing cardiovascular disease in a patient in need thereof. In such embodiments, the method comprises administering to the patient an LPA RNAi construct described herein at a dose of about 9 mg to about 675 mg with an administration interval of at least 8 weeks. Cardiovascular diseases that may be treated, ameliorated, attenuated, or prevented by the methods of the present invention may include coronary artery disease, carotid artery disease, peripheral artery disease, myocardial infarction, cerebrovascular disease, stroke, aortic stenosis, stable or unstable angina, atrial fibrillation, heart failure, hyperlipidemia, heterozygous familial hypercholesterolemia, or homozygous familial hypercholesterolemia.

[0011] The present invention also includes methods for reducing the risk of cardiovascular events in patients with atherosclerotic cardiovascular disease. In some embodiments, the methods include administering to a patient an LPA RNAi construct described herein at a dose of about 9 mg to about 675 mg with an administration interval of at least 8 weeks. The cardiovascular event can be a major cardiovascular event, such as cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, or hospitalization for unstable angina. In some embodiments, the cardiovascular event can be a major adverse limb event, such as acute limb ischemia, major amputation surgery, or peripheral revascularization for ischemia. In certain embodiments, the cardiovascular event is cardiovascular death, myocardial infarction, stroke, and / or coronary revascularization. The patient with atherosclerotic cardiovascular disease to whom the LPA RNAi construct is to be administered may have a history of coronary revascularization, a history of coronary artery bypass grafting, a diagnosis of coronary artery disease, a diagnosis of atherosclerotic cerebrovascular disease, a diagnosis of peripheral artery disease, and / or a history of myocardial infarction. In one embodiment, the patient to whom the LPA RNAi construct is to be administered has recently experienced a myocardial infarction event, for example, the patient has experienced a myocardial infarction within one year prior to the first administration of the LPA RNAi construct. In another embodiment, the patient to whom the LPA RNAi construct is to be administered has been hospitalized due to acute coronary syndrome or unstable angina.

[0012] Patients to whom an LPA RNAi construct is to be administered according to the methods of the present invention have elevated serum or plasma levels of Lp(a). In some embodiments, the patient has a serum or plasma Lp(a) level of about 70 nmol / L or higher before the first administration of an LPA RNAi construct. In other embodiments, the patient has a serum or plasma Lp(a) level of about 150 nmol / L or higher before the first administration of an LPA RNAi construct. In certain embodiments, the patient has a serum or plasma Lp(a) level of about 175 nmol / L or higher before the first administration of an LPA RNAi construct. In certain other embodiments, the patient has a serum or plasma Lp(a) level of about 200 nmol / L or higher before the first administration of an LPA RNAi construct.

[0013] In some embodiments of the methods of the present invention, the patient to whom the LPA RNAi construct is to be administered is undergoing lipid-lowering therapy, for example, to reduce the patient's LDL-LC level. The lipid-lowering therapy can be a PCSK9 inhibitor, such as a PCSK9 antagonist monoclonal antibody (e.g., evolocumab, alirocumab), a statin (e.g., atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin), a cholesterol absorption inhibitor (e.g., ezetimibe), bempedoic acid, nicotinic acid (e.g., niacin), fibric acid (e.g., gemfibrozil, fenofibrate), a bile acid sequestrant (e.g., cholestyramine, colestipol, colesevelam), LDL apheresis, or a combination thereof. In these and other embodiments, the patient may have a serum LDL-C level of about 100 mg / dL or less, or about 70 mg / dL or less, prior to the first administration of the LPA RNAi construct.

[0014] In certain embodiments of the methods of the present invention, a fixed dose of an LPA RNAi construct is administered to a patient once every 12 weeks or once every three months. In some such embodiments, the fixed dose may be about 10 mg to about 225 mg, about 75 mg to about 225 mg, about 50 mg to about 100 mg, or about 150 mg to about 225 mg. In one embodiment, the LPA RNAi construct is administered to a patient at a fixed dose of about 10 mg once every 12 weeks or once every three months. In another embodiment, the LPA RNAi construct is administered to a patient at a fixed dose of about 75 mg once every 12 weeks or once every three months. In yet another embodiment, the LPA RNAi construct is administered to a patient at a fixed dose of about 150 mg once every 12 weeks or once every three months. In yet another embodiment, the LPA RNAi construct is administered to a patient at a fixed dose of about 225 mg once every 12 weeks or once every three months.

[0015] In certain other embodiments of the methods of the present invention, a fixed dose of an LPA RNAi construct is administered to a patient once every 24 weeks or once every six months. In some such embodiments, the fixed dose can be about 225 mg to about 675 mg, about 225 mg to about 450 mg, or about 200 mg to about 300 mg. In some embodiments, the LPA RNAi construct is administered to a patient at a fixed dose of about 225 mg once every 24 weeks or once every six months. In other embodiments, the LPA RNAi construct is administered to a patient at a fixed dose of about 300 mg once every 24 weeks or once every six months. In certain embodiments, the LPA RNAi construct is administered to a patient at a fixed dose of about 450 mg once every 24 weeks or once every six months. In certain other embodiments, the LPA RNAi construct is administered to a patient at a fixed dose of about 675 mg once every 24 weeks or once every six months.

[0016] Administration of an LPA RNAi construct to a patient according to the methods of the present invention substantially reduces the patient's plasma or serum Lp(a) levels over an extended period of time. For example, in some embodiments of the methods of the present invention, administration of an LPA RNAi construct reduces the patient's serum or plasma Lp(a) levels by more than 80% for at least 12 weeks, at least 16 weeks, or at least 24 weeks, compared to the patient's baseline serum or plasma Lp(a) levels. In other embodiments of the methods of the present invention, administration of an LPA RNAi construct reduces the patient's serum or plasma Lp(a) levels by more than 90% for at least 12 weeks, at least 16 weeks, or at least 24 weeks, compared to the patient's baseline serum or plasma Lp(a) levels. In certain embodiments, administration of an LPA RNAi construct to a patient according to the methods of the present invention reduces the patient's plasma or serum Lp(a) levels to about 100 nmol / L or less. In some embodiments, administration of an LPA RNAi construct to a patient according to the methods of the present invention reduces the patient's plasma or serum Lp(a) levels to about 75 nmol / L or less. In another embodiment, administration of an LPA RNAi construct to a patient according to the methods of the invention reduces the patient's plasma or serum Lp(a) levels to about 50 nmol / L or below.

[0017] In any embodiment of the methods disclosed herein, the LPA RNAi construct administered to a patient can be a double-stranded RNA molecule, such as an siRNA molecule, comprising a sense strand and an antisense strand, where the antisense strand comprises a region having a sequence complementary to the LPA mRNA sequence. Preferably, the sense strand comprises a sequence sufficiently complementary to that of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length. In certain embodiments of the methods of the present invention, the LPA RNAi construct administered to a patient comprises a sense strand and an antisense strand, each of which is about 19 to about 23 nucleotides in length, where the antisense strand comprises a sequence complementary to the LPA mRNA sequence and the sense strand comprises a sequence complementary to that of the antisense strand. In one such embodiment, the sense and antisense strands of the LPA RNAi construct can each be 21 nucleotides in length and can hybridize to each other to form a 21-base pair duplex region such that the RNAi construct has two blunt ends. In another such embodiment, the sense and antisense strands of the LPA RNAi construct can each be 19 nucleotides in length and can hybridize to each other to form a 19 base pair long duplex region such that the RNAi construct has two blunt ends.

[0018] In some embodiments of the methods of the present invention, the LPA RNAi construct administered to a patient further comprises a targeting moiety comprising an asialoglycoprotein receptor ligand, the targeting moiety being covalently attached to the sense strand, e.g., to the 5' end of the sense strand. The targeting moiety may comprise a trivalent GalNAc moiety, e.g., a moiety having the structure of Structure 1 described herein. In certain embodiments, the LPA RNAi construct administered to a patient according to the methods of the present invention comprises a sense strand comprising the sequence of SEQ ID NO: 1 and an antisense strand comprising the sequence of SEQ ID NO: 2. In some embodiments, the LPA RNAi construct comprises a sense strand comprising or consisting of the sequence of SEQ ID NO: 3 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 4. In certain preferred embodiments, the sense and / or antisense strands of the LPA RNAi construct comprise one or more modified nucleotides. In such embodiments, the LPA RNAi construct comprises a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 5 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 6. In a preferred embodiment, the LPA RNAi construct administered to a patient according to the methods of the present invention is olpaciran.

[0019] The present invention also provides pharmaceutical compositions comprising an LPA RNAi construct, e.g., olpaciran, for use in the methods of the present invention described herein. The pharmaceutical composition may include one or more pharmaceutically acceptable diluents, carriers, or excipients. In certain embodiments, the pharmaceutical composition comprises an LPA RNAi construct (e.g., olpaciran), potassium phosphate buffer, and sodium chloride, and the composition has a pH of about 6.6 to about 7.0, preferably about 6.8. Any of the pharmaceutical compositions described herein may be incorporated into an injection device, such as a prefilled syringe, autoinjector, injection pump, on-body injector, or injection pen, for administration (e.g., subcutaneous administration) to a patient according to the methods described herein. In some embodiments, the LPA RNAi construct (e.g., olpaciran) or a pharmaceutical composition comprising an LPA RNAi construct (e.g., olpaciran) to a patient according to the methods of the present invention is administered by subcutaneous injection. In such embodiments, the injection volume is about 2 mL or less, or about 1 mL or less, e.g., about 1 mL.

[0020] Use of an LPA RNAi construct in any of the methods disclosed herein or for preparation of a medicament for administration according to any of the methods disclosed herein is specifically contemplated. For example, the present invention includes an LPA RNAi construct for use in a method of treating, attenuating, or preventing atherosclerosis or cardiovascular disease in a patient in need thereof, the method comprising administering to the patient an LPA RNAi construct at a dose of about 9 mg to about 675 mg with a dosing interval of at least 8 weeks. The present invention also includes an LPA RNAi construct for use in a method of reducing serum or plasma Lp(a) levels in a patient, the method comprising administering to the patient an LPA RNAi construct at a dose of about 9 mg to about 675 mg with a dosing interval of at least 8 weeks. In certain embodiments, the present invention provides an LPA RNAi construct for use in a method for reducing the risk of cardiovascular events in a patient with atherosclerotic cardiovascular disease, the method comprising administering to the patient the LPA RNAi construct at a dose of about 9 mg to about 675 mg with an administration interval of at least 8 weeks.

[0021] The invention also encompasses the use of an LPA RNAi construct in the preparation of a medicament for treating, attenuating, or preventing atherosclerosis or cardiovascular disease in a patient in need thereof, wherein the medicament is administered or formulated for administration at a dose of about 9 mg to about 675 mg with a dosing interval of at least 8 weeks. In some embodiments, the invention provides the use of an LPA RNAi construct in the preparation of a medicament for reducing serum or plasma Lp(a) levels in a patient, wherein the medicament is administered or formulated for administration at a dose of about 9 mg to about 675 mg with a dosing interval of at least 8 weeks. In another embodiment, the present invention provides use of an LPA RNAi construct in the preparation of a medicament for reducing the risk of cardiovascular events in patients with atherosclerotic cardiovascular disease, wherein the medicament is administered or formulated for administration at a dose of about 9 mg to about 675 mg with a dosing interval of at least 8 weeks. [Brief explanation of the drawings]

[0022] [Figure 1] The structure of the LPA RNAi construct, olpaciran, is shown schematically. The top strand, listed in a 5' to 3' direction, is the sense strand (SEQ ID NO: 5), and the bottom strand, listed in a 3' to 5' direction, is the antisense strand (SEQ ID NO: 6). Black circles represent nucleotides with a 2'-O-methyl modification, white circles represent nucleotides with a 2'-deoxy-2'-fluoro modification, and gray circles represent deoxyadenosine nucleotides linked to adjacent nucleotides via a 3'-3' bond (i.e., inverted). Gray lines connecting the circles represent phosphodiester bonds, while black lines connecting the circles represent phosphorothioate linkages. A trivalent GalNAc moiety having the structure shown is represented by R1 and is covalently attached to the 5' end of the sense strand via a phosphorothioate linkage. [Figure 2]1 is a line graph showing the percent change from baseline in plasma Lp(a) levels in human subjects after a single subcutaneous dose of placebo or olpaciran at the indicated doses in each of cohorts 1-7 across study days. Baseline values ​​were the average of screening Lp(a) and day 1 predose Lp(a) levels. If only one value was available, that value was used as the baseline. [Figure 3-1] Predicted Lp(a) levels of baseline are shown as a percentage for quarterly (Q3M) administration of olpaciran at doses of 10 mg (FIG. 3A), 30 mg (FIG. 3B), 50 mg (FIG. 3C), 75 mg (FIG. 3D), 150 mg (FIG. 3E), and 225 mg (FIG. 3F) for subjects with baseline Lp(a) levels ≥ 150 nmol / L. The horizontal line in each graph represents an 80% reduction in Lp(a) levels from baseline. Predicted Lp(a) levels are based on PK / PD model simulations for 10,000 subjects. Predicted data are shown as median values ​​(solid lines), with 95% prediction intervals represented by shading. The solid circles in FIG. 3D represent observed data for Cohort 7, as described in Example 1. [Figure 3-2] Predicted Lp(a) levels of baseline are shown as a percentage for quarterly (Q3M) administration of olpaciran at doses of 10 mg (FIG. 3A), 30 mg (FIG. 3B), 50 mg (FIG. 3C), 75 mg (FIG. 3D), 150 mg (FIG. 3E), and 225 mg (FIG. 3F) for subjects with baseline Lp(a) levels ≥ 150 nmol / L. The horizontal line in each graph represents an 80% reduction in Lp(a) levels from baseline. Predicted Lp(a) levels are based on PK / PD model simulations for 10,000 subjects. Predicted data are shown as median values ​​(solid lines), with 95% prediction intervals represented by shading. The solid circles in FIG. 3D represent observed data for Cohort 7, as described in Example 1. [Figure 4-1]Predicted Lp(a) levels of baseline are shown as a percentage for twice-yearly (Q6M) administration of olpaciran at doses of 10 mg (FIG. 4A), 75 mg (FIG. 4B), 150 mg (FIG. 4C), 225 mg (FIG. 4D), 450 mg (FIG. 4E), and 675 mg (FIG. 4F) for subjects with baseline Lp(a) levels ≥ 150 nmol / L. The horizontal line in each graph represents an 80% reduction in Lp(a) levels from baseline. Predicted Lp(a) levels are based on PK / PD model simulations for 10,000 subjects. Predicted data are shown as median values ​​(solid lines), with 95% prediction intervals represented by shading. The filled circles in FIG. 4B represent observed data for Cohort 7, as described in Example 1. [Figure 4-2] Predicted Lp(a) levels of baseline are shown as a percentage for twice-yearly (Q6M) administration of olpaciran at doses of 10 mg (FIG. 4A), 75 mg (FIG. 4B), 150 mg (FIG. 4C), 225 mg (FIG. 4D), 450 mg (FIG. 4E), and 675 mg (FIG. 4F) for subjects with baseline Lp(a) levels ≥ 150 nmol / L. The horizontal line in each graph represents an 80% reduction in Lp(a) levels from baseline. Predicted Lp(a) levels are based on PK / PD model simulations for 10,000 subjects. Predicted data are shown as median values ​​(solid lines), with 95% prediction intervals represented by shading. The filled circles in FIG. 4B represent observed data for Cohort 7, as described in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0023] Lp(a) has been reported to be a causative risk factor for various forms of cardiovascular disease, including myocardial infarction, stroke, peripheral arterial disease, and aortic stenosis. Lp(a) levels are genetically determined and, unlike LDL cholesterol (LDL-C) levels, cannot be altered by diet, exercise, or other lifestyle changes. Currently, there are no approved therapies that selectively target apo(a) to substantially lower Lp(a) levels. The present invention provides a novel administration regimen of an RNAi construct targeting mRNA transcribed from the LPA gene, which encodes the apo(a) protein, to sustained suppression of Lp(a) levels for the treatment or prevention of atherosclerosis and related cardiovascular conditions. A specific LPA-targeting RNAi construct, olpaciran, was observed to reduce Lp(a) concentrations by 71% to 96% after a single dose in human subjects with baseline Lp(a) levels of ≥70 nmol / L, with a maximum percent reduction of >90%, and the effect persisting for more than 6 months at single doses of 9 mg or higher (see Example 1). Specifically, a single dose of olpaciran as low as 9 mg reduced Lp(a) levels by more than 80% for more than 3 months in human subjects, while single doses of 75 mg and 225 mg suppressed Lp(a) levels by more than 80% for more than 6 months. Furthermore, olpaciran was well tolerated at these doses, with no serious treatment-related adverse events (see Example 1). The robust and sustained suppression of Lp(a) at this dosage range was unexpected. This is because, based on allometric scaling of olpaciran dose evaluated in cynomolgus monkeys, it was predicted that an 8-fold higher dose (e.g., 75 mg vs. 9 mg) would be required to produce an 80% reduction in Lp(a) over one month.

[0024] Furthermore, the depth and duration of Lp(a) suppression achieved by olpaciran in human subjects was surprising given the results reported in human subjects with other nucleic acid therapeutics targeting apo(a). AKCEA-APO(a)-LRx, an antisense oligonucleotide targeting apo(a), has been reported to reduce Lp(a) levels by 35% to 80% in human subjects after 6 months of treatment. However, a 20 mg weekly dose or a 60 mg monthly dose of AKCEA-APO(a)-LRx was required to achieve an 80% and 72% reduction in Lp(a), respectively (see Tsimikas et al., New England Journal of Medicine, Vol. 382:244-255, 2020). In contrast, as described herein, a single dose of olpaciran resulted in a greater than 80% reduction in Lp(a) levels for more than six months, thereby allowing olpaciran to be administered at lower doses and at longer dosing intervals, for example, once every three months or once every six months. Thus, the methods of the present invention provide significant improvements in human treatment for atherosclerotic cardiovascular disease, including, for example, improved patient adherence, reduced drug costs, and reduced injection volume and frequency. Accordingly, in certain embodiments, the present invention provides a method of treating, preventing, or reducing the risk of developing cardiovascular disease in a patient in need thereof, comprising administering to the patient an effective amount of an LPA RNAi construct according to the specific dosing regimen described herein.

[0025] Atherosclerosis is a disease in which plaque, composed of fatty substances, cholesterol, calcium, fibrin, and cellular waste products, builds up in various arteries throughout the body. Over time, the plaque hardens and narrows the arterial lumen, restricting blood flow to organs and tissues within the body. Atherosclerosis can lead to the development of several other diseases, such as cardiovascular disease, cerebrovascular disease, or chronic kidney disease, depending on the specific artery affected by atherosclerotic plaque buildup. For example, coronary artery disease occurs when plaque builds up in a coronary artery and partially blocks blood flow to the heart, which can lead to angina and myocardial infarction. Atherosclerotic plaque buildup in the carotid artery, which supplies oxygen-rich blood to the brain, can lead to carotid artery disease and, if blood flow is reduced or blocked, can cause transient ischemic attack or stroke. When plaques build up in the major arteries supplying blood to the limbs and pelvis, peripheral arterial disease can occur, leading to abdominal aortic aneurysms and limb ischemia (causing paralysis and pain). When atherosclerotic plaques accumulate in the renal arteries, chronic kidney disease can develop, leading over time to decreased kidney function, which can result in renal failure. Lp(a) is an atherogenic lipoprotein. Elevated levels have been associated with increased risk of, among other things, coronary artery disease, peripheral artery disease, myocardial infarction, and stroke. The methods of the present invention are useful for treating, attenuating, or preventing atherosclerosis in patients by lowering circulating Lp(a) levels. Thus, in some embodiments, the present invention provides a method of treating, attenuating, or preventing atherosclerosis in a patient in need thereof, comprising administering to the patient an effective amount of an LPA RNAi construct according to any of the dosing regimens described herein. In one embodiment, the invention includes the use of any of the LPA RNAi constructs described herein for the preparation of a medicament for treating, attenuating, or preventing atherosclerosis in a patient in need thereof, wherein the medicament is administered according to, or formulated for administration according to, any of the dosing regimens described herein.In another embodiment, the present invention provides an LPA RNAi construct, such as any of the LPA RNAi constructs described herein, for use in a method of treating, alleviating, or preventing atherosclerosis in a patient in need thereof, the method comprising administering the LPA RNAi construct according to any of the dosing regimens described herein.

[0026] In certain embodiments, the present invention also provides a method of treating, alleviating, ameliorating, or preventing cardiovascular disease in a patient in need thereof, comprising administering to the patient an effective amount of an LPA RNAi construct according to any of the dosage regimens described herein. In some embodiments, the present invention includes the use of any of the LPA RNAi constructs described herein for the preparation of a medicament for treating, alleviating, or preventing cardiovascular disease in a patient in need thereof, wherein the medicament is administered according to or formulated for administration according to any of the dosage regimens described herein. In other embodiments, the present invention provides an LPA RNAi construct, such as any of the LPA RNAi constructs described herein, for use in a method of treating, alleviating, or preventing cardiovascular disease in a patient in need thereof, wherein the method comprises administering an LPA RNAi construct according to any of the dosage regimens described herein. Cardiovascular disease is a class of diseases and conditions that affect blood vessels or the heart, including, but not limited to, myocardial infarction, heart failure, transient ischemic attack, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (e.g., peripheral arterial disease), aneurysm (e.g., abdominal aortic aneurysm), carotid artery disease, cerebrovascular disease, stable or unstable angina, atrial fibrillation, hyperlipidemia, familial hypercholesterolemia (heterozygous and homozygous), vulnerable plaque, and aortic stenosis. Thus, in certain embodiments, patients to be treated according to the methods of the present invention are diagnosed with or at risk of developing cardiovascular disease. Patients at risk of developing cardiovascular disease may have a family history of cardiovascular disease and / or may have one or more risk factors for cardiovascular disease. Such risk factors include, but are not limited to, high blood pressure, elevated levels of non-HDL cholesterol, elevated levels of triglycerides, diabetes, obesity, or smoking.Diagnosis of atherosclerosis and cardiovascular disease can be made using a variety of methods known to those skilled in the art, which may include one or more of the following: a patient's medical examination and family history, the patient's risk factors, a physical examination, blood tests measuring various biomarkers such as lipid levels (e.g., LDL-C, triglycerides, Lp(a), glycated hemoglobin A1C, C-reactive protein, apolipoprotein B, cardiac troponin T, etc.), an electrocardiogram, an echocardiogram, a stress test, a chest x-ray, a computed tomography (CT) scan (e.g., a cardiac CT scan), and angiography.

[0027] In some embodiments, the cardiovascular disease to be treated, alleviated, ameliorated, or prevented in accordance with the methods of the present invention is coronary artery disease. Signs and symptoms of coronary artery disease may include chest pain (e.g., angina), shortness of breath, myocardial infarction, narrowing of one or more coronary arteries, pain or discomfort in the arm or shoulder, weakness, dizziness, nausea, and a history of coronary artery bypass and / or percutaneous coronary intervention. In a related embodiment, the cardiovascular disease to be treated, alleviated, ameliorated, or prevented in accordance with the methods of the present invention is myocardial infarction.

[0028] In another embodiment, the cardiovascular disease to be treated, alleviated, ameliorated, or prevented according to the method of the present invention is coronary artery disease, particularly atherosclerotic cerebrovascular disease.Cerebrovascular disease refers to a disorder in which an area of ​​the brain is temporarily or permanently affected by ischemia or hemorrhage due to one or more malfunctions or complications of cerebral blood vessels.Cerebrovascular disease includes, but is not limited to, transient ischemic attack, stroke (ischemic or hemorrhagic), carotid artery stenosis, vertebral artery stenosis, intracranial artery stenosis, aneurysm, and vascular malformation. Signs and symptoms of cerebrovascular disease may include dizziness, nausea, vomiting, very severe headache, confusion, disorientation, memory loss, numbness or weakness of an arm, leg, or face (especially one side), abnormal or slurred speech, difficulty understanding, loss of vision or difficulty seeing, loss of balance, coordination, or ability to walk, carotid stenosis, and a history of transient ischemic attack and / or carotid revascularization. In one embodiment, the cardiovascular disease to be treated, alleviated, ameliorated, or prevented in accordance with the methods of the present invention is stroke.

[0029] In certain other embodiments, the cardiovascular disease that is treated or prevented by the method of the present invention is peripheral arterial disease.The signs and symptoms of peripheral arterial disease can include: pain or muscle cramps in legs or arms when walking (claudication), numbness or weakness in legs, coldness in lower legs or feet, pain in toes, non-healing feet or legs, change in leg color, hair loss or slower hair growth in feet and legs, slower growth of toenails, shiny skin in legs, no pulse or weak pulse in legs or feet, ankle-brachial index≦0.90, and history of abdominal aortic aneurysm, abdominal aortic treatment (percutaneous or surgical), and / or peripheral arterial revascularization (percutaneous or surgical).

[0030] In some embodiments, the administration of LPA RNAi constructs according to the method of the present invention is for the treatment of atherosclerosis and other cardiovascular diseases and conditions.The term " treatment " or " treating " used herein refers to the application or administration of LPA RNAi constructs to patients who have or have been diagnosed with atherosclerosis or other cardiovascular diseases, have signs of atherosclerosis or other cardiovascular diseases, are at risk of developing atherosclerosis or other cardiovascular diseases, or have a predisposition to atherosclerosis or other cardiovascular diseases, one or more symptoms of atherosclerosis or other cardiovascular diseases, are at risk of developing atherosclerosis or other cardiovascular diseases, or are prone to atherosclerosis or other cardiovascular diseases, for the purpose of curing, curing, alleviating, alleviating, modifying, ameliorating or improving atherosclerosis or other cardiovascular diseases. The term "treatment" encompasses any improvement in a patient's condition, including slowing or stopping the progression of atherosclerosis or other cardiovascular disease, reducing the number or severity of symptoms of atherosclerosis or other cardiovascular disease, or increasing the frequency or length of time during which the patient is free of symptoms of atherosclerosis or other cardiovascular disease. As used herein, the term "patient" refers to a mammal, including a human, and may be used interchangeably with the term "subject." In a preferred embodiment, the patient is a human patient.

[0031] In certain preferred embodiments, administration of an LPA RNAi construct to a patient according to any of the methods of the present invention reduces circulating Lp(a) levels or concentrations (e.g., serum or plasma Lp(a) levels / concentrations) in the patient compared to the circulating Lp(a) levels (e.g., baseline Lp(a) levels / concentrations) in the patient before administration of the LPA RNAi construct, or compared to the circulating Lp(a) levels / concentrations in a patient not receiving the LPA RNAi construct. Thus, in some embodiments, the present invention provides methods of reducing serum or plasma Lp(a) levels (or concentrations) in a patient in need thereof, comprising administering an LPA RNAi construct to the patient according to any of the dosing regimens described herein. In one embodiment, the present invention includes the use of any of the LPA RNAi constructs described herein for the preparation of a medicament for reducing serum or plasma Lp(a) levels (or concentrations) in a patient in need thereof, wherein the medicament is administered according to or formulated for administration according to any of the dosing regimens described herein. In another embodiment, the present invention provides an LPA RNAi construct, e.g., any of the LPA RNAi constructs described herein, for use in a method of reducing serum or plasma Lp(a) levels (or concentrations) in a patient in need thereof, the method comprising administering the LPA RNAi construct according to any of the dosing regimens described herein. In some embodiments, the patient in need of reducing serum or plasma Lp(a) levels (or concentrations) is a patient diagnosed with or at risk of cardiovascular disease, such as any of the cardiovascular diseases described above. In some such embodiments, the cardiovascular disease is coronary artery disease, carotid artery disease, peripheral artery disease, myocardial infarction, cerebrovascular disease, stroke, aortic stenosis, stable or unstable angina, atrial fibrillation, heart failure, hyperlipidemia, heterozygous familial hypercholesterolemia, or homozygous familial hypercholesterolemia. In a particular embodiment, the patient in need of reducing serum or plasma Lp(a) levels (or concentrations) has a history of myocardial infarction or a family history of myocardial infarction.

[0032] In certain embodiments, patients who need to reduce serum or plasma Lp(a) levels (or concentrations) are diagnosed with acute coronary syndrome. Acute coronary syndrome refers to symptoms associated with a sudden reduction in blood flow to the heart, often caused by the rupture of atherosclerotic plaques and partial or complete thrombosis of the coronary arteries. Acute coronary syndromes include acute myocardial ischemia or infarction, such as non-ST elevation myocardial infarction (NSTEMI) and ST elevation MI (STEMI), and unstable angina. Even if acute coronary syndrome does not initially result in infarction, this is a sign of a high risk of infarction occurring and must be diagnosed and treated promptly. Signs and symptoms of acute coronary syndrome typically begin suddenly and may include chest pain (angina) or discomfort, pain radiating from the chest to the shoulders, arms, upper abdomen, back, neck, or jaw, nausea or vomiting, indigestion, shortness of breath, sudden heavy sweating, dizziness, or fainting, unusual or inexplicable fatigue, and feelings of restlessness or anxiety.

[0033] In certain other embodiments, the patient in need of a reduction in serum or plasma Lp(a) levels (or concentrations) is diagnosed with chronic kidney disease. Chronic kidney disease generally refers to the gradual damage to and loss of function of the kidneys. Chronic kidney disease can worsen over time, putting the patient at increased risk for other cardiovascular diseases. In one embodiment, the patient to be treated according to the methods of the present invention has stage 3 chronic kidney disease. The stage of kidney disease is determined by the estimated glomerular filtration rate (eGFR), which is a value based on the amount of creatinine in the blood. Stage 3 chronic kidney disease is characterized by a creatinine level of approximately 30 mL / min / 1.73 m 2 ~Approx. 59mL / min / 1.73m 2 Stage 3 chronic kidney disease is characterized by an eGFR of about 15 mL / min / 1.73 m, which may be more or less than normal, and may be accompanied by some initial symptoms, such as swelling in the hands and feet, back pain, and urination. Stage 3 chronic kidney disease patients may also have other health-related problems, such as high blood pressure, anemia, and bone disease. In another embodiment, patients to be treated according to the methods of the present invention have stage 4 chronic kidney disease. Stage 4 chronic kidney disease patients have an eGFR of about 15 mL / min / 1.73 m. 2~Approx. 29mL / min / 1.73m 2 and typically presents with more or less symptomatic swelling in the hands and feet, back pain, and urination than normal.

[0034] In some embodiments, administration of an LPA RNAi construct to a patient according to the methods of the invention reduces the level (or concentration) of Lp(a) in serum or plasma in the patient by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, compared to the level (or concentration) of Lp(a) in serum or plasma in the patient before administration of the RNAi construct (e.g., a baseline Lp(a) level or concentration), or compared to the level (or concentration) of Lp(a) in serum or plasma in a patient who has not received the RNAi construct. In these and other embodiments, following administration of the LPA RNAi construct (e.g., administration of a single dose of the LPA RNAi construct), circulating Lp(a) levels or concentrations are reduced in the patient for at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 18 weeks, at least 20 weeks, at least 22 weeks, at least 24 weeks, at least 26 weeks, at least 28 weeks, at least 30 weeks, at least 32 weeks, at least 36 weeks, or at least 48 weeks.

[0035] In one embodiment of the methods of the present invention, administration of an LPA RNAi construct (e.g., a single dose of an LPA RNAi construct) reduces serum or plasma Lp(a) levels (or concentrations) in a patient by more than 50% for at least 12 weeks, compared to the patient's baseline serum or plasma Lp(a) level (or concentration). Baseline serum or plasma Lp(a) levels (or concentrations) refer to the serum or plasma Lp(a) levels (or concentrations) in a patient before administration of the LPA RNAi construct (i.e., pretreatment levels or concentrations). The baseline level / concentration may be a single measurement taken before the patient receives the LPA RNAi construct, or the baseline level / concentration may be the average of two or more measurements taken before the patient receives the LPA RNAi construct. In another embodiment of the method of the present invention, administration of an LPA RNAi construct (e.g., a single dose of an LPA RNAi construct) reduces serum or plasma Lp(a) levels (or concentrations) in a patient by more than 50% for at least 24 weeks compared to the patient's baseline serum or plasma Lp(a) levels (or concentrations). In yet another embodiment of the method of the present invention, administration of an LPA RNAi construct (e.g., a single dose of an LPA RNAi construct) reduces serum or plasma Lp(a) levels (or concentrations) in a patient by more than 80% for at least 12 weeks compared to the patient's baseline serum or plasma Lp(a) levels (or concentrations). In yet another embodiment of the method of the present invention, administration of an LPA RNAi construct (e.g., a single dose of an LPA RNAi construct) reduces serum or plasma Lp(a) levels (or concentrations) in a patient by more than 80% for at least 24 weeks compared to the patient's baseline serum or plasma Lp(a) levels (or concentrations). In certain embodiments of the methods of the present invention, administration of an LPA RNAi construct (e.g., a single dose of an LPA RNAi construct) reduces serum or plasma Lp(a) levels (or concentrations) in a patient by more than 80% compared to the patient's baseline serum or plasma Lp(a) levels (or concentrations) for at least 32 weeks.In some embodiments of the methods of the present invention, administration of an LPA RNAi construct (e.g., a single dose of an LPA RNAi construct) reduces serum or plasma Lp(a) levels (or concentrations) in a patient by more than 90% for at least 12 weeks, compared to the patient's baseline serum or plasma Lp(a) levels (or concentrations). In other embodiments of the methods of the present invention, administration of an LPA RNAi construct (e.g., a single dose of an LPA RNAi construct) reduces serum or plasma Lp(a) levels (or concentrations) in a patient by more than 90% for at least 16 weeks, compared to the patient's baseline serum or plasma Lp(a) levels (or concentrations).

[0036] In certain embodiments, administration of an LPA RNAi construct to a patient according to the methods of the invention reduces the absolute Lp(a) level (or concentration) in the patient's serum or plasma to about 150 nmol / L or less, about 125 nmol / L or less, about 100 nmol / L or less, about 75 nmol / L or less, about 70 nmol / L or less, about 65 nmol / L or less, about 60 nmol / L or less, about 55 nmol / L or less, about 50 nmol / L, about 45 nmol / L or less, about 40 nmol / L or less, about 35 nmol / L or less, or about 30 nmol / L or less. In one embodiment, administration of an LPA RNAi construct to a patient according to the methods of the invention reduces the absolute Lp(a) level (or concentration) in the patient's serum or plasma to about 125 nmol / L or less. In another embodiment, administration of an LPA RNAi construct to a patient according to the methods of the invention reduces the absolute Lp(a) level (or concentration) in the patient's serum or plasma to about 100 nmol / L or less. In another embodiment, administration of an LPA RNAi construct to a patient according to the methods of the invention reduces the absolute Lp(a) level (or concentration) in the patient's serum or plasma to about 75 nmol / L or less. In yet another embodiment, administration of an LPA RNAi construct to a patient according to the methods of the invention reduces the absolute Lp(a) level (or concentration) in the patient's serum or plasma to about 50 nmol / L or less.

[0037] Although there is a preference for measuring Lp(a) levels / concentrations in units of particle concentration (e.g., nmol / L) (see, e.g., Wilson et al., Journal of Clinical Lipidology, Vol. 13; 374-392, 2019), Lp(a) levels may also be measured in units of mass concentration (e.g., mg / dL). In such embodiments, administration of an LPA RNAi construct to a patient according to the methods of the present invention may reduce the Lp(a) level (or concentration) in the patient's serum or plasma to about 100 mg / dL or less, about 90 mg / dL or less, about 80 mg / dL or less, about 70 mg / dL or less, about 60 mg / dL or less, about 50 mg / dL or less, about 45 mg / dL or less, about 40 mg / dL or less, about 35 mg / dL or less, about 30 mg / dL or less, about 25 mg / dL or less, about 20 mg / dL or less, or about 15 mg / dL or less.

[0038] Lp(a) levels in plasma or serum samples can be measured using commercially available kits, such as the Lp(a) ELISA assay kit from Mercodia AB (Uppsala, Sweden), the Lp(a) immunoturbidimetric assay from Randox Laboratories Ltd. (Crumlin, United Kingdom), or the Tina-quant® Lp(a) Gen.2 assay from F. Hoffmann-La Roche Ltd. (Basel, Switzerland), or other methods known in the art, such as those described in Marcovina and Albers, J. Lipid Res., Vol. 57:526-537, 2016. In certain embodiments, Lp(a) levels are measured using a turbidimetric immunoassay that is standardized to detect and quantify Lp(a) particles independent of apo(a) isoform size. In these and other embodiments, the assay used to measure Lp(a) levels is normalized to the IFCC reference material SRM2B in nmol / L (Marcovina et al., Clin. Chem., Vol. 46:1946-1967, 2000).

[0039] As previously described, elevated levels of circulating Lp(a) are associated with an increased risk of cardiovascular disease. Therefore, the methods of the present invention are also useful for reducing the risk of cardiovascular events in patients with elevated serum or plasma levels of Lp(a). Accordingly, in certain embodiments, the present invention provides a method for reducing the risk of cardiovascular events in patients with atherosclerotic cardiovascular disease, comprising administering to the patient an effective amount of an LPA RNAi construct according to any of the dosing regimens described herein. In one embodiment, the present invention includes the use of any of the LPA RNAi constructs described herein for the preparation of a medicament for reducing the risk of cardiovascular events in patients with atherosclerotic cardiovascular disease, wherein the medicament is administered according to or formulated for administration according to any of the dosing regimens described herein. In another embodiment, the present invention provides an LPA RNAi construct, such as any of the LPA RNAi constructs described herein, for use in a method for reducing the risk of cardiovascular events in patients with atherosclerotic cardiovascular disease, the method comprising administering the LPA RNAi construct according to any of the dosing regimens described herein.

[0040] In some embodiments, the cardiovascular event is one or more of the following: cardiovascular death, myocardial infarction, stroke (e.g., ischemic stroke), coronary revascularization, hospitalization for unstable angina, hospitalization for heart failure, peripheral revascularization, acute limb ischemia, transient ischemic attack, major limb amputation surgery for ischemia, cerebral revascularization (all resulting in death). In certain embodiments, the cardiovascular event is cardiovascular death, myocardial infarction, stroke (e.g., ischemic stroke), and / or coronary revascularization. In some such embodiments, the cardiovascular event is cardiovascular death, myocardial infarction, and / or coronary revascularization. In other such embodiments, the cardiovascular event is myocardial infarction and / or coronary revascularization. In other embodiments, the cardiovascular event is a major cardiovascular event selected from cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, and hospitalization for unstable angina. In yet another embodiment, the cardiovascular event is a major adverse limb event selected from acute limb ischemia, major amputation surgery, and peripheral revascularization for ischemia. In one embodiment, the cardiovascular event is cardiovascular death. In another embodiment, the cardiovascular event is non-fatal myocardial infarction. In yet another embodiment, the cardiovascular event is non-fatal stroke (e.g., ischemic stroke). In yet another embodiment, the cardiovascular event is coronary revascularization.

[0041] In certain embodiments, patients administered an LPA RNAi construct according to the methods of the present invention have a relative risk reduction of at least 15%, at least 20%, at least 25%, or at least 30% for any of the above-mentioned cardiovascular events compared to patients not receiving an LPA RNAi construct. In one embodiment, patients administered an LPA RNAi construct according to the methods of the present invention have a relative risk reduction of about 15% to about 25% for any one of cardiovascular death, myocardial infarction, and ischemic stroke compared to patients not receiving an LPA RNAi construct. In another embodiment, patients administered an LPA RNAi construct according to the methods of the present invention have a relative risk reduction of about 20% to about 30% for any one of cardiovascular death, myocardial infarction, and ischemic stroke compared to patients not receiving an LPA RNAi construct.

[0042] In certain other embodiments, patients administered an LPA RNAi construct according to the methods of the invention have an absolute risk reduction of at least 1.5%, at least 1.8%, at least 2.0%, at least 2.2%, at least 2.5%, at least 2.8%, at least 3.0%, at least 3.2%, or at least 3.5% for any of the cardiovascular events described above. In one embodiment, patients administered an LPA RNAi construct according to the methods of the invention have an absolute risk reduction of about 1.5% to about 3.0% for any one of cardiovascular death, myocardial infarction, and ischemic stroke. In another embodiment, patients administered an LPA RNAi construct according to the methods of the invention have an absolute risk reduction of about 2.0% to about 3.5% for any one of cardiovascular death, myocardial infarction, and ischemic stroke. In yet another embodiment, a patient administered an LPA RNAi construct according to the methods of the present invention has an absolute risk reduction of about 2.0% to about 3.0% for any one of cardiovascular death, myocardial infarction, and ischemic stroke.

[0043] In any of the above embodiments, patients to whom an LPA RNAi construct is administered according to the methods of the present invention to reduce cardiovascular events may have a history of coronary revascularization, a history of coronary artery bypass grafting, a diagnosis of coronary artery disease, a diagnosis of atherosclerotic cerebrovascular disease, a diagnosis of peripheral artery disease, and / or a history of myocardial infarction. In certain embodiments, patients to whom an LPA RNAi construct is administered according to the methods of the present invention to reduce cardiovascular events have experienced a myocardial infarction. For example, in some such embodiments, patients to whom an LPA RNAi construct is administered according to the methods of the present invention to reduce cardiovascular events have experienced a myocardial infarction within one year, two years, three years, four years, or five years prior to receiving the initial administration of the LPA RNAi construct. In one such embodiment, patients to whom an LPA RNAi construct is administered according to the methods of the present invention to reduce cardiovascular events have experienced a myocardial infarction within one year prior to receiving the initial administration of the LPA RNAi construct. In certain other embodiments, patients to whom an LPA RNAi construct is administered according to the methods of the present invention to reduce cardiovascular events are or have recently been hospitalized for acute coronary syndrome or unstable angina.

[0044] In certain preferred embodiments, patients to whom an LPA RNAi construct is administered according to the methods of the present invention are patients with elevated circulating levels or concentrations of Lp(a) (e.g., elevated serum or plasma levels / concentrations of Lp(a)). Patients to whom an LPA RNAi construct is administered according to the methods of the present invention may have baseline circulating Lp(a) levels or concentrations of about 50 nmol / L or higher, about 55 nmol / L or higher, about 60 nmol / L or higher, about 65 nmol / L or higher, about 70 nmol / L or higher, about 75 nmol / L or higher, about 100 nmol / L or higher, about 125 nmol / L or higher, about 150 nmol / L or higher, about 175 nmol / L or higher, about 200 nmol / L or higher, about 225 nmol / L or higher, or about 250 nmol / L or higher. In one embodiment, a patient is administered an LPA RNAi construct according to the methods of the present invention if the serum or plasma Lp(a) level (or concentration) before the first administration of the LPA RNAi construct is about 70 nmol / L or higher. In another embodiment, a patient is administered an LPA RNAi construct according to the methods of the present invention if the serum or plasma Lp(a) level (or concentration) before the first administration of the LPA RNAi construct is about 100 nmol / L or higher. In yet another embodiment, a patient is administered an LPA RNAi construct according to the methods of the present invention if the serum or plasma Lp(a) level (or concentration) before the first administration of the LPA RNAi construct is about 125 nmol / L or higher. In yet another embodiment, a patient is administered an LPA RNAi construct according to the methods of the present invention if the serum or plasma Lp(a) level (or concentration) before the first administration of the LPA RNAi construct is about 150 nmol / L or higher. In some embodiments, a patient is administered an LPA RNAi construct according to the methods of the present invention if the serum or plasma Lp(a) level (or concentration) before the first administration of the LPA RNAi construct is about 175 nmol / L or higher. In other embodiments, a patient is administered an LPA RNAi construct according to the methods of the present invention if the serum or plasma Lp(a) level (or concentration) before the first administration of the LPA RNAi construct is about 200 nmol / L or higher.In certain other embodiments, a patient is administered an LPA RNAi construct according to the methods of the invention if the patient has a serum or plasma Lp(a) level (or concentration) of about 225 nmol / L or greater prior to the first administration of the LPA RNAi construct.

[0045] In less preferred embodiments in which circulating Lp(a) levels (or concentrations) are measured in mass concentration units, patients to whom an LPA RNAi construct is to be administered in accordance with the methods of the present invention may have circulating Lp(a) levels (or concentrations) of about 30 mg / dL or greater, about 35 mg / dL or greater, about 40 mg / dL or greater, about 45 mg / dL or greater, about 50 mg / dL or greater, about 55 mg / dL or greater, about 60 mg / dL or greater, about 65 mg / dL or greater, about 70 mg / dL or greater, about 75 mg / dL or greater, about 90 mg / dL or greater, or about 100 mg / dL or greater. In one embodiment, patients are administered an RNAi construct of the present invention if their serum or plasma Lp(a) levels (or concentrations) prior to the first administration of an LPA RNAi construct are about 50 mg / dL or greater. In another embodiment, a patient is administered an RNAi construct of the present invention if the serum or plasma Lp(a) level (or concentration) before the first administration of an LPA RNAi construct is about 60 mg / dL or greater. In yet another embodiment, a patient is administered an RNAi construct of the present invention if the serum or plasma Lp(a) level (or concentration) before the first administration of an LPA RNAi construct is about 70 mg / dL or greater. In yet another embodiment, a patient is administered an RNAi construct of the present invention if the serum or plasma Lp(a) level (or concentration) before the first administration of an LPA RNAi construct is about 90 mg / dL or greater.

[0046] As discussed above, Lp(a) levels (or concentrations) in plasma or serum samples can be measured using commercially available kits, such as the Lp(a) ELISA assay kit from Mercodia AB (Uppsala, Sweden), the Lp(a) immunoturbidimetric assay from Randox Laboratories Ltd. (Crumlin, United Kingdom), or the Tina-quant® Lp(a) Gen.2 assay from F. Hoffmann-La Roche Ltd. (Basel, Switzerland), or other methods known in the art, such as those described in Marcovina and Albers, J. Lipid Res., Vol. 57:526-537, 2016. In certain embodiments, Lp(a) levels are measured using a turbidimetric immunoassay that is standardized to detect and quantify Lp(a) particles independent of apo(a) isoform size. In these and other embodiments, the assay used to measure Lp(a) levels is normalized to the IFCC reference material SRM2B in nmol / L (Marcovina et al., Clin. Chem., Vol. 46:1946-1967, 2000).

[0047] In some embodiments, a patient to whom an LPA RNAi construct is to be administered according to the methods of the present invention may have a serum low-density lipoprotein cholesterol (LDL-C) level within the normal range or be adjusted to within the normal range through treatment with one or more lipid-lowering therapies. For example, in one embodiment, a patient to whom an LPA RNAi construct is to be administered according to the methods of the present invention has a serum LDL-C level of about 100 mg / dL or less before the first administration of the LPA RNAi construct. In another embodiment, a patient to whom an LPA RNAi construct is to be administered according to the methods of the present invention has a serum LDL-C level of about 70 mg / dL or less before the first administration of the LPA RNAi construct. In a related embodiment, a patient to whom an LPA RNAi construct is to be administered according to the methods of the present invention is undergoing one or more lipid-lowering therapies. Lipid-lowering therapies include, but are not limited to, PCSK9 inhibitors, such as PCSK9 antagonist monoclonal antibodies (e.g., evolocumab, alirocumab) and PCSK9-targeted siRNA (e.g., inclisiran), statins (e.g., atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin), cholesterol absorption inhibitors (e.g., ezetimibe), bempedoic acid, nicotinic acid (e.g., niacin), fibric acids (e.g., gemfibrozil, fenofibrate), bile acid sequestrants (e.g., cholestyramine, colestipol, colesevelam), LDL apheresis, or combinations thereof. In certain embodiments, the patient to whom an LPA RNAi construct is to be administered in accordance with the methods of the present invention is receiving a lipid-lowering therapy selected from the group consisting of a PCSK9 antagonist monoclonal antibody, a statin, ezetimibe, bempedoic acid, or a combination thereof.

[0048] In certain embodiments, the patient who will be administered with LPA RNAi construct according to the method of the present invention has a serum triglyceride level of less than about 500 mg / dL before the first administration of LPA RNAi construct.For example, the patient's serum triglyceride level at baseline (for example, before the first administration of LPA RNAi construct) can be less than about 400 mg / dL, less than about 375 mg / dL, less than about 350 mg / dL, less than about 325 mg / dL, less than about 300 mg / dL, less than about 275 mg / dL, less than about 250 mg / dL, less than about 225 mg / dL, less than about 200 mg / dL, less than about 175 mg / dL, or less than about 150 mg / dL.In one embodiment, the patient who will be administered with LPA RNAi construct according to the method of the present invention has a serum triglyceride level of less than about 400 mg / dL before the first administration of LPA RNAi construct. In another embodiment, a patient to whom an LPA RNAi construct is to be administered according to the methods of the present invention has a serum triglyceride level of about 50 mg / dL to about 400 mg / dL before the first administration of the LPA RNAi construct. In yet another embodiment, a patient to whom an LPA RNAi construct is to be administered according to the methods of the present invention has a serum triglyceride level of about 150 mg / dL to about 375 mg / dL before the first administration of the LPA RNAi construct.

[0049] Measurements of LDL-C, triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), very-low-density lipoprotein cholesterol (VLDL-C), and other lipid biomarkers, such as apolipoprotein A1 and apolipoprotein B, can be measured using a standard lipid panel using a blood sample from the patient. In some embodiments, the patient fasts for at least 9 hours, preferably 12 hours, before the blood sample is drawn. Thus, the levels / concentrations of the above-mentioned lipid biomarkers (e.g., LDL-C, triglycerides) can be fasting levels.

[0050] In some embodiments, the patient who will be administered with LPA RNAi construct according to the method of the present invention has glycohemoglobin A1C level that does not indicate untreated or inadequately controlled type 2 diabetes.For example, the patient who will be administered with LPA RNAi construct according to the method of the present invention has a glycohemoglobin A1C level at baseline (for example, before the first administration of LPA RNAi construct) that is less than about 10.0%, less than about 9.5%, less than about 9.0%, less than about 8.5%, less than about 8.0%, less than about 7.5%, less than about 7.0%, less than about 6.5%, less than about 6.0%, or less than about 5.5%.In one embodiment, the patient who will be administered with LPA RNAi construct according to the method of the present invention has a glycohemoglobin A1C level that is less than about 8.5% before the first administration of LPA RNAi construct. In another embodiment, a patient to whom an LPA RNAi construct is to be administered in accordance with the methods of the present invention has a glycated hemoglobin A1C level of less than about 7.0% prior to the first administration of the LPA RNAi construct.

[0051] In other embodiments, the patient to whom the LPA RNAi construct is administered according to the methods of the present invention does not have uncontrolled systolic and / or diastolic blood pressure. For example, the patient to whom the LPA RNAi construct is administered according to the methods of the present invention has a mean resting systolic blood pressure at baseline (e.g., before the first administration of the LPA RNAi construct) of less than about 180 mmHg, less than about 160 mmHg, less than about 140 mmHg, less than about 135 mmHg, less than about 130 mmHg, less than about 125 mmHg, or less than about 120 mmHg, and a mean resting diastolic blood pressure at baseline (e.g., before the first administration of the LPA RNAi construct) of less than about 120 mmHg, less than about 110 mmHg, less than about 100 mmHg, less than about 85 mmHg, less than about 90 mmHg, or less than about 80 mmHg. In one embodiment, a patient to whom an LPA RNAi construct is to be administered according to the methods of the present invention has a resting mean systolic blood pressure of less than about 180 mmHg and a mean diastolic blood pressure of less than about 110 mmHg before the first administration of an LPA RNAi construct. In another embodiment, a patient to whom an LPA RNAi construct is to be administered according to the methods of the present invention has a resting mean systolic blood pressure of less than about 160 mmHg and a mean diastolic blood pressure of less than about 100 mmHg before the first administration of an LPA RNAi construct. In yet another embodiment, a patient to whom an LPA RNAi construct is to be administered according to the methods of the present invention has a resting mean systolic blood pressure of less than about 140 mmHg and a mean diastolic blood pressure of less than about 90 mmHg before the first administration of an LPA RNAi construct.

[0052] In some embodiments, patients to whom an LPA RNAi construct is administered according to the methods of the present invention have no signs of severe renal dysfunction. Thus, in certain embodiments, patients to whom an LPA RNAi construct is administered according to the methods of the present invention have an eGFR at baseline (e.g., before the first administration of an LPA RNAi construct) of at least about 30 mL / min / 1.73 m 2 , at least about 45 mL / min / 1.73 m 2 , at least about 60 mL / min / 1.73 m 2, at least about 75 mL / min / 1.73 m 2 , or at least about 90 mL / min / 1.73 m 2 In one particular embodiment, a patient to whom an LPA RNAi construct is to be administered according to the methods of the present invention has an eGFR of about 30 mL / min / 1.73 m before the first administration of the LPA RNAi construct. 2 That's all.

[0053] In other embodiments, patients to whom an LPA RNAi construct is to be administered according to the methods of the present invention have no signs of active liver disease or liver dysfunction. Active liver disease can be determined by measuring one or more biomarkers of liver function, such as those included in a liver function test or liver panel, including albumin, alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), bilirubin, and lactate dehydrogenase (LD). In certain embodiments, patients to whom an LPA RNAi construct is to be administered according to the methods of the present invention have baseline ALT levels (e.g., before the first administration of the LPA RNAi construct) that are no greater than three times the upper limit of normal (ULN). In a related embodiment, patients to whom an LPA RNAi construct is to be administered according to the methods of the present invention have baseline ALT levels (e.g., before the first administration of the LPA RNAi construct) that are no greater than three times the ULN. In these and other embodiments, patients to whom an LPA RNAi construct is to be administered according to the methods of the invention have a baseline total bilirubin level (e.g., before the first administration of the LPA RNAi construct) of less than or equal to two times the ULN. In some embodiments, patients to whom an LPA RNAi construct is to be administered according to the methods of the invention have, at baseline (e.g., before the first administration of the LPA RNAi construct): (i) a serum AST level of less than about 170 units / L, (ii) a serum AST level of less than about 150 units / L, and / or (iii) a total bilirubin level of less than about 2.0 mg / dL.

[0054] In one aspect, the method of the present invention comprises administering to a patient an effective amount of an LPA RNAi construct. "Effective amount" refers to an amount sufficient to treat, alleviate, or ameliorate cardiovascular disease or one or more symptoms of cardiovascular disease, particularly conditions or symptoms associated with cardiovascular disease, or otherwise prevent, hinder, delay, or halt the progression of cardiovascular disease or any other undesirable symptoms associated with cardiovascular disease in any way. An effective amount may also refer to an amount sufficient to reduce the occurrence or severity of sequelae resulting from cardiovascular disease. For example, in some embodiments, an effective amount of an LPA RNAi construct is an amount sufficient to reduce the occurrence or severity of cardiovascular events, such as myocardial infarction, stroke, or revascularization of coronary, cerebral, or peripheral arteries, in patients with atherosclerosis or other cardiovascular disease.

[0055] In certain embodiments of the methods of the present invention, the LPA RNAi construct is administered to a patient at a fixed dose. A "fixed dose" refers to a dose administered to all patients regardless of patient-specific factors, such as body weight. Thus, a fixed dose is not adjusted by the patient based on the patient's body weight. In some embodiments of the methods of the present invention, the LPA RNAi construct may be administered to a patient at a fixed dose of about 9 mg to about 675 mg with an administration interval of at least 8 weeks. For example, the fixed dose of the LPA RNAi construct can be about 9 mg, about 10 mg, about 15 mg, about 30 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, or about 675 mg, wherein the doses are administered at an interval of at least 8 weeks. Ranges between any and all of these endpoints are also contemplated; for example, a fixed dose of an LPA RNAi construct administered to a patient in the methods of the present invention can be about 10 mg to about 225 mg, about 50 mg to about 100 mg, about 150 mg to about 225 mg, about 225 mg to about 675 mg, about 75 mg to about 150 mg, about 225 mg to about 450 mg, about 75 mg to about 225 mg, about 10 mg to about 75 mg, or about 200 mg to about 300 mg, with the dose administered at an interval of at least 8 weeks.

[0056] Any of the doses of the LPA RNAi construct described herein are preferably administered at a dosing interval of at least 8 weeks (i.e., the dose is not administered to the patient more frequently than once every 8 weeks (or once every 2 months)). For example, the dosing interval may be about 8 weeks, about 12 weeks, about 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks, or about 32 weeks. In certain embodiments, the dosing interval is about 12 weeks, e.g., a fixed dose of the LPA RNAi construct is administered to the patient once every 12 weeks (or once every 3 months). In certain other embodiments, the dosing interval is about 24 weeks, e.g., a fixed dose of the LPA RNAi construct is administered to the patient once every 24 weeks (or once every 6 months).

[0057] The fixed dose of LPA RNAi construct can be administered at each dosing interval as a single bolus injection (for example, by a single subcutaneous injection), or as two or more consecutive bolus injections (for example, two or more subcutaneous injections).In some embodiments, the entire amount of the fixed dose of LPA RNAi construct is administered to the patient at each dosing interval by a single bolus injection, for example, by using a pre-filled syringe or injection device as further described herein.For example, a fixed dose of 225 mg of LPA RNAi construct can be administered to the patient at each dosing interval (for example, once every 12 weeks) as a single bolus injection of 225 mg, optionally by an autoinjector, pen injector, or pre-filled syringe containing a 225 mg dose.In other embodiments, the entire amount of the fixed dose of LPA RNAi construct is administered to the patient as two or more consecutive bolus injections. As an example, a 225 mg fixed dose of an LPA RNAi construct may be administered to a patient at each dosing interval (e.g., once every 12 weeks) by three sequential injections of 75 mg each, optionally by three injection devices (e.g., an autoinjector, pen injector, or pre-filled syringe) each containing a 75 mg dose. Sequential injections given within a single day's period are considered to be a single administration within the context of the present invention. In other words, as an example, a 225 mg fixed dose administration once every 12 weeks may be given as a single bolus injection of 225 mg administered to a patient once every 12 weeks, or as three sequential bolus injections of 75 mg each administered to a patient within a single day's period, once every 12 weeks.

[0058] In certain embodiments of the methods of the present invention, a fixed dose of the LPA RNAi construct described herein is administered once every 12 weeks or once every three months. In some such embodiments, the methods of the present invention comprise administering to a patient an LPA RNAi construct at a fixed dose of about 10 mg to about 225 mg once every 12 weeks or once every three months. In other embodiments, the methods of the present invention comprise administering to a patient an LPA RNAi construct at a fixed dose of about 50 mg to about 100 mg once every 12 weeks or once every three months. In yet other embodiments, the methods of the present invention comprise administering to a patient an LPA RNAi construct at a fixed dose of about 75 mg to about 225 mg once every 12 weeks or once every three months. In yet other embodiments, the methods of the present invention comprise administering to a patient an LPA RNAi construct at a fixed dose of about 150 mg to about 225 mg once every 12 weeks or once every three months. In one embodiment, the method of the present invention comprises administering to a patient an LPA RNAi construct at a fixed dose of about 10 mg once every 12 weeks or once every three months. In another embodiment, the method of the present invention comprises administering to a patient an LPA RNAi construct at a fixed dose of about 30 mg once every 12 weeks or once every three months. In another embodiment, the method of the present invention comprises administering to a patient an LPA RNAi construct at a fixed dose of about 75 mg once every 12 weeks or once every three months. In another embodiment, the method of the present invention comprises administering to a patient an LPA RNAi construct at a fixed dose of about 100 mg once every 12 weeks or once every three months. In another embodiment, the method of the present invention comprises administering to a patient an LPA RNAi construct at a fixed dose of about 125 mg once every 12 weeks or once every three months. In yet another embodiment, the method of the present invention comprises administering to a patient an LPA RNAi construct at a fixed dose of about 150 mg once every 12 weeks or once every three months. In another embodiment, the method of the present invention comprises administering to a patient an LPA RNAi construct at a fixed dose of about 175 mg once every 12 weeks or once every three months, hi another embodiment, the method of the present invention comprises administering to a patient an LPA RNAi construct at a fixed dose of about 200 mg once every 12 weeks or once every three months.In yet another embodiment, the methods of the present invention comprise administering to the patient an LPA RNAi construct at a fixed dose of about 225 mg once every 12 weeks or once every three months.

[0059] In certain other embodiments of the methods of the invention, a fixed dose of the LPA RNAi construct described herein is administered once every 24 weeks or once every six months. In some such embodiments, the methods of the invention comprise administering to a patient an LPA RNAi construct at a fixed dose of about 225 mg to about 675 mg once every 24 weeks or once every six months. In other embodiments, the methods of the invention comprise administering to a patient an LPA RNAi construct at a fixed dose of about 225 mg to about 450 mg once every 24 weeks or once every six months. In still other embodiments, the methods of the invention comprise administering to a patient an LPA RNAi construct at a fixed dose of about 200 mg to about 300 mg once every 24 weeks or once every six months. In one embodiment, the methods of the invention comprise administering to a patient an LPA RNAi construct at a fixed dose of about 225 mg once every 24 weeks or once every six months. In another embodiment, the method of the present invention comprises administering to a patient an LPA RNAi construct at a fixed dose of about 300 mg once every 24 weeks or once every six months. In yet another embodiment, the method of the present invention comprises administering to a patient an LPA RNAi construct at a fixed dose of about 450 mg once every 24 weeks or once every six months. In yet another embodiment, the method of the present invention comprises administering to a patient an LPA RNAi construct at a fixed dose of about 675 mg once every 24 weeks or once every six months.

[0060] In some embodiments of the methods of the present invention, the LPA RNAi construct is administered to a patient over a set treatment period. The "treatment period" begins with the administration of the first dose of the LPA RNAi construct and ends with the administration of the final dose of the LPA RNAi construct. The treatment period may be from about 12 weeks to about 240 weeks, from about 24 weeks to about 144 weeks, from about 3 months to about 60 months, or from about 6 months to about 48 months, for example, about 12 weeks, about 24 weeks, about 36 weeks, about 48 weeks, about 60 weeks, about 72 weeks, about 84 weeks, about 96 weeks, about 108 weeks, about 120 weeks, about 132 weeks, about 144 weeks, about 156 weeks, about 168 weeks, about 180 weeks, about 192 weeks, about 20 weeks, or about 30 weeks. The treatment period may be 4 weeks, about 216 weeks, about 228 weeks, about 240 weeks, about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 27 months, about 30 months, about 33 months, about 36 months, about 39 months, about 42 months, about 45 months, about 48 months, about 51 months, about 54 months, about 57 months, or about 60 months. In some embodiments, the treatment period is about 48 weeks. In other embodiments, the treatment period is about 192 weeks. In yet other embodiments, the treatment period is about 12 months. In yet other embodiments, the treatment period is about 48 months. In certain embodiments, the treatment period may be longer than 240 weeks or 60 months, for example, the treatment period may be greater than 5 years, e.g., 6, 7, 8, 9, or 10 years or more. In one specific embodiment, the LPA RNAi construct is administered for a treatment period of at least about 36 weeks to result in a statistically significant percent reduction from baseline in serum or plasma Lp(a) levels compared to subjects not receiving the LPA RNAi construct. In another specific embodiment, the LPA RNAi construct is administered for a treatment period of at least about 48 weeks to result in a statistically significant percent reduction from baseline in serum or plasma Lp(a) levels compared to subjects not receiving the LPA RNAi construct.

[0061] The method described herein comprises administering an LPA RNAi construct to a patient. As used herein, the term "LPA RNAi construct" refers to an agent comprising an RNA molecule that, when introduced into a cell, can downregulate the expression of the LPA gene via the RNA interference mechanism. RNA interference is a process in which a nucleic acid molecule induces the cleavage and degradation of a target RNA molecule (e.g., a messenger RNA or mRNA molecule) in a sequence-specific manner, for example, via the RNA-induced silencing complex (RISC) pathway. In some embodiments, the LPA RNAi construct comprises a double-stranded RNA molecule comprising two antiparallel strands of consecutive nucleotides that are sufficiently complementary to each other to hybridize and form a duplex region. "Hybridizing" or "hybridization" typically refers to the pairing of complementary polynucleotides via hydrogen bonds (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds) between complementary bases in two polynucleotides. A strand that includes a region having a sequence that is substantially complementary to a target LPA sequence (e.g., a target LPA mRNA) is referred to as an "antisense strand." A "sense strand" refers to a strand that includes a region that is substantially complementary to a region of the antisense strand. In some embodiments, the sense strand may include a region that has a sequence that is substantially identical to the target sequence.

[0062] Double-stranded RNA molecules can contain chemical modifications to ribonucleotides, including modifications to the ribose sugar, base, or backbone components of the ribonucleotides, such as those described herein or known in the art. All modifications used in double-stranded RNA molecules (e.g., siRNA, shRNA, etc.) are encompassed by the term "double-stranded RNA" for purposes of this disclosure.

[0063] As used herein, a first sequence is "complementary" to a second sequence if, under certain conditions, such as physiological conditions, a polynucleotide comprising the first sequence can hybridize to a polynucleotide comprising the second sequence to form a duplex region. Other such conditions may include moderate or stringent hybridization conditions known to those skilled in the art. A first sequence is considered to be fully complementary (100% complementary) to a second sequence if the polynucleotide comprising the first sequence base pairs with the polynucleotide comprising the second sequence without any mismatches over the entire length of one or both nucleotide sequences. A sequence is "substantially complementary" to a target sequence if it is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence. The percent complementarity can be calculated by dividing the number of bases in a first sequence that are complementary to the bases at corresponding positions in a second sequence or target sequence by the total length of the first sequence. Also, if there are 5, 4, 3, or 2 or fewer mismatches throughout the entire 30-base pair duplex region when the two sequences hybridize, one sequence can be said to be substantially complementary to another sequence. Generally, if any nucleotide overhangs exist as defined herein, the sequence of such overhangs is not considered when determining the degree of complementarity between two sequences. For example, a 21-nucleotide sense strand and a 21-nucleotide antisense strand that hybridize to form a 19-base pair duplex region with a 2-nucleotide overhang at the 3' end of each strand would be considered fully complementary as the term is used herein.

[0064] In some embodiments, a region of the antisense strand comprises a sequence that is substantially or completely complementary to a region of the target LPA RNA sequence (e.g., LPA mRNA). In such embodiments, the sense strand may comprise a sequence that is completely complementary to the sequence of the antisense strand. In other such embodiments, the sense strand may comprise a sequence that is substantially complementary to the sequence of the antisense strand, for example, a sequence that has 1, 2, 3, 4, or 5 mismatches within the duplex region formed by the sense strand and the antisense strand. In certain embodiments, it is preferred that any mismatches occur within the terminal region (e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5'-end and / or 3'-end of the strand). In one embodiment, any mismatches within the duplex region formed by the sense strand and the antisense strand occur within 6, 5, 4, 3, or 2 nucleotides of the 5'-end of the antisense strand.

[0065] In certain embodiments, the sense strand and antisense strand of double-stranded RNA can hybridize to form a duplex region, but otherwise can be two separate molecules that are separate.This double-stranded RNA molecule that is formed by two separate strands is called " small interfering RNA " or " short interfering RNA " (siRNA).Therefore, in some embodiments, the LPA RNAi construct used in the method of the present invention comprises siRNA.

[0066] In other embodiments, the sense and antisense strands that hybridize to form a duplex region can be part of a single RNA molecule; i.e., the sense and antisense strands are part of a self-complementary region of a single RNA molecule. In such cases, the single RNA molecule comprises a duplex region (also referred to as a stem region) and a loop region. The 3' end of the sense strand is connected to the 5' end of the antisense strand by a contiguous sequence of unpaired nucleotides, thereby forming a loop region. The loop region is typically long enough to allow the RNA molecule to refold on itself so that the antisense strand can base pair with the sense strand to form a duplex or stem region. The loop region may contain about 3 to about 25, about 5 to about 15, or about 8 to about 12 unpaired nucleotides. Such RNA molecules with at least a partially self-complementary region are referred to as "short hairpin RNAs" (shRNAs). In certain embodiments, the LPA RNAi construct used in the methods of the present invention comprises an shRNA. The length of the single, at least partially self-complementary RNA molecule can be from about 40 nucleotides to about 100 nucleotides, from about 45 nucleotides to about 85 nucleotides, or from about 50 nucleotides to about 60 nucleotides, and can include a duplex region and a loop region, each having a length as described herein.

[0067] The LPA RNAi construct used in the method of the present invention comprises a sense strand and an antisense strand, and the antisense strand comprises a region having a sequence substantially or completely complementary to the LPA messenger RNA (mRNA) sequence. As used herein, "LPA mRNA sequence" refers to the messenger RNA sequence encoding apo(a) protein, including allelic variants and splice variants, including variants or isoforms of apo(a) protein from any species (e.g., non-human primates, humans). The LPA gene (also known as AK38, APOA, and LP) encodes apo(a) protein, which is the main component of low-density lipoprotein particles known as lipoprotein(a) or Lp(a). In humans, the LPA gene is found on chromosome 6 at locus 6q25.3-q26. The LPA gene is highly polymorphic due to differences between individuals in the copy number of the kringle IV type 2 (KIV-2) domain, which can range from 2 to over 40 copies of the gene alleles (see, e.g., Kronenberg and Utermann, J. Intern. Med., Vol. 273:6-30, 2013).

[0068] The LPA mRNA sequence also includes the transcript sequence expressed as its complementary DNA (cDNA) sequence. The cDNA sequence refers to the sequence of the mRNA transcript expressed as DNA bases (e.g., guanine, adenine, thymine, and cytosine) rather than RNA bases (e.g., guanine, adenine, uracil, and cytosine). Therefore, the antisense strand of the LPA RNAi construct used in the method of the present invention can comprise a region having a sequence that is substantially or completely complementary to the target LPA mRNA sequence or LPA cDNA sequence. The LPA mRNA or cDNA sequence may include, but is not limited to, any LPA mRNA or cDNA sequence selected from the NCBI reference sequences NM_005577.4 (human), XM_015448520.1 (cynomolgus monkey), XM_028847001.1 (rhesus monkey), XM_024357489.1 (chimpanzee), and XM_031012244.1 (gorilla). In certain embodiments, the LPA mRNA sequence is the human transcript listed in the NCBI database as reference sequence NM_005577.4.

[0069] The sense strand of an LPA RNAi construct typically contains a sequence sufficiently complementary to that of the antisense strand such that the two strands hybridize to form a duplex region under physiological conditions. A "duplex region" refers to a region within two complementary or substantially complementary polynucleotides that base-pair with each other through either Watson-Crick base pairing or other hydrogen-bonding interactions to create a duplex between the two polynucleotides. The duplex region of an LPA RNAi construct should be of sufficient length to allow, for example, binding of the Dicer enzyme and / or the RISC complex, thereby allowing the LPA RNAi construct to enter the RNA interference pathway. For example, in some embodiments, the duplex region is about 15 to about 30 base pairs in length. Other lengths of the duplex region within this range are also suitable, such as about 15 to about 28 base pairs, about 15 to about 26 base pairs, about 15 to about 24 base pairs, about 15 to about 22 base pairs, about 17 to about 28 base pairs, about 17 to about 26 base pairs, about 17 to about 24 base pairs, about 17 to about 23 base pairs, about 17 to about 21 base pairs, about 19 to about 25 base pairs, about 19 to about 23 base pairs, or about 19 to about 21 base pairs. In certain embodiments, the duplex region is about 17 to about 26 base pairs in length. In other embodiments, the duplex region is about 19 to about 21 base pairs in length. In one embodiment, the duplex region is about 19 base pairs in length. In another embodiment, the duplex region is about 21 base pairs in length.

[0070] For embodiments in which the sense and antisense strands are two separate molecules (e.g., an LPA RNAi construct comprises an siRNA), the sense and antisense strands need not be the same length as the duplex region. For example, one or both strands may be longer than the duplex region and may have one or more unpaired nucleotides or mismatches flanking the duplex region. Thus, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, a "nucleotide overhang" refers to an unpaired nucleotide or nucleotides at the end of a strand that extend beyond the duplex region. A nucleotide overhang is typically created when the 3' end of one strand extends beyond the 5' end of the other strand, or when the 5' end of one strand extends beyond the 3' end of the other strand. The length of a nucleotide overhang is generally 1 to 6 nucleotides, 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, or 2 to 4 nucleotides. In some embodiments, the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In a particular embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. In certain other embodiments, the nucleotide overhang comprises a single nucleotide. When the nucleotide overhang is present in the antisense strand, the nucleotides in the overhang may be complementary to the target gene sequence, may form a mismatch with the target gene sequence, or may comprise some other sequence (e.g., a polypyrimidine or polypurine sequence, e.g., UU, TT, AA, GG, etc.).

[0071] The nucleotide overhangs may be present at the 5'-end or 3'-end of one or both strands. For example, in one embodiment, the LPA RNAi construct comprises nucleotide overhangs at the 5'-end and 3'-end of the antisense strand. In another embodiment, the LPA RNAi construct comprises nucleotide overhangs at the 5'-end and 3'-end of the sense strand. In some embodiments, the LPA RNAi construct comprises nucleotide overhangs at the 5'-end of the sense strand and the 5'-end of the antisense strand. In other embodiments, the LPA RNAi construct comprises nucleotide overhangs at the 3'-end of the sense strand and the 3'-end of the antisense strand.

[0072] The RNAi construct may comprise a nucleotide overhang at one end of the double-stranded RNA molecule and a blunt end at the other. "Blunt end" means that the sense strand and the antisense strand are perfectly base-paired at the ends of the molecule, and there are no unpaired nucleotides extending beyond the duplex region. In some embodiments, the LPA RNAi construct comprises a nucleotide overhang at the 3' end of the sense strand and a blunt end at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the LPA RNAi construct comprises a nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, the LPA RNAi construct comprises blunt ends at both ends of the double-stranded RNA molecule. In such embodiments, the sense strand and the antisense strand have the same length, and the duplex region is the same length as the sense strand and the antisense strand (i.e., the molecule is double-stranded throughout its entire length).

[0073] The sense strand and antisense strand in the LPA RNAi construct used in the methods of the present invention can each independently be about 15 to about 30 nucleotides, about 19 to about 30 nucleotides, about 18 to about 28 nucleotides, about 19 to about 27 nucleotides, about 19 to about 25 nucleotides, about 19 to about 23 nucleotides, about 19 to about 21 nucleotides, about 21 to about 25 nucleotides, or about 21 to about 23 nucleotides in length. In certain embodiments, the sense strand and antisense strand each independently are about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. In some embodiments, the sense strand and antisense strand form a duplex region of the same length but shorter than the other strands, such that the LPA RNAi construct has a two-nucleotide overhang. For example, in one embodiment, the LPA RNAi construct comprises (i) a sense strand and an antisense strand each 21 nucleotides long, (ii) a duplex region 19 base pairs long, and (iii) a nucleotide overhang of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the LPA RNAi construct comprises (i) a sense strand and an antisense strand each 23 nucleotides long, (ii) a duplex region 21 base pairs long, and (iii) a nucleotide overhang of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the sense strand and the antisense strand have the same length and form a duplex region over their entire length such that there are no nucleotide overhangs at either end of the double-stranded molecule. In a specific embodiment, the LPA RNAi construct used in the method of the present invention is blunt-ended and comprises (i) a sense strand and an antisense strand each 21 nucleotides long, and (ii) a duplex region 21 base pairs long. In another specific embodiment, the LPA RNAi construct used in the methods of the present invention is blunt-ended and comprises (i) sense and antisense strands, each 19 nucleotides in length, and (ii) a duplex region 19 base pairs in length.

[0074] In another embodiment, the sense strand or antisense strand is longer than the other strand, and the two strands form a duplex region having a length equal to the length of the shorter strand, so that the LPA RNAi construct comprises at least one nucleotide overhang.For example, in one embodiment, the LPA RNAi construct used in the method of the present invention comprises (i) a sense strand having a length of 19 nucleotides, (ii) an antisense strand having a length of 21 nucleotides, (iii) a duplex region having a length of 19 base pairs, and (iv) a nucleotide overhang of two unpaired nucleotides at the 3'-end of the antisense strand.In another embodiment, the LPA RNAi construct used in the method of the present invention comprises (i) a sense strand having a length of 21 nucleotides, (ii) an antisense strand having a length of 23 nucleotides, (iii) a duplex region having a length of 21 base pairs, and (iv) a nucleotide overhang of two unpaired nucleotides at the 3'-end of the antisense strand.

[0075] The LPA RNAi construct used in the method of the present invention may contain one or more modified nucleotides. "Modified nucleotide" refers to a nucleotide having one or more chemical modifications to the nucleoside, nucleobase, pentose ring, or phosphate group. As used herein, modified nucleotides do not include ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate. However, the LPA RNAi construct may contain a combination of modified nucleotides and ribonucleotides. Incorporation of modified nucleotides into one or both strands of a double-stranded RNA molecule can improve the in vivo stability of the RNA molecule, for example, by reducing the molecule's susceptibility to nucleases and other degradation processes. In addition, incorporation of modified nucleotides can enhance the efficacy of the LPA RNAi construct for reducing the expression of the LPA gene.

[0076] In certain embodiments, modified nucleotides have modifications of the ribose sugar. Such sugar modifications may include modifications at the 2' and / or 5' positions of the pentose ring, as well as bicyclic sugar modifications. A 2'-modified nucleotide refers to a nucleotide having a pentose ring with a substituent at the 2' position other than OH. Such 2' modifications include 2'H (e.g., deoxyribonucleotides), 2'-O-alkyl (e.g., O-C1-C2). 10 Or O-C1~C 10 Modifications at the 5' position of the pentose ring include, but are not limited to, 5'-methyl (R or S); 5'-vinyl, and 5'-methoxy.

[0077] "Bicyclic sugar modification" refers to a modification of a pentose ring in which two atoms of the ring are linked by a bridge to form a second ring, resulting in a bicyclic sugar structure. In some embodiments, a bicyclic sugar modification comprises a bridge between the 4' and 2' carbons of the pentose ring. Nucleotides comprising a sugar moiety having a bicyclic sugar modification are referred to herein as bicyclic nucleic acids or BNAs. Exemplary bicyclic sugar modifications include α-L-methyleneoxy (4'-CH2-O-2') bicyclic nucleic acids (BNAs); β-D-methyleneoxy (4'-CH2-O-2') BNAs (also referred to as locked nucleic acids or LNAs); ethyleneoxy (4'-(CH2)2-O-2') BNAs; aminooxy (4'-CH2-ON(R)-2') BNAs; oxyamino (4'-CH2-N(R)-O-2') BNAs; methyl(methyleneoxy) (4'-CH(CH3)-O-2') BNAs (constrained Examples of sugar-modified nucleotides that can be incorporated into the LPA RNAi constructs used in the methods of the present invention include, but are not limited to, methylene-thio (4'-CH2-S-2') BNAs, methylene-amino (4'-CH2-N(R)-2') BNAs, methyl carbocyclic (4'-CH2-CH(CH3)-2') BNAs, propylene carbocyclic (4'-(CH2)3-2') BNAs, and methoxy(ethyleneoxy) (4'-CH(CHOMe)-O-2') BNAs (also referred to as constrained MOE or cMOE). These and other sugar-modified nucleotides that can be incorporated into the LPA RNAi constructs used in the methods of the present invention are described in U.S. Patent No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012.

[0078] In some embodiments, the LPA RNAi construct comprises one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNAs), deoxyribonucleotides, or combinations thereof. In certain embodiments, the LPA RNAi construct comprises one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, deoxyribonucleotides, or combinations thereof. In a specific embodiment, the LPA RNAi construct used in the methods of the present invention comprises one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, deoxyribonucleotides, or combinations thereof. In some such embodiments, the deoxyribonucleotides may be terminal nucleotides at the 3'-end and / or 5'-end of the sense strand or antisense strand. In embodiments where a deoxyribonucleotide is a terminal nucleotide, the terminal nucleotide may be an inverted nucleotide, i.e., linked to the adjacent nucleotide via a 3'-3' internucleotide bond (if on the 3' end of the strand) or via a 5'-5' internucleotide bond (if on the 5' end of the strand) rather than the natural 3'-5' internucleotide bond.

[0079] Both the sense strand and the antisense strand of the LPA RNAi construct can contain one or more modified nucleotides. For example, in some embodiments, the sense strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In certain embodiments, all nucleotides in the sense strand are modified nucleotides. In some embodiments, the antisense strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In other embodiments, all nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides can be 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or a combination thereof.

[0080] In certain embodiments, the modified nucleotides incorporated into one or both strands of the LPA RNAi construct used in the methods of the present invention have a modified nucleobase (also referred to herein as "base"). "Modified nucleobase" or "modified base" refers to a base other than the naturally occurring purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases may be synthetic or naturally occurring modifications, including but not limited to the universal bases 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine (X), hypoxanthine (I), 2-aminoadenine, 6-methyladenine, 6-methylguanine, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, 2 ... These include, but are not limited to, cytosine, thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0081] In some embodiments, the modified base is a universal base. "Universal base" refers to a base analog that indiscriminately base pairs with all natural bases in RNA and DNA without changing the resulting double-stranded region's double helix structure. Universal bases are known to those skilled in the art and include, but are not limited to, inosine, C-phenyl, C-naphthyl, and other aromatic derivatives, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole.

[0082] Other suitable modified bases that can be incorporated into the LPA RNAi construct include those described in Herdewijn, Antisense Nucleic Acid Drug Dev., Vol. 10: 297-310, 2000 and Peacock et al., J. Org. Chem., Vol. 76: 7295-7300, 2011. Those skilled in the art will be well aware that guanine, cytosine, adenine, thymine, and uracil can be substituted by other nucleobases, such as the modified nucleobases described above, without substantially altering the base pairing properties of a polynucleotide containing a nucleotide having such a substituted nucleobase.

[0083] In some embodiments, the sense strand and antisense strand of the LPA RNAi construct used in the method of the present invention may contain one or more abasic nucleotides. An "abasic nucleotide" or "abasic nucleoside" is a nucleotide or nucleoside lacking a nucleobase at the 1' position of the ribose sugar. In certain embodiments, an abasic nucleotide is incorporated at the end of the sense strand and / or antisense strand of the RNAi construct. In one embodiment, the sense strand contains an abasic nucleotide as the terminal nucleotide at its 3' end, its 5' end, or both its 3' and 5' ends. In another embodiment, the antisense strand contains an abasic nucleotide as the terminal nucleotide at its 3' end, its 5' end, or both its 3' and 5' ends. In embodiments where the abasic nucleotide is a terminal nucleotide, the terminal nucleotide may be an inverted nucleotide, i.e., linked to the adjacent nucleotide via a 3'-3' internucleotide linkage (if on the 3' end of the strand) or a 5'-5' internucleotide linkage (if on the 5' end of the strand) rather than the natural 3'-5' internucleotide linkage. The abasic nucleotide may also include a sugar modification, such as any of the sugar modifications described above. In certain embodiments, the abasic nucleotide includes a 2' modification, such as a 2'-fluoro modification, a 2'-O-methyl modification, or a 2'-H (deoxy) modification. In one embodiment, the abasic nucleotide includes a 2'-O-methyl modification. In another embodiment, the abasic nucleotide includes a 2'-H modification (i.e., a deoxy abasic nucleotide).

[0084] The LPA RNAi construct used in the methods of the present invention may also contain one or more modified internucleotide linkages. As used herein, the term "modified internucleotide linkage" refers to an internucleotide linkage other than a natural 3'-5' phosphodiester linkage. In some embodiments, the modified internucleotide linkage is a phosphorus-containing internucleotide linkage, such as a phosphotriester, an aminoalkylphosphotriester, an alkylphosphonate (e.g., methylphosphonate, 3'-alkylenephosphonate), a phosphinate, a phosphoramidate (e.g., 3'-aminophosphoramidate and aminoalkylphosphoramidate), a phosphorothioate (P=S), a chiral phosphorothioate, a phosphorodithioate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, and a boranophosphate. In one embodiment, the modified internucleotide linkage is a 2'-5' phosphodiester linkage. In other embodiments, the modified internucleotide linkage is a non-phosphorus-containing internucleotide linkage, and may therefore be referred to as a modified internucleoside linkage. Such non-phosphorus-containing linkages include, but are not limited to, morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane linkages (—O—Si(H)—O—); sulfide, sulfoxide, and sulfone linkages; formacetyl and thioformacetyl linkages; alkene-containing backbones; sulfamate backbones; methylenemethylimino (—CH—N(CH)—O—CH—) and methylenehydrazino linkages; sulfonate and sulfonamide linkages; amide linkages; and others with mixed N, O, S, and CH component moieties. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g., aminoethylglycine) to create peptide nucleic acids or PNAs, such as those described in U.S. Pat. Nos. 5,539,082, 5,714,331, and 5,719,262.Other suitable modified internucleotide and internucleoside linkages that can be used in LPA RNAi constructs are described in U.S. Pat. No. 6,693,187, U.S. Pat. No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012.

[0085] In certain embodiments, the LPA RNAi construct used in the methods of the present invention contains one or more phosphorothioate internucleotide linkages. The phosphorothioate internucleotide linkages may be present in the sense strand, the antisense strand, or both strands of the LPA RNAi construct. For example, in some embodiments, the sense strand contains one, two, three, four, five, six, seven, eight, or more phosphorothioate internucleotide linkages. In other embodiments, the antisense strand contains one, two, three, four, five, six, seven, eight, or more phosphorothioate internucleotide linkages. In still other embodiments, both strands contain one, two, three, four, five, six, seven, eight, or more phosphorothioate internucleotide linkages. The LPA RNAi construct may contain one or more phosphorothioate internucleotide linkages at the 3' end, the 5' end, or both the 3' and 5' ends of the sense strand, the antisense strand, or both strands. For example, in certain embodiments, the LPA RNAi construct comprises from about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 3'-end of the sense strand, the antisense strand, or both strands. In other embodiments, the LPA RNAi construct comprises from about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand, the antisense strand, or both strands. In any of the embodiments in which one or both strands comprise one or more phosphorothioate internucleotide linkages, the remaining internucleotide linkages within the strands can be natural 3' to 5' phosphodiester linkages. For example, in some embodiments, each internucleotide linkage in the sense strand and the antisense strand is selected from phosphodiester and phosphorothioate, and at least one internucleotide linkage is phosphorothioate.

[0086] In some embodiments of the RNAi construct of the present invention, the 5'-end of the sense strand, the antisense strand, or both the antisense strand and the sense strand comprises a phosphate moiety. As used herein, the term "phosphate moiety" refers to a terminal phosphate group, including unmodified phosphate (-OP=O)(OH)OH) and modified phosphate. Modified phosphates include phosphates in which one or more of the O and OH groups are replaced with H, O, S, N(R), or alkyl (wherein R is H, an amino-protecting group, or unsubstituted or substituted alkyl). Exemplary phosphate moieties include, but are not limited to, 5'-monophosphate; 5'-diphosphate; 5'-triphosphate; 5'-guanosine cap (7-methylated or unmethylated); 5'-adenosine cap, or any other modified or unmodified nucleotide cap structure; 5'-monothiophosphate (phosphorothioate); 5'-monodithiophosphate (phosphorodithioate); 5'-alpha-thiotriphosphate; 5'-gamma-thiotriphosphate; 5'-phosphoramidate; 5'-vinylphosphate; 5'-alkylphosphonates (e.g., alkyl = methyl, ethyl, isopropyl, propyl, etc.); 5'-cyclopropylphosphonate, and 5'-alkyl ether phosphonates (e.g., alkyl ether = methoxymethyl, ethoxymethyl, etc.).

[0087] Modified nucleotides that can be incorporated into LPA RNAi constructs suitable for use in the methods of the present invention can have multiple chemical modifications described herein. For example, modified nucleotides can have modifications to the ribose sugar and modifications to the nucleobase. For example, modified nucleotides can include a 2' sugar modification (e.g., 2'-fluoro or 2'-O-methyl) and a modified base (e.g., 5-methylcytosine or pseudouracil). In other embodiments, modified nucleotides can include a sugar modification combined with a modification to the 5' phosphate, resulting in a modified internucleotide or internucleoside linkage when the modified nucleotide is incorporated into a polynucleotide. For example, in some embodiments, modified nucleotides can include sugar modifications such as a 2'-fluoro modification, a 2'-O-methyl modification, or a bicyclic sugar modification, and a 5' phosphorothioate group. Thus, in some embodiments, one or both strands of an RNAi construct of the present invention include a combination of 2'-modified nucleotides or BNAs and a phosphorothioate internucleotide linkage. In certain embodiments, both the sense and antisense strands of an RNAi construct of the invention comprise a combination of 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, and phosphorothioate internucleotide linkages.

[0088] The LPA gene is primarily expressed in the liver. Therefore, in certain embodiments, it is desirable to specifically deliver the LPA RNAi construct to liver cells. Thus, in some embodiments, the LPA RNAi construct used in the methods of the present invention can include a targeting moiety that specifically directs the LPA RNAi construct to liver cells (e.g., hepatocytes) using various approaches, as described in more detail below. In certain embodiments, the LPA RNAi construct includes a targeting moiety that includes a ligand that binds to the surface-expressed asialoglycoprotein receptor (ASGR) or its components (e.g., ASGR1, ASGR2).

[0089] In some embodiments, the LPA RNAi construct can be specifically targeted to the liver by using a ligand that binds to or interacts with a protein expressed on the surface of liver cells. For example, in certain embodiments, the ligand can comprise an antigen-binding protein (e.g., an antibody or a binding fragment thereof (e.g., Fab, scFv)) that specifically binds to a receptor expressed on liver cells, such as the asialoglycoprotein receptor and the LDL receptor. In a specific embodiment, the ligand comprises an antibody or a binding fragment thereof that specifically binds to ASGR1 and / or ASGR2. In another embodiment, the ligand comprises a Fab fragment of an antibody that specifically binds to ASGR1 and / or ASGR2. A "Fab fragment" is composed of one immunoglobulin light chain (i.e., the light chain variable region (VL) and constant region (CL)) and the CH1 region and variable region (VH) of one immunoglobulin heavy chain. In another embodiment, the ligand comprises a single-chain variable antibody fragment (scFv fragment) of an antibody that specifically binds to ASGR1 and / or ASGR2. An "scFv fragment" comprises the VH and VL regions of an antibody, these regions being present in a single polypeptide chain, optionally including a peptide linker between the VH and VL regions that enables the Fv to form the desired structure for antigen binding. Exemplary antibodies and binding fragments thereof that specifically bind to ASGR1 and can be used as the asialoglycoprotein receptor in the targeting moiety of the LPA RNAi construct used in the methods of the present invention are described in WO 2017 / 058944 (incorporated herein by reference in its entirety). Other antibodies or binding fragments thereof that specifically bind to ASGR1, LDL receptor, or other liver surface-expressed proteins suitable for use as the targeting moiety in the LPA RNAi construct are commercially available.

[0090] In certain embodiments, the targeting moiety comprises a carbohydrate. "Carbohydrate" refers to a compound composed of one or more monosaccharide units (which may be linear, branched, or cyclic) having at least six carbon atoms, with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. In some embodiments, the carbohydrate incorporated into the targeting moiety is a monosaccharide selected from pentose, hexose, or heptose, as well as disaccharides and trisaccharides containing such monosaccharide units. In other embodiments, the carbohydrate incorporated into the targeting moiety is an amino sugar, such as galactosamine, glucosamine, N-acetylgalactosamine, and N-acetylglucosamine.

[0091] In some embodiments, the targeting moiety comprises an asialoglycoprotein receptor ligand comprising glucose, galactose, galactosamine, glucosamine, N-acetylglucosamine, N-acetyl-galactosamine, or a derivative of any of the foregoing. In certain embodiments, the asialoglycoprotein receptor ligand comprises N-acetyl-galactosamine (GalNAc) or a derivative thereof. Ligands comprising glucose, galactose, and GalNAc are particularly effective in targeting compounds to liver cells because such ligands bind to ASGR, which is expressed on the surface of liver cells. See, for example, D'Souza and Devarajan, J. Control Release, Vol. 203:126-139, 2015. Examples of GalNAc- or galactose-containing ligands that can be incorporated into the targeting moiety of the LPA RNAi constructs used in the methods of the present invention are described in U.S. Pat. Nos. 7,491,805; 8,106,022; 8,877,917; and 10,246,709; U.S. Patent Application Publication No. 20030130186; and WO 2013166155, all of which are incorporated by reference herein in their entireties.

[0092] In certain embodiments, the targeting moiety in the LPA RNAi construct comprises a multivalent carbohydrate moiety. As used herein, a "multivalent carbohydrate moiety" refers to a moiety containing two or more carbohydrate units that can independently bind or interact with other molecules. For example, a multivalent carbohydrate moiety contains two or more binding domains composed of carbohydrates that can bind to two or more different molecules or to two or more different sites on the same molecule. The valency of a carbohydrate moiety indicates the number of individual binding domains within the carbohydrate moiety. For example, the terms "monovalent," "divalent," "trivalent," and "tetravalent" in reference to a carbohydrate moiety refer to carbohydrate moieties having one, two, three, and four binding domains, respectively. A multivalent carbohydrate moiety can comprise a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetyl-galactosamine moiety, a multivalent N-acetyl-glucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the targeting moiety comprises a multivalent galactose moiety. In other embodiments, the targeting moiety comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the polyvalent carbohydrate moiety can be divalent, trivalent, or tetravalent. In such embodiments, the polyvalent carbohydrate moiety can be biantennary or triantennary. In one particular embodiment, the polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another particular embodiment, the polyvalent galactose moiety is trivalent or tetravalent.

[0093] The targeting moiety can be directly or indirectly linked or conjugated to the RNA molecule of the LPA RNAi construct. For example, in some embodiments, the targeting moiety is directly covalently linked to the sense strand or antisense strand of the LPA RNAi construct. In other embodiments, the targeting moiety is covalently linked to the sense strand or antisense strand of the LPA RNAi construct via a linker. The targeting moiety can be linked to the nucleobase, sugar moiety, or internucleotide bond of the polynucleotide (e.g., sense strand or antisense strand) of the LPA RNAi construct used in the method of the present invention. Conjugation or binding to a purine nucleobase or its derivative can occur at any position, including endocyclic and exocyclic atoms. In certain embodiments, the 2-, 6-, 7-, or 8-position of a purine nucleobase is linked to the targeting moiety. Conjugation or binding to a pyrimidine nucleobase or its derivative can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be linked to the targeting moiety. Conjugation or attachment to the sugar moiety of a nucleotide can occur at any carbon atom. Exemplary carbon atoms of the sugar moiety that can be attached to a targeting moiety include the 2', 3', and 5' carbon atoms. Also, for example, in abasic nucleotides, the 1' position can be attached to a targeting moiety. The internucleotide linkage can also assist in the attachment of the targeting moiety. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the targeting moiety can be attached directly to the phosphorus atom or to an O atom, N atom, or S atom attached to the phosphorus atom. For amine- or amide-containing internucleoside linkages (e.g., PNA), the targeting moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0094] In some embodiments, the targeting moiety can be attached to the 3' or 5' end of either the sense strand or the antisense strand. In certain preferred embodiments, the targeting moiety is covalently attached to the 5' end of the sense strand. In such embodiments, the targeting moiety is attached to the 5'-terminal nucleotide of the sense strand. In these and other embodiments, the targeting moiety is attached at the 5' position of the 5'-terminal nucleotide of the sense strand. In embodiments in which an inverted abasic nucleotide or inverted deoxyribonucleotide is the 5'-terminal nucleotide of the sense strand and is linked to the adjacent nucleotide via a 5'-5' internucleotide bond, the targeting moiety can be attached at the 3' position of the inverted abasic nucleotide or inverted deoxyribonucleotide. In other embodiments, the targeting moiety is covalently attached to the 3' end of the sense strand. For example, in some embodiments, the targeting moiety is attached to the 3'-terminal nucleotide of the sense strand. In certain such embodiments, the targeting moiety is attached at the 3' position of the 3'-terminal nucleotide of the sense strand. In embodiments in which the inverted abasic nucleotide or inverted deoxyribonucleotide is the 3'-terminal nucleotide of the sense strand and is linked to the adjacent nucleotide via a 3'-3' internucleotide linkage, the targeting moiety can be attached at the 5'-position of the inverted abasic nucleotide or inverted deoxyribonucleotide. In alternative embodiments, the targeting moiety is attached near the 3'-end of the sense strand but before one or more terminal nucleotides (i.e., before one, two, three, or four terminal nucleotides). In some embodiments, the targeting moiety is attached at the 2'-position of the sugar of the 3'-terminal nucleotide of the sense strand. In other embodiments, the targeting moiety is attached at the 2'-position of the sugar of the 5'-terminal nucleotide of the sense strand.

[0095] In certain embodiments, the targeting moiety is attached to the sense or antisense strand via a linker. A "linker" is an atom or group of atoms that covalently attaches a ligand to a polynucleotide component of an LPA RNAi construct. Linkers can be about 1 to about 30 atoms in length, about 2 to about 28 atoms in length, about 3 to about 26 atoms in length, about 4 to about 24 atoms in length, about 6 to about 20 atoms in length, about 7 to about 20 atoms in length, about 8 to about 20 atoms in length, about 8 to about 18 atoms in length, about 10 to about 18 atoms in length, and about 12 to about 18 atoms in length. In some embodiments, the linker can comprise a bifunctional linking moiety, which generally comprises an alkyl moiety bearing two functional groups. One of the functional groups is selected to bind to a compound of interest (e.g., the sense or antisense strand of an RNAi construct), and the other is selected to essentially bind to any selected group, such as a targeting moiety or component thereof, as described herein. In certain embodiments, the linker comprises a chain structure or oligomer of repeating units, such as ethylene glycol units or amino acid units. Examples of functional groups typically used in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups, and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double or triple bonds), and the like.

[0096] Linkers that can be used to attach a targeting moiety to the sense or antisense strand in the LPA RNAi constructs used in the methods of the invention include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, substituted C1-C 10 Alkyl, substituted or unsubstituted C2-C 10 Alkenyl, or substituted or unsubstituted C2-C 10The preferred substituents for such linker include, but are not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.Other types of linker that are suitable for connecting targeting moiety to the sense strand or antisense strand in the LPA RNAi construct used in the method of the present invention are known in the art, and can include the linker described in United States Patent (USP) 7,723,509; United States Patent (USP) 8,017,762; United States Patent (USP) 8,828,956; United States Patent (USP) 8,877,917; and United States Patent (USP) 9,181,551.

[0097] In certain embodiments, the targeting moiety covalently linked to the sense strand or antisense strand of the LPA RNAi construct used in the methods of the present invention comprises a GalNAc moiety, for example, a multivalent GalNAc moiety. In some embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is linked to the 3'-end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is linked to the 5'-end of the sense strand. In yet other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is linked to the 3'-end of the sense strand. In yet other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is linked to the 5'-end of the sense strand.

[0098] In certain embodiments, the LPA RNAi construct used in the methods of the present invention comprises a targeting moiety having the following structure [Structure 1]: [ka] In a preferred embodiment, a targeting moiety having this structure is covalently attached to the 5' end of the sense strand via a phosphorothioate or phosphodiester bond.

[0099] In certain embodiments, the LPA RNAi construct suitable for use in the methods of the present invention is: a sense strand and an antisense strand, each of which is about 19 to about 23 nucleotides in length, wherein the antisense strand comprises a sequence complementary to the LPA mRNA sequence, and the sense strand comprises a sequence complementary to the sequence of the antisense strand; a targeting moiety comprising an asialoglycoprotein receptor ligand, the targeting moiety being covalently attached to the 5' end of the sense strand; In some embodiments, the LPA RNAi construct has two blunt ends. For example, in some such embodiments, the sense strand and the antisense strand are each 21 nucleotides in length and hybridize to each other to form a 21-base pair long duplex region. In other such embodiments, the sense strand and the antisense strand are each 19 nucleotides in length and hybridize to each other to form a 19-base pair long duplex region. In other embodiments, the LPA RNAi construct has two nucleotide overhangs. In one such embodiment, the LPA RNAi construct comprises (i) a sense strand and an antisense strand each 21 nucleotides in length, (ii) a 19-base pair long duplex region, and (iii) nucleotide overhangs of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand.

[0100] In some embodiments, the targeting moiety comprises a trivalent GalNAc moiety, such as any of the trivalent GalNAc moieties described in U.S. Patent No. 10,246,709, which is incorporated herein by reference in its entirety. In a preferred embodiment, the targeting moiety has the structure of Structure 1 described above.

[0101] In certain embodiments, the antisense strand of the LPA RNAi construct comprises a sequence substantially complementary to or fully complementary to nucleotides 2706-2726 of the human LPA mRNA transcript set forth in NCBI Reference Sequence NM_005577.4, nucleotides 2697-2726 of the human LPA mRNA transcript set forth in NCBI Reference Sequence NM_005577.4, or nucleotides 2708-2725 of the human LPA mRNA transcript set forth in NCBI Reference Sequence NM_005577.4. In such embodiments, the LPA RNAi construct may comprise a sense strand that is substantially complementary to or fully complementary to the antisense strand targeting this region. Thus, in these embodiments, the sense strand may comprise a sequence identical to nucleotides 2706-2726, nucleotides 2697-2726, or nucleotides 2725-2708 of the human LPA mRNA transcript set forth in NCBI Reference Sequence NM_005577.4.

[0102] In some embodiments, the sense strand of the LPA RNAi construct used in the methods of the invention comprises the sequence 5'-GCCCCUUAUUGUUAUACG-3' (SEQ ID NO: 1). In a related embodiment, the antisense strand of the LPA RNAi construct used in the methods of the invention comprises the sequence 5'-CGUAUAACAAUAAGGGGC-3' (SEQ ID NO: 2).

[0103] Examples of LPA RNAi constructs suitable for use in the methods of the invention are described in WO 2017 / 059223 (incorporated herein by reference in its entirety). The duplexes AD03851, AD03853, and AD03536 described in WO 2017 / 059223 are particularly useful in the methods of the invention. In certain preferred embodiments, the LPA RNAi construct used in the methods of the invention comprises a sense strand comprising or consisting of the sequence 5'-CAGCCCCUUAUUGUUAUACGA-3' (SEQ ID NO: 3) and an antisense strand comprising or consisting of the sequence 5'-UCGUAUAACAAUAAGGGGCUG-3' (SEQ ID NO: 4). In related embodiments, the LPA RNAi construct used in the methods of the invention comprises a sense strand comprising or consisting of the sequence 5'-CAGCCCCUUAUUGUUAUACGA-3' (SEQ ID NO: 4). The RNAi construct comprises a sense strand comprising or consisting of a sequence of modified nucleotides according to the sequence 5'-csagccccuUfAfUfuguuauacgs(invdA)-3' (SEQ ID NO:5), and an antisense strand comprising or consisting of a sequence of modified nucleotides according to the sequence 5'-usCfsgUfaUfaacaaUfaAfgGfgGfcsUfsg-3' (SEQ ID NO:6), where a, g, c, and u are each a 2'-O Af, Gf, Cf, and Uf are 2'-deoxy-2'-fluoro ("2'-fluoro") adenosine, 2'-fluoroguanosine, 2'-fluorocytidine, and 2'-fluorouridine, respectively; invdA is inverted deoxyadenosine (a 3'-3' linked nucleotide), and s is a phosphorothioate linkage. In some such embodiments, a targeting moiety having the structure of Structure 1 described herein is covalently attached to the 5'-end of the sense strand via a phosphorothioate linkage.

[0104] In another embodiment, the LPA RNAi construct used in the methods of the invention comprises a sense strand comprising the sequence 5'-GCCCCUUAUUGUUAUACGAUU-3' (SEQ ID NO:7) and an antisense strand comprising the sequence 5'-UCGUAUAACAAUAAGGGGCUU-3' (SEQ ID NO:8). In a related embodiment, the LPA RNAi construct used in the methods of the invention comprises a sense strand comprising or consisting of a sequence of modified nucleotides according to the sequence 5'-gsccccuUfAfUfuguuauacgauus(invAb)-3' (SEQ ID NO:9) and an antisense strand comprising or consisting of a sequence of modified nucleotides according to the sequence 5'-usCfsgUfaUfaacaaUfaAfgGfgGfcsusu-3' (SEQ ID NO:10), where a, g, c, and u are 2'- where Af, Gf, Cf, and Uf are 2'-deoxy-2'-fluoro ("2'-fluoro") adenosine, 2'-fluoroguanosine, 2'-fluorocytidine, and 2'-fluorouridine, respectively; invAb is an inverted abasic nucleotide (a 3'-3' linked nucleotide), and s is a phosphorothioate linkage. In some such embodiments, a targeting moiety having the structure of Structure 1 described herein is covalently attached to the 5'-end of the sense strand via a phosphorothioate linkage.In another related embodiment, the LPA RNAi construct used in the methods of the invention comprises a sense strand comprising or consisting of a sequence of modified nucleotides according to the sequence 5'-(invAb)GfcCfcCfuUfAfUfuGfuUfaUfaCfgausu(invAb)-3' (SEQ ID NO: 11), and an antisense strand comprising or consisting of a sequence of modified nucleotides according to the sequence 5'-usCfsgsUfaUfaAfCfAfauaAfgGfgGfcusu-3' (SEQ ID NO: 12), where a, g, c, and u are 2'-O-methyladenosine, 2'-amino adenosine, 2'-amino hydroxybenzoates ... Af, Gf, Cf, and Uf are 2'-deoxy-2'-fluoro ("2'-fluoro") adenosine, 2'-fluoroguanosine, 2'-fluorocytidine, and 2'-fluorouridine, respectively; invAb is an inverted abasic nucleotide (a 5'-5' linked nucleotide if on the 5' end of the strand, and a 3'-3' linked nucleotide if on the 3' end of the strand), and s is a phosphorothioate linkage. In some of these embodiments, a targeting moiety having the structure of Structure 1 described herein is covalently attached to the 5' end of the sense strand via a phosphodiester bond.

[0105] In a particularly preferred embodiment, the LPA RNAi construct administered to a patient according to the methods of the present invention is olpaciran. The structure of olpaciran is also shown schematically in FIG. 1 and described in WO 2017 / 059223, where olpaciran is designated duplex no. AD03851. Olpaciran is a double-stranded siRNA molecule comprising two separate strands, a sense strand and an antisense strand, each 21 nucleotides in length. The nucleobase sequences of the sense and antisense strands are fully complementary to each other and hybridize to form a 21-base pair long duplex. The nucleotide sequences for the sense and antisense strands of olpaciran are set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Both the sense and antisense strands of olpaciran are composed of modified nucleotides, and the modified sequences for each strand are set forth in SEQ ID NO: 5 (sense strand) and SEQ ID NO: 6 (antisense strand). A trivalent GalNAc moiety having the structure of Structure 1 (and represented as R1 in Figure 1) is covalently attached to the 5' end of the sense strand of olpaciran by a phosphorothioate linkage. The term olpaciran refers to the free acid of the compound shown in Figure 1, as well as pharmaceutically acceptable salts thereof, such as the sodium salt.

[0106] The LPA RNAi construct used in the method of the present invention can be easily produced using techniques known in the art, for example, by conventional solid-phase nucleic acid synthesis. The polynucleotide of the RNAi construct can be assembled using standard nucleotide or nucleoside precursors (e.g., phosphoramidites) on a suitable nucleic acid synthesizer. Automated nucleic acid synthesizers are commercially available from several vendors, including the DNA / RNA synthesizer from Applied Biosystems (Foster City, CA), the MerMade synthesizer from BioAutomation (Irving, TX), and the OligoPilot synthesizer from GE Healthcare Life Sciences (Pittsburgh, PA). Exemplary methods for synthesizing LPA RNAi constructs and selecting targeting moieties are described in the examples of WO 2017 / 059223 and U.S. Pat. No. 10,246,709, both of which are incorporated herein by reference in their entirety.

[0107] The 2' silyl protecting group can be used in conjunction with acid-labile dimethoxytrityl (DMT) at the 5' position of ribonucleosides to synthesize oligonucleotides via phosphoramidite chemistry. Final deprotection conditions are known not to significantly degrade the RNA product. All syntheses can be performed on large-, medium-, or small-scale scales using any automated or manual synthesizer. Synthesis can also be performed in multiwell plates, columns, or glass slides.

[0108] The 2'-O-silyl group can be removed by exposure to fluoride ions, which can include any source of fluoride ions, such as salts containing fluoride ions paired with inorganic counterions, such as cesium fluoride and potassium fluoride, or salts containing fluoride ions paired with organic counterions, such as tetraalkylammonium fluoride.Crown ether catalysts can be used in combination with inorganic fluorides in the deprotection reaction.Preferred fluoride ion sources include tetrabutylammonium fluoride or aminohydrofluorides (for example, aqueous HF in a dipolar aprotic solvent, such as dimethylformamide, in combination with triethylamine).

[0109] The choice of protecting groups used for the phosphite triesters and phosphotriesters can modify the stability of the triesters to fluoride. Methyl protection of the phosphotriester or phosphite triester can stabilize the bond to fluoride ions and improve process yields.

[0110] Because ribonucleosides have a reactive 2' hydroxyl substituent, it may be desirable to protect the reactive 2' position in the RNA with a protecting group that is orthogonal to the 5'-O-dimethoxytrityl protecting group (e.g., a protecting group that is stable to acid treatment). Silyl protecting groups meet this requirement and can be easily removed in a final fluoride deprotection step, thereby minimizing RNA degradation.

[0111] Tetrazole catalysts can be used in standard phosphoramidite coupling reactions. Preferred catalysts include, for example, tetrazole, S-ethyl-tetrazole, benzylthiotetrazole, and p-nitrophenyltetrazole.

[0112] As can be understood by those skilled in the art, further methods for synthesizing the LPA RNAi constructs described herein will be apparent to those skilled in the art. In addition, various synthetic steps can be performed in an alternative order or sequence to obtain the desired compound. Other synthetic chemical transformations, protecting groups (e.g., for hydroxyl, amino, and other bases) and protecting group methodologies (protection and deprotection) useful in the synthesis of RNAi constructs are known in the art, including, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Buts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley & Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley & Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley & Sons (1995), and subsequent editions thereof. Custom synthesis of RNAi agents is also available from several commercial vendors, including Agilent Technologies (Santa Clara, CA), Nitto Denko Avecia (Milford, MA), Dharmacon, Inc. (Lafayette, CO), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, CA).

[0113] The LPA RNAi construct is usually administered to a patient in a pharmaceutical composition that may contain a pharmaceutically acceptable carrier, excipient, or diluent. Thus, the present invention also includes pharmaceutical compositions and formulations that contain the LPA RNAi construct and a pharmaceutically acceptable carrier, excipient, or diluent used in the methods of the present invention described herein. For clinical use, pharmaceutical compositions and formulations will be prepared in a form appropriate for the intended use. Generally, this involves preparing compositions that are essentially free of pyrogens as well as other impurities that may be harmful to humans or animals.

[0114] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carriers, excipients, or diluents" include solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, and absorption delaying agents that are acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for human administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent is contemplated for use in therapeutic compositions, except insofar as it is incompatible with the LPA RNAi construct described herein. Supplementary active ingredients may also be incorporated into the compositions, provided that they do not inactivate the LPA RNAi construct of the composition.

[0115] Compositions and methods for formulating pharmaceutical compositions depend on several criteria, including, but not limited to, the route of administration, the type and severity of the disease or disorder to be treated, or the dose to be administered. In some embodiments, pharmaceutical compositions are formulated based on the intended delivery route. For example, in certain embodiments, pharmaceutical compositions are formulated for parenteral delivery. Parenteral delivery forms include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal, or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In some embodiments, the pharmaceutical composition comprises an effective amount of an LPA RNAi construct. The effective amount of the LPA RNAi construct, particularly olpaciran, may be any of the doses described herein.

[0116] Administration of a pharmaceutical composition comprising an LPA RNAi construct according to the method of the present invention can be via any common route, as long as the target tissue is accessible via that route. Such routes include, but are not limited to, parenteral (e.g., subcutaneous, intramuscular, intraperitoneal, or intravenous), oral, nasal, buccal, intradermal, transdermal, and sublingual routes, or direct injection into liver tissue or delivery via the hepatic portal vein. In some embodiments of the method of the present invention, an LPA RNAi construct or a pharmaceutical composition comprising an LPA RNAi construct is administered parenterally to a patient. For example, in certain embodiments, an LPA RNAi construct or a pharmaceutical composition comprising an LPA RNAi construct is administered intravenously. In other embodiments, an LPA RNAi construct or a pharmaceutical composition comprising an LPA RNAi pharmaceutical composition is administered subcutaneously, for example, by subcutaneous injection. In such embodiments, the subcutaneous injection volume is about 2 mL or less, e.g., about 2 mL, about 1.8 mL, about 1.7 mL, about 1.6 mL, about 1.5 mL, about 1.4 mL, about 1.3 mL, about 1.2 mL, about 1.1 mL, about 1 mL, about 0.9 mL, about 0.8 mL, about 0.7 mL, about 0.6 mL, or about 0.5 mL. In one embodiment, the subcutaneous injection volume is about 1 mL or less. In another embodiment, the subcutaneous injection volume is about 1 mL. In yet another embodiment, the subcutaneous injection volume is about 1.5 mL.

[0117] In the embodiment in which pharmaceutical composition is administered by parenteral injection, the pharmaceutical composition can be administered to the patient by syringe.In some embodiments, the syringe is pre-filled with the pharmaceutical composition.In other embodiments in which pharmaceutical composition is administered to the patient by parenteral injection, for example, subcutaneous injection, the pharmaceutical composition is administered by injection device, including self-administration device.Such device is commercially available, and includes, but is not limited to, auto-injector, administration pen, microinjection pump, on-body injector and pre-filled syringe. Exemplary devices for administering a pharmaceutical composition comprising an effective amount of an LPA RNAi construct (e.g., olpaciran) according to the methods of the present invention include autoinjectors (e.g., SureClick®, EverGentle®, Avanti®, DosePro®, Molly®, and Leva®), pen injection devices (e.g., Madie® pen injector, DCP™ pen injector, BD Vystra™ disposable pen, BD™ reusable pen), and pre-filled syringes (BD Sterifill™, BD Hypak™, Baxter pre-filled syringes). In some embodiments, a pharmaceutical composition comprising an effective amount of an LPA RNAi construct (e.g., olpaciran) is administered to a patient via a pre-filled syringe. In other embodiments, a pharmaceutical composition comprising an effective amount of an LPA RNAi construct (e.g., olpaciran) is administered to a patient via an autoinjector. In certain such embodiments, the injection volume of the syringe, autoinjector, or other injection device is about 2 mL or less, e.g., about 2 mL, about 1.8 mL, about 1.7 mL, about 1.6 mL, about 1.5 mL, about 1.4 mL, about 1.3 mL, about 1.2 mL, about 1.1 mL, about 1 mL, about 0.9 mL, about 0.8 mL, about 0.7 mL, about 0.6 mL, or about 0.5 mL. In one embodiment, the injection volume of the syringe, autoinjector, or other injection device is about 1 mL or less. In another embodiment, the injection volume of the syringe, autoinjector, or other injection device is about 1 mL.In yet another embodiment, the injection volume of the syringe, autoinjector, or other injection device is about 1.5 mL.

[0118] Colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes, can be used as delivery vehicles for the LPA RNAi constructs of the present invention. Commercially available lipid emulsions suitable for delivering the nucleic acids of the present invention include Intralipid® (Baxter International Inc.), Liposyn® (Abbott Pharmaceuticals), Liposyn® II (Hospira), Liposyn® III (Hospira), Nutrilipid (B. Braun Medical Inc.), and other similar lipid emulsions. A preferred colloidal system used as an in vivo delivery vehicle is a liposome (i.e., an artificial membrane vesicle). The LPA RNAi construct may be encapsulated within a liposome or complexed to a liposome, particularly a cationic liposome. Alternatively, the LPA RNAi construct may be complexed to a lipid, particularly a cationic lipid. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC)), distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). The preparation and use of such colloidal dispersion systems is well known in the art. Exemplary formulations are also disclosed in U.S. Pat. Nos. 5,981,505, 6,217,900; 6,383,512; 5,783,565; 7,202,227; 6,379,965; 6,127,170; 5,837,533; 6,747,014, and WO 03 / 093449.

[0119] In some embodiments, the LPA RNAi construct is fully encapsulated within a lipid formulation, for example, to form a SNALP or other nucleic acid-lipid particle. As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle. SNALP typically contains a cationic lipid, a non-cationic lipid, and a lipid (e.g., a PEG-lipid conjugate) that prevents particle aggregation. SNALP is highly useful for systemic administration because it exhibits a long circulatory life after intravenous injection and accumulates at distal sites (e.g., sites physically separated from the administration site). Nucleic acid-lipid particles typically have an average diameter of about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm and are substantially non-toxic. Additionally, nucleic acids present within nucleic acid-lipid particles are resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Pat. Nos. 5,976,567, 5,981,501, 6,534,484, 6,586,410, 6,815,432, and WO 96 / 40964.

[0120] Pharmaceutical compositions containing LPA RNAi constructs suitable for injection include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that they are easily syringable. Preparations must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Suitable solvents or dispersion media may contain, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained by, for example, using a coating such as lecithin to maintain the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0121] Sterile injectable solutions can be prepared by incorporating the active compound in an appropriate amount into a solvent with any other ingredients as needed (for example, as listed above), and then sterilizing by filtration.Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other desired ingredients, for example, the ingredients listed above.In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation methods include vacuum drying and freeze-drying techniques, which yield powders of the active ingredient plus any additional desired ingredients from the previously sterile-filtered solution.

[0122] The compositions used in the methods of the present invention can generally be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups) derived from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.). Sodium salts of LPA RNAi constructs are particularly useful for therapeutic administration to human subjects. Thus, in certain preferred embodiments, the LPA RNAi construct, particularly olpaciran, is in the form of a sodium salt. In other embodiments, the LPA RNAi construct (e.g., olpaciran) is in the form of a potassium salt.

[0123] For parenteral administration in an aqueous solution, for example, the solution is usually suitably buffered and the liquid diluent first rendered isotonic, for example, with sufficient saline or glucose. Such aqueous solutions can be used, for example, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. It is preferable to use a sterile aqueous medium, as known to those skilled in the art, particularly in light of the present disclosure. By way of illustration, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed site of infusion or injection (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). For human administration, preparations must meet sterility, pyrogenicity, general safety, and purity standards required by FDA standards. In certain embodiments, the pharmaceutical composition used in the methods of the present invention comprises or consists of sterile saline and an LPA RNAi construct (e.g., olpaciran) described herein. In other embodiments, the pharmaceutical compositions used in the methods of the invention comprise or consist of an LPA RNAi construct described herein (e.g., olpaciran) and sterile water (e.g., water for injection, WFI). In yet other embodiments, the pharmaceutical compositions used in the methods of the invention comprise or consist of an LPA RNAi construct described herein (e.g., olpaciran) and phosphate buffered saline (PBS).

[0124] In certain embodiments, pharmaceutical compositions useful for treating, ameliorating, preventing, or reducing the risk of cardiovascular disease according to the methods of the present invention comprise an effective amount of an LPA RNAi construct (e.g., olpaciran), potassium phosphate buffer, and sodium chloride. In some such embodiments, the pharmaceutical composition comprises about 10 mg / mL to about 200 mg / mL of an LPA RNAi construct (e.g., olpaciran), about 5 mM to about 30 mM potassium phosphate, and about 20 mM to about 160 mM sodium chloride. In other embodiments, the pharmaceutical composition comprises about 65 mg / mL to about 85 mg / mL of an LPA RNAi construct (e.g., olpaciran), about 15 mM to about 25 mM potassium phosphate, and about 70 mM to about 90 mM sodium chloride. In yet another embodiment, the pharmaceutical composition comprises about 140 mg / ml to about 160 mg / ml of an LPA RNAi construct (e.g., olpaciran), about 15 mM to about 25 mM potassium phosphate, and about 30 mM to about 50 mM sodium chloride. The pH of any of these pharmaceutical compositions can be within the range of about 6.4 to about 7.2 (e.g., a pH of about 6.4, about 6.6, about 6.8, about 7.0, or about 7.2).

[0125] In some embodiments, a pharmaceutical composition to be administered according to the methods of the present invention comprises about 10 mg / ml of an LPA RNAi construct (e.g., olpaciran), about 5 mM to about 15 mM potassium phosphate, and about 135 mM to about 155 mM sodium chloride at a pH of 6.8±0.2. In one embodiment, a pharmaceutical composition comprises about 10 mg / ml of an LPA RNAi construct (e.g., olpaciran), about 10 mM potassium phosphate, and about 145 mM sodium chloride at a pH of 6.8. In another embodiment, a pharmaceutical composition to be administered according to the methods of the present invention comprises about 75 mg / ml of an LPA RNAi construct (e.g., olpaciran), about 15 mM to about 25 mM potassium phosphate, and about 70 mM to about 90 mM sodium chloride at a pH of 6.8±0.2. In one such embodiment, the pharmaceutical composition comprises about 75 mg / ml of an LPA RNAi construct (e.g., olpaciran), about 20 mM potassium phosphate, and about 80 mM sodium chloride at pH 6.8. In a specific embodiment, a pharmaceutical composition to be administered in accordance with the methods of the present invention comprises about 150 mg / ml of an LPA RNAi construct (e.g., olpaciran), about 15 mM to about 25 mM potassium phosphate, and about 30 mM to about 50 mM sodium chloride at pH 6.8±0.2. In a specific embodiment, the pharmaceutical composition comprises about 150 mg / ml of an LPA RNAi construct (e.g., olpaciran), about 20 mM potassium phosphate, and about 40 mM sodium chloride at pH 6.8.

[0126] Any of the LPA RNAi constructs described herein can be incorporated into any of the pharmaceutical compositions described above and administered to patients according to the methods of the present invention. In a specific embodiment, the LPA RNAi construct incorporated into any of the pharmaceutical compositions described above and administered to patients according to the methods of the present invention is olpaciran.

[0127] In some embodiments, the pharmaceutical compositions of the present invention are packaged with or stored in an administration device, such as any of the injection devices described above (e.g., pre-filled syringes, auto-injectors, injection pumps, on-body injectors, and injection pens). Devices for aerosolized or powdered formulations include, but are not limited to, inhalers, insufflators, inhalers, and the like. Thus, the present invention includes administration devices comprising the pharmaceutical compositions of the present invention for treating or preventing one or more of the disorders or diseases described herein.

[0128] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the scope of the appended claims. [Example]

[0129] Example 1. A Phase 1, Randomized, Double-Blind, Placebo-Controlled, Single Ascending Dose Study to Evaluate the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Olpaciran in Subjects With Elevated Plasma Lipoprotein(a) Mendelian and epidemiological randomized studies have recently established lipoprotein(a) (Lp(a)) as a strong causative risk factor for myocardial infarction and other atherosclerotic complications. There are currently no approved medications that selectively target Lp(a) and demonstrate a reduction in cardiovascular events. Olpaciran (also known as AMG 890) is an siRNA designed to reduce Lp(a) production by targeting mRNA transcribed from the LPA gene. The structure of olpaciran is shown in Figure 1.

[0130] This Phase 1 study was a randomized, double-blind, placebo-controlled, single-ascending-dose study in subjects with elevated plasma lipoprotein(a) conducted at eight sites in the United States and Australia. Approximately 80 subjects were planned for enrollment in nine single-ascending-dose cohorts; within each cohort, subjects were randomized 3:1 to receive olpaciran or placebo.

[0131] Eligible subjects were non-fertile women and men, both aged 18 to 60 years (inclusive) for Cohorts 1-5; and 18 to 65 years (inclusive) for Cohorts 6-9. For Cohorts 1-5, plasma Lp(a) concentrations were ≥ 70 nmol / L and ≤ 199 nmol / L at screening; for Cohorts 6-9, plasma Lp(a) concentrations were ≥ 200 nmol / L at screening; for Cohorts 6-9, at least six subjects in each cohort had a stable statin dose for at least 6 weeks at the time of enrollment. Subjects were free of any clinically significant abnormalities in their medical history at the time of randomization.

[0132] After providing informed consent, subjects were screened for eligibility criteria over 28 days and entered the study site on Day -1. After completing pre-dose procedures, subjects received study medication (olpaciran or placebo). Subjects in Cohorts 1-5 spent Days -1 through 4 at the study site and returned to the site for evaluation until the end of the study. Subjects received single subcutaneous doses of 3 mg, 9 mg, 30 mg, 75 mg, and 225 mg for Cohorts 1-5, respectively; and 9 mg, 75 mg, 225 mg, and 675 mg for Cohorts 6-9, respectively (see Table 1 below).

[0133] [Table 1]

[0134] For Cohorts 1-5 and Cohort 9, the first two enrolled subjects in each cohort were randomized to receive olpaciran or placebo in a 1:1 ratio (sentinel pair), administered in a blinded manner at the same study site on the same day. The remaining cohort subjects received the same dose as deemed safe by the investigator and at least 24 hours after the sentinel pair administration. Enrollment into Cohorts 1-5 was staggered. After the dosing regimen in the preceding cohort was found to be safe and reasonably well tolerated by the Dose Level Review Team (DLRT), all subjects were dosed in the subsequent cohort based on available safety data through study day 15. Enrollment into Cohorts 5-7 began after the dosing regimen in Cohort 4 was found to be safe and reasonably well tolerated by the DLRT based on available safety data through study day 15. Subjects returned to the study site for post-treatment evaluation on Day 113 for Cohorts 1 and 2 and on Day 225 for Cohorts 3-7. Subjects returned for follow-up (approximately every 2 weeks for Cohorts 1 and 2 and monthly for Cohorts 3-7) until Lp(a) levels were at least 80% of baseline. Blood and urine samples were collected throughout the study for evaluation of olpaciran pharmacokinetics (PK) and pharmacodynamics (PD). Safety variables were also assessed periodically.

[0135] The primary endpoints were safety and tolerability as measured by treatment-emergent adverse events (TEAEs), safety laboratory analytes, vital signs, and electrocardiogram (ECG). Secondary endpoints were maximum observed concentration (C max ), the time point of maximum observed concentration (t maxolpaciran PK parameters, including, but not limited to, PK (proportionality) and area under the concentration-time curve (AUC); and PD parameters, including the change and percentage change in plasma Lp(a) levels at each scheduled visit. The baseline value for Lp(a) was defined as the average of screening and 1 day before dosing. If, for any reason, only one value was available, that value was used as the baseline. Exploratory endpoints included the percentage change in low-density lipoprotein cholesterol (LDL-C) and total apolipoprotein B (ApoB) at each scheduled visit.

[0136] Sixty-four subjects were enrolled in the study and received olpaciran or placebo (Cohorts 1-5: olpaciran, n=30, doses: 3 mg, 9 mg, 30 mg, 75 mg, 225 mg; placebo, n=10; Cohorts 6-7: olpaciran, n=18, doses: 9 mg and 75 mg; placebo, n=6). For subjects receiving olpaciran in Cohorts 1-5 (n=30), the mean (SD) age was 43.9 (13.5) years, 30.0% were female, 63.3% were Hispanic or Latino, 30.0% were Black or African American, and 70.0% were White. For subjects receiving placebo in cohorts 1-5 (n=10), subjects had a mean (SD) age of 46.3 (8.5) years, 30.0% were female, 50.0% were Hispanic or Latino, 30.0% were Black or African American, and 70.0% were Caucasian. For subjects receiving olpaciran in cohorts 6 and 7 (n=18), subjects had a mean (SD) age of 52.7 (9.4) years, 33.3% were female, 27.8% were Hispanic or Latino, and 88.9% were Caucasian. For subjects receiving placebo in Cohorts 6 and 7 (n = 6), subjects had a mean (SD) age of 57.8 (5.8) years, 66.7% were female, 33.3% were Hispanic or Latino, and 83.3% were white. Sixty-seven percent of all subjects enrolled in Cohorts 6 and 7 (n = 24) used statins at baseline. Subjects had few comorbidities. There was no use of lipid-modifying medications in Cohorts 1-5, but a significantly larger proportion of subjects in Cohorts 6 and 7 took statins and / or ezetimibe. The median (Q1, Q3) baseline Lp(a) concentration was 124 nmol / L (104, 137) for subjects receiving placebo in Cohorts 1-5 and 122 nmol / L (97, 146) for subjects receiving olpaciran in Cohorts 1-5.The median (Q1, Q3) baseline Lp(a) concentration was 272 nmol / L (233, 307) for subjects receiving placebo in cohorts 6 and 7 and 253 nmol / L (224, 334) for subjects receiving olpaciran in cohorts 6 and 7.

[0137] Olpaciran appeared to be well tolerated. There were no treatment-related serious adverse events. One placebo subject had a serious non-cardiac chest pain adverse event, which was considered unrelated to treatment. In cohorts 1-5, the most common TEAE was upper respiratory tract infection (10% placebo, 13% olpaciran). See Table 2 below. In cohorts 6-7, the most common TEAEs were headache (50% placebo, 28% olpaciran) and upper respiratory tract infection (17% placebo, 17% olpaciran) (Table 2). Only one subject in the study experienced an injection site reaction. There was no apparent dose-related relationship with the frequency of adverse events. No clinically relevant changes in liver tests, platelet or coagulation parameters, or renal function were observed.

[0138] [Table 2]

[0139] Lp(a) suppression occurred in a dose-responsive manner. As shown in Figure 2, in cohorts 1–5, a single dose of olpaciran effectively reduced mean Lp(a) levels from baseline by 71–96% (based on dose) at day 43 and by 80–94% at day 113 (cohorts 2–5). In cohorts 6 and 7, a single dose of olpaciran effectively reduced mean Lp(a) levels from baseline by 75% and 89%, respectively, at day 43 and by 61% and 80%, respectively, at day 113 (Figure 2). A steep decline in Lp(a) was observed beginning on day 15, with maximum Lp(a) suppression observed between days 43 and 71. Lp(a) levels gradually recovered but remained well below placebo levels at day 225. A single dose of 9 mg or greater caused a reduction in Lp(a) levels that persisted for 3–6 months.

[0140] The pharmacokinetic parameters of olpaciran in each of the seven dosing cohorts are shown below in Table 3. Following single doses of 3, 9, 30, 75, and 225 mg (Cohorts 1-5), olpaciran was rapidly absorbed, with geometric mean C max occurred within 7.5 hours after administration. The geometric mean half-life (t 1 / 2 ) values ​​ranged from 3 to 8 hours, with the majority of olpaciran being cleared from serum within 2 to 3 days. Systemic exposure increased approximately dose-proportionally at doses up to 225 mg. Olpaciran AUC exposure in subjects with baseline Lp(a) ≥ 200 nmol / L (Cohorts 6 and 7) was approximately 18 to 33% lower than in subjects with baseline Lp(a) ≥ 70 to ≤ 199 nmol / L (Cohorts 2 and 4).

[0141] [Table 3]

[0142] Results of a phase 1 study demonstrated that single-dose treatment with olpaciran was well tolerated and significantly reduced Lp(a) in adults with elevated Lp(a), with an approximate observed median percent reduction of >90%, and effects persisting for 3 to 6 months at doses of 9 mg or higher. The 75 and 225 mg doses in the high Lp(a) group (Lp(a) ≥ 70 to ≤ 199 nmol / L) were superimposable in terms of their effect on Lp(a) concentrations; similarly, the 9 and 30 mg doses were superimposable. However, the 9 and 75 mg doses in the very high Lp(a) group (Lp(a) ≥ 200 nmol / L) demonstrated a reduction in percent suppression of Lp(a) from baseline compared with the same dose in the high Lp(a) cohort.

[0143] The depth and duration of Lp(a) suppression observed with this low single dose of olpaciran was significantly better than expected from the proposed human dose based on studies of olpaciran in cynomolgus monkeys. Based on efficacy data for olpaciran in cynomolgus monkeys (see, e.g., Example 18 in WO 2017 / 059223), a proposed human dose of 75 mg was predicted to reduce Lp(a) levels by approximately 80% for at least one month. Notably, as previously mentioned, a single dose of olpaciran as low as 9 mg reduced Lp(a) levels in human subjects by more than 80% for more than three months. Single olpaciran doses of 75 mg and 225 mg suppressed Lp(a) levels by more than 80% for more than six months. Thus, olpaciran can be administered at lower doses and at longer dosing intervals (including up to once every six months) to human patients in need of Lp(a) reduction. Such a dosing regimen has several distinct benefits, including increased patient loyalty, reduced drug costs, and reduced injection volume and frequency.

[0144] Example 2. Olpaciran PK / PD Model for Design of Dosage Regimen for Optimal Lp(a) Reduction Based on the Phase 1 data described in Example 1, a mathematical model was developed to characterize olpaciran pharmacokinetics and Lp(a) suppression in healthy volunteers with elevated plasma Lp(a) (≥70 nmol / L to <200 nmol / L for Cohorts 1-5 and ≥200 nmol / L for Cohorts 6-7). The pharmacokinetics (PK) of olpaciran was described using a PK model with first-order absorption from the subcutaneous administration site into the circulation, distribution to the liver via asialoglycoprotein receptor (ASGPR) absorption, recycling of olpaciran from the liver back to the circulation, and elimination via systemic circulation and liver degradation. Olpaciran serum exposure was found to correlate with baseline Lp(a). Therefore, the modulation of olpaciran bioavailability by baseline Lp(a) was also included in the model. By describing the suppression of Lp(a) from baseline using the PK / PD model, the model-predicted olpaciran liver concentration promoted the degradation of LPA mRNA, leading to a decrease in Lp(a) production and suppression during the period when olpaciran concentrations in the liver were sufficient. The relationship between olpaciran liver concentration and LPA mRNA degradation was evaluated using E max The model was used. Changes in LPA mRNA concentrations were predicted based on the degree of Lp(a) suppression. Baseline Lp(a) levels informed Lp(a) synthesis and degradation rates. Higher baseline values ​​were associated with greater production rates.

[0145] The following assumptions were made during model development and for clinical dosing regimen simulation: a) Simulations for Phase 2 dose selection were performed for a target population with baseline Lp(a) levels ≥ 150 nmol / L. However, model parameters were estimated from subjects with baseline Lp(a) values ​​≥ 70 nmol / L to < 200 nmol / L and ≥ 200 nmol / L; b) the inter- and intra-subject variability after multiple doses for the Phase 2 population was assumed to be the same as that estimated for the Phase 1 study subjects; and c) Duration of Lp(a) suppression is based on model-predicted olpaciran PK / PD half-life in the liver, and estimates of response after multiple doses are based on observed suppression from the Phase 1 study and accumulation of effect at the end of the dosing interval.

[0146] The model was able to adequately predict the significant observed reduction in olpaciran exposure in subjects with higher baseline Lp(a) (≥ 150 nmol / L). This suggested that higher doses of olpaciran may be necessary to achieve target Lp(a) suppression in this patient population. Simulations of the model were performed to explore Q3M and Q6M dosing regimens and to extrapolate predicted Lp(a) suppression after multiple doses of olpaciran. For each candidate dosing regimen, the proportion of subjects achieving target Lp(a) suppression (≥ 80% reduction from baseline) and the proportion of subjects achieving absolute Lp(a) values ​​≤ 50 nmol / L at the end of the dosing interval were calculated.

[0147] Figures 3A-3F show predicted Lp(a) levels as a percentage of baseline for Q3M administration of olpaciran at doses of 10 mg, 30 mg, 50 mg, 75 mg, 150 mg, and 225 mg for subjects with baseline Lp(a) levels of ≥ 150 nmol / L. The model predicts that doses of 10 mg and above will suppress Lp(a) levels by ≥ 80% throughout the 3-month dosing interval. Table 4 below shows the predicted proportion of subjects achieving at least an 80% reduction from baseline in Lp(a) levels with different doses of olpaciran administered once every 3 months (Q3M administration) at each dosing interval, while Table 5 shows the predicted proportion of subjects achieving absolute Lp(a) levels of ≤ 50 nmol / L with the same olpaciran dosing regimen.

[0148] [Table 4]

[0149] [Table 5]

[0150] Model simulations based on Phase 1 data predict that a 10 mg dose of olpaciran administered quarterly (Q3M) will result in approximately 42% of subjects with baseline Lp(a) levels of ≥ 150 nmol / L achieving at least an 80% reduction in Lp(a) from baseline by Month 12. Olpaciran doses of ≥ 75 mg administered quarterly are predicted to achieve ≥ 80% Lp(a) suppression in at least 90% of subjects with baseline Lp(a) levels of ≥ 150 nmol / L as early as 3 months after receiving a single dose of olpaciran. Similar percentages of subjects are predicted to achieve absolute Lp(a) levels of ≤ 50 nmol / L with these same dosing regimens.

[0151] Simulations were also performed on a model for a twice-yearly (Q6M) dosing regimen of olpaciran. Figures 4A-4F show predicted Lp(a) levels as a percentage of baseline for Q6M administration of olpaciran at doses of 10 mg, 75 mg, 150 mg, 225 mg, 450 mg, and 675 mg for subjects with baseline Lp(a) levels of ≥ 150 nmol / L. The model predicts that doses of at least 75 mg will suppress Lp(a) levels by ≥ 80% throughout the entire 6-month dosing interval. Table 6 below shows the predicted proportion of subjects achieving at least an 80% reduction from baseline in Lp(a) levels with different doses of olpaciran administered once every 6 months (Q6M administration) at each dosing interval, while Table 7 shows the predicted proportion of subjects achieving absolute Lp(a) levels of ≤ 50 nmol / L with the same olpaciran dosing regimen.

[0152] [Table 6]

[0153] [Table 7]

[0154] Modeling data indicate that a dose of at least 75 mg of olpaciran administered once every six months (Q6M) is predicted to reduce Lp(a) levels by at least 80% from baseline in approximately 50% of subjects with baseline Lp(a) levels of ≥ 150 nmol / L after only two doses (i.e., after 12 months of treatment). The same proportion of patients is predicted to achieve absolute Lp(a) levels of less than 50 nmol / L with 75 mg of olpaciran administered once every six months after one year of treatment. A dose of 225 mg of olpaciran administered once every six months is predicted to suppress Lp(a) levels by at least 80% in approximately 76% of subjects after one year of treatment, while doses of 450 mg or greater administered once every six months are predicted to suppress Lp(a) levels above this threshold in approximately 90% of subjects after one year of treatment.

[0155] Based on recent Mendelian randomization studies, a reduction in Lp(a) levels of 80% or more from baseline is expected to result in clinically meaningful cardiovascular benefits in patients with atherosclerotic cardiovascular disease (Burgess et al., JAMA Cardiol., Vol. 3:619-627, 2018; Lamina et al., JAMA Cardiol., Vol. 4:575-579, 2019; and Madsen et al., Arterioscler. Thromb. Vasc. Biol., 40:255-266, 2020). Therefore, olpaciran PK / PD modeling focused on identifying an olpaciran dosing regimen that could reduce Lp(a) levels below this threshold. The results of olpaciran PK / PD modeling and the simulations described in this example demonstrate the following: The 10 mg dose administered once every 3 months or once every 12 weeks achieves an Lp(a) reduction of ≥ 80% in approximately half (42%) of subjects with baseline Lp(a) levels of ≥ 150 nmol / L by 12 months, and a median Lp(a) reduction from baseline of approximately 77% at 6 and 12 months; A 75 mg dose administered once every 3 months or once every 12 weeks is expected to achieve an Lp(a) reduction of ≥ 80% from baseline within 2–3 doses in the majority of subjects (94%), and approximately 90% of subjects are expected to achieve absolute Lp(a) concentrations of ≤ 50 nmol / L with this dosing regimen; A dose of 225 mg administered once every 3 months or once every 12 weeks is expected to achieve an Lp(a) reduction of ≥ 80% in 98% of subjects and reduce Lp(a) levels to an absolute concentration of ≤ 50 nmol / L in 96% of subjects; For doses of 10 mg or greater, a dosing frequency of once every 3 months or once every 12 weeks results in suppression of Lp(a) levels to less than 20% of baseline throughout the 3-month dosing interval in the majority (≥90%) of subjects with baseline Lp(a) levels of ≥150 nmol / L; and The 225 mg dose administered once every 6 months or once every 24 weeks resulted in a median Lp(a) reduction from baseline of 88%, with approximately 74% of subjects achieving absolute Lp(a) levels below 50 nmol / L.

[0156] Example 3. A double-blind, randomized, placebo-controlled phase 2 study to evaluate the efficacy, safety, and tolerability of olpaciran in subjects with elevated lipoprotein(a) The primary objective of this Phase 2 study is to evaluate the effect of subcutaneous administration of olpaciran once every 12 weeks (Q12W) compared to placebo on the percent change from baseline in Lp(a) levels after 36 weeks of treatment in subjects with atherosclerotic cardiovascular disease and elevated Lp(a). Secondary objectives of the study include the effect of olpaciran administered subcutaneously Q12W compared to placebo on the percent change from baseline in: (i) Lp(a) levels after 48 weeks of treatment, (ii) low-density lipoprotein cholesterol (LDL-C) levels after 36 and 48 weeks of treatment, and (iii) apolipoprotein B (ApoB) levels after 36 and 48 weeks of treatment, as well as characterization of the pharmacokinetic properties of olpaciran. Administration of olpaciran once every 24 weeks (Q24W) will also be evaluated.

[0157] Approximately 240 subjects will be randomized in a 1:1:1:1:1 ratio. Four arms will be treated with olpaciran and one arm with placebo. Randomization will be stratified by screening for Lp(a) ≦200 nmol / L vs. >200 nmol / L and by region (Japan vs. non-Japan). The study treatment period will be 48 weeks, with dosing at Day 1, Week 12, Week 24, and Week 36. After Week 48, safety follow-up will continue for ≧40 weeks without further administration of olpaciran or placebo until Lp(a) returns to 80% of baseline (whichever occurs later). Subjects will continue standard of care (including stable lipid-altering therapy) according to local guidelines during the treatment and long-term safety follow-up periods.

[0158] After signing informed consent, subjects enter a screening phase (up to 4 weeks) during which the subject's eligibility criteria are assessed. Eligible subjects include adults aged 18-80 years with atherosclerotic cardiovascular disease who have an Lp(a) >150 nmol / L during screening. Specifically, subjects are enrolled in the study if they meet all of the following key inclusion criteria: Ages 18-80 Lp(a) >150nmol / L during central laboratory screening Atherosclerotic cardiovascular disease based on one of the following: · History of coronary revascularization by percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG); · Diagnosis of coronary artery disease with or without previous myocardial infarction; A diagnosis of atherosclerotic cerebrovascular disease; or Diagnosis of peripheral arterial disease For subjects not on lipid-altering therapy (not required for eligibility criteria), lipid-altering therapy, including statin dose, must remain stable according to local guidelines for ≥ 4 weeks prior to screening and during screening Subjects will be excluded from the study if they meet any of the following key exclusion criteria: Estimated glomerular filtration rate (eGFR) < 30 mL / min / 1.73 m during screening 2 Severe renal insufficiency defined as History or clinical evidence of liver dysfunction, defined as aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >3 x upper limit of normal (ULN) or total bilirubin (TBL) >2 x ULN during screening Malignant tumors within the past 5 years before Day 1 (excluding non-melanoma skin cancer, cervical intraepithelial carcinoma, breast intraepithelial carcinoma, or stage 1 prostate cancer) Uncontrolled hypertension on Day 1 (defined as mean systolic blood pressure ≥ 160mmHg or mean diastolic blood pressure ≥ 100mmHg at rest) Fasting triglycerides of ≥ 400 mg / dL (4.5 mmol / L) during screening Type 1 diabetes or uncontrolled type 2 diabetes as determined by a glycosylated hemoglobin (HbA1c) of ≥ 8.5% as determined by a core laboratory at the time of screening

[0159] Eligible subjects had a baseline Lp(a) of >150 nmol / L. This threshold was based on available epidemiological data indicating that an Lp(a) of >125 nmol / L is considered elevated from general population data (Averna et al., Atheroscler Suppl., Vol. 26:16-24, 2017; Nordestgaard and Langsted, J. Lipid Res., Vol. 57:1953-75, 2016; Ohro-Melander, J. Intern Med., Vol. 278:433-46, 2015; Leebmann et al., Circulation, Vol. 128:2567-2576, 2013). In addition, based on the magnitude of absolute Lp(a) reduction required to demonstrate a corresponding effect on cardiovascular events, the enrolled population must have a high baseline Lp(a). Thus, the entry criterion of Lp(a) > 150 nmol / L yields a study population with a median Lp(a) of approximately 200 nmol / L and allows for evaluation of the efficacy and safety of olpaciran in subjects with very high Lp(a).

[0160] Eligible enrolled subjects will be randomized in a 1:1:1:1:1 ratio to one of the following five treatment arms (with approximately 48 subjects in each arm): Group 1: 10 mg olpaciran Q12W Group 2: 75 mg olpaciran Q12W Group 3: 225 mg olpaciran Q12W Group 4: 225 mg olpaciran Q24W Group 5: Placebo Q12W As described in Example 2, these olpaciran dosing regimens are predicted to suppress Lp(a) levels by at least 80% from baseline throughout the entire dosing interval (3 or 6 months) in human subjects with baseline Lp(a) levels >150 nmol / L. Olpaciran will be administered by subcutaneous injection at doses of 10 mg, 75 mg, or 225 mg once every 12 weeks (treatment groups 1-3) or once every 24 weeks (treatment group 4), depending on the assigned treatment group. Samples for assessing serum Lp(a), LDL-C, and ApoB, as well as other clinical laboratory analytes, will be collected from enrolled subjects during screening, prior to administration of the first dose of olpaciran, and at weeks 12, 24, 36, and 48, and at various other time points during the study. Blood samples will be collected for measurement of olpaciran serum concentrations at various time points during the study to assess olpaciran pharmacokinetic parameters.

[0161] Screening for Lp(a) is performed in a central laboratory using approved or investigational turbidimetric immunoassays standardized to detect and quantify Lp(a) particles independent of apo(a) isoform size, such as the Tina-quant® Lipoprotein(a) Gen. 2 assay available from Roche Diagnostics. The assay is validated for nmol / L measurement of Lp(a) in serum samples with a detection limit of 7 nmol / L and normalized to nmol / L against the IFCC reference material SRM2B (Marcovina et al., Clin. Chem., Vol. 46:1946-1967, 2000). Lipid panels and assays for other clinical analytes, such as ApoB, hemoglobin A1C, ALT, AST, and bilirubin, are performed by the central laboratory using standard methods.

[0162] The primary analysis will be conducted when all randomized subjects have had an opportunity to complete the Week 36 assessment or terminated early. Treatment-duration analyses will be completed when all subjects have had an opportunity to complete the Week 48 assessment or terminated early. The final analysis will be conducted after the last subject has completed the long-term safety follow-up and has either completed the study or terminated early. The primary endpoint (percent change from baseline in Lp(a) at Week 36) will be compared between groups using a repeated measures linear effects model that includes treatment group duration, stratification factors, scheduled visits, and the interaction of treatment with scheduled visits. The Hochberg procedure will be used to control for type I error due to multiple comparisons between the active and placebo arms. Secondary endpoints, percent change from baseline in Lp(a) at Week 48, ApoB, and LDL-C at Weeks 36 and 48, will be analyzed similarly to the primary endpoint. Safety endpoints (e.g., treatment-emergent adverse events) will be summarized narratively. Baseline Lp(a) is defined as the mean of the two most recent non-missing Lp(a) values ​​measured by a central laboratory before or on Study Day 1. If, for any reason, only one value is available, that value will be used as the baseline.

[0163] A reduction in Lp(a) of 80% or more from baseline was observed with a single dose of olpaciran lasting for more than three months (see Example 1). It is anticipated that this sustained reduction in Lp(a) may result in clinically meaningful cardiovascular benefits in patients with atherosclerotic cardiovascular disease by reducing the risk of cardiovascular events. Recent Mendelian randomization studies suggest that in individuals with very high baseline Lp(a) concentrations, an 80% to 90% reduction in Lp(a) would be expected to translate into a clinically meaningful reduction in the risk of cardiovascular events (Burgess et al., JAMA Cardiol., Vol. 3:619-627, 2018; Lamina et al., JAMA Cardiol., Vol. 4:575-579, 2019; and Madsen et al., Arterioscler. Thromb. Vasc. Biol., 40:255-266, 2020). Thus, the results of this study are expected to demonstrate that olpaciran, compared with placebo, will provide a significant percent reduction in Lp(a) from baseline in subjects with elevated Lp(a) and atherosclerotic cardiovascular disease at all doses tested. In particular, olpaciran is expected to effectively reduce Lp(a) levels to less than 50 nmol / L in the majority of subjects at doses as low as 10 mg administered once every 12 weeks. This is expected to result in a reduced risk of cardiovascular events in such subjects. Olpaciran administered at a dose of 75 mg once every 12 weeks is expected to be a particularly effective dosing regimen based on the PK / PD modeling described in Example 2.

[0164] All publications, patents, and patent applications discussed and cited herein are incorporated herein by reference in their entirety. It is understood that the disclosed invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the appended claims.

[0165] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims. The present invention provides, for example, the following items. (Item 1) 1. A method of treating, attenuating, or preventing atherosclerosis in a patient in need thereof, comprising administering to the patient an LPA RNAi construct at a dose of about 9 mg to about 675 mg with an administration interval of at least 8 weeks, wherein the LPA RNAi construct comprises a sense strand comprising the sequence of SEQ ID NO: 1, an antisense strand comprising the sequence of SEQ ID NO: 2, and a targeting moiety comprising an asialoglycoprotein receptor ligand, wherein the targeting moiety is covalently attached to the 5' end of the sense strand. (Item 2) 1. A method for reducing serum or plasma Lp(a) levels in a patient in need thereof, comprising administering to the patient an LPA RNAi construct at a dose of about 9 mg to about 675 mg with an administration interval of at least 8 weeks, wherein the LPA RNAi construct comprises a sense strand comprising the sequence of SEQ ID NO: 1, an antisense strand comprising the sequence of SEQ ID NO: 2, and a targeting moiety comprising an asialoglycoprotein receptor ligand, wherein the targeting moiety is covalently attached to the 5' end of the sense strand. (Item 3) 3. The method of item 2, wherein the patient is diagnosed with or at risk for cardiovascular disease. (Item 4) Item 4. The method according to Item 3, wherein the cardiovascular disease is coronary artery disease, carotid artery disease, peripheral artery disease, myocardial infarction, cerebrovascular disease, stroke, aortic stenosis, stable or unstable angina, atrial fibrillation, heart failure, hyperlipidemia, heterozygous familial hypercholesterolemia, or homozygous familial hypercholesterolemia. (Item 5) 3. The method of claim 2, wherein the patient is diagnosed with coronary artery disease. (Item 6) 3. The method of claim 2, wherein the patient has a history of myocardial infarction. (Item 7) 3. The method of claim 2, wherein the patient is diagnosed with acute coronary syndrome. (Item 8) 1. A method for treating, attenuating, or preventing cardiovascular disease in a patient in need thereof, comprising administering to the patient an LPA RNAi construct at a dose of about 9 mg to about 675 mg with an administration interval of at least 8 weeks, wherein the LPA RNAi construct comprises a sense strand comprising the sequence of SEQ ID NO: 1, an antisense strand comprising the sequence of SEQ ID NO: 2, and a targeting moiety comprising an asialoglycoprotein receptor ligand, wherein the targeting moiety is covalently attached to the 5' end of the sense strand. (Item 9) Item 9. The method according to Item 8, wherein the cardiovascular disease is coronary artery disease, carotid artery disease, peripheral artery disease, myocardial infarction, cerebrovascular disease, stroke, aortic stenosis, stable or unstable angina, atrial fibrillation, heart failure, hyperlipidemia, heterozygous familial hypercholesterolemia, or homozygous familial hypercholesterolemia. (Item 10) 1. A method for reducing the risk of cardiovascular events in a patient with atherosclerotic cardiovascular disease, comprising administering to the patient an LPA RNAi construct at a dose of about 9 mg to about 675 mg with an administration interval of at least 8 weeks, wherein the LPA RNAi construct comprises a sense strand comprising the sequence of SEQ ID NO: 1, an antisense strand comprising the sequence of SEQ ID NO: 2, and a targeting moiety comprising an asialoglycoprotein receptor ligand, the targeting moiety being covalently attached to the 5' end of the sense strand. (Item 11) 11. The method of item 10, wherein the cardiovascular event is cardiovascular death, myocardial infarction, stroke, and / or coronary revascularization. (Item 12) 12. The method of item 10 or 11, wherein the patient has a history of coronary revascularization, a history of coronary artery bypass grafting, a diagnosis of coronary artery disease, a diagnosis of atherosclerotic cerebrovascular disease, a diagnosis of peripheral artery disease, and / or a history of myocardial infarction. (Item 13) 13. The method of any one of items 10 to 12, wherein the patient has experienced a myocardial infarction within one year prior to the first administration of the LPA RNAi construct. (Item 14) 13. The method according to any one of items 10 to 12, wherein the patient is hospitalized due to acute coronary syndrome or unstable angina. (Item 15) 15. The method of any one of items 1 to 14, wherein the patient has a serum or plasma Lp(a) level of about 70 nmol / L or greater before the first administration of the LPA RNAi construct. (Item 16) 15. The method of any one of items 1 to 14, wherein the patient has a serum or plasma Lp(a) level of about 100 nmol / L or greater before the first administration of the LPA RNAi construct. (Item 17) 15. The method of any one of items 1 to 14, wherein the patient has a serum or plasma Lp(a) level of about 125 nmol / L or greater before the first administration of the LPA RNAi construct. (Item 18) 15. The method of any one of items 1 to 14, wherein the patient has a serum or plasma Lp(a) level of about 150 nmol / L or greater before the first administration of the LPA RNAi construct. (Item 19) 15. The method of any one of items 1 to 14, wherein the patient has a serum or plasma Lp(a) level of about 175 nmol / L or greater before the first administration of the LPA RNAi construct. (Item 20) 15. The method of any one of items 1 to 14, wherein the patient has a serum or plasma Lp(a) level of about 200 nmol / L or greater before the first administration of the LPA RNAi construct. (Item 21) 15. The method of any one of items 1 to 14, wherein the patient has a serum or plasma Lp(a) level of about 225 nmol / L or greater before the first administration of the LPA RNAi construct. (Item 22) 22. The method of any one of items 1 to 21, wherein the administration interval is about 12 weeks. (Item 23) 22. The method of any one of items 1 to 21, wherein the administration interval is about 24 weeks. (Item 24) 22. The method according to any one of items 1 to 21, wherein the LPA RNAi construct is administered to the patient at a dose of about 10 mg to about 225 mg once every 12 weeks. (Item 25) 25. The method according to item 24, wherein the LPA RNAi construct is administered to the patient at a dose of about 50 mg to about 100 mg once every 12 weeks. (Item 26) 25. The method of claim 24, wherein the LPA RNAi construct is administered to the patient at a dose of about 150 mg to about 225 mg once every 12 weeks. (Item 27) 25. The method of claim 24, wherein the LPA RNAi construct is administered to the patient at a dose of about 75 mg once every 12 weeks. (Item 28) 25. The method of claim 24, wherein the LPA RNAi construct is administered to the patient at a dose of about 150 mg once every 12 weeks. (Item 29) 25. The method of claim 24, wherein the LPA RNAi construct is administered to the patient at a dose of about 225 mg once every 12 weeks. (Item 30) 22. The method of any one of items 1 to 21, wherein the LPA RNAi construct is administered to the patient at a dose of about 225 mg to about 675 mg once every 24 weeks. (Item 31) 31. The method of claim 30, wherein the LPA RNAi construct is administered to the patient at a dose of about 225 mg once every 24 weeks. (Item 32) 32. The method of any one of items 1 to 31, wherein the patient is undergoing lipid-lowering therapy. (Item 33) 33. The method of item 32, wherein the lipid-lowering therapy is a statin, ezetimibe, a PCSK9 inhibitor, bempedoic acid, or a combination thereof. (Item 34) 34. The method of any one of items 1 to 33, wherein the patient has a serum low-density lipoprotein cholesterol (LDL-C) level of about 100 mg / dL or less before the first administration of the LPA RNAi construct. (Item 35) 34. The method of any one of items 1 to 33, wherein the patient has a serum low-density lipoprotein cholesterol (LDL-C) level of about 70 mg / dL or less before the first administration of the LPA RNAi construct. (Item 36) The patient had an estimated glomerular filtration rate of about 30 mL / min / 1.73 m before the first administration of the LPA RNAi construct. 2 The method according to any one of items 1 to 35, wherein (Item 37) 37. The method of any one of items 1 to 36, wherein the patient has a resting mean systolic blood pressure of less than about 160 mmHg and a mean diastolic blood pressure of less than about 100 mmHg before the first administration of the LPA RNAi construct. (Item 38) 38. The method of any one of items 1 to 37, wherein the patient has a glycated hemoglobin A1c level of less than about 8.5% before the first administration of the LPA RNAi construct. (Item 39) 39. The method of any one of items 1 to 38, wherein the patient has a serum triglyceride level of less than about 400 mg / dL before the first administration of the LPA RNAi construct. (Item 40) 40. The method according to any one of items 1 to 39, wherein the sense strand of the LPA RNAi construct comprises or consists of the sequence of SEQ ID NO: 3, and the antisense strand of the LPA RNAi construct comprises or consists of the sequence of SEQ ID NO: 4. (Item 41) 41. The method of any one of items 1 to 40, wherein the sense strand of the LPA RNAi construct comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 5, and the antisense strand of the LPA RNAi construct comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 6. (Item 42) The targeting portion of the LPA RNAi construct:

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Claims

1. 1. A pharmaceutical composition for treating, attenuating, or preventing atherosclerosis in a human patient in need thereof, comprising an LPA RNAi construct, wherein the LPA RNAi construct is administered to the patient at a dose of 10 mg to 225 mg once every 12 weeks, the LPA RNAi construct comprising a sense strand comprising the sequence of SEQ ID NO: 3, an antisense strand comprising the sequence of SEQ ID NO: 4, and a targeting moiety comprising an asialoglycoprotein receptor ligand, the targeting moiety being covalently attached to the 5' end of the sense strand; 【Chemistry 2】 A pharmaceutical composition having the structure:

2. 1. A pharmaceutical composition for reducing serum or plasma lipoprotein(a) (Lp(a)) levels in a human patient in need thereof, comprising an LPA RNAi construct, wherein the LPA RNAi construct is administered to the patient at a dose of 10 mg to 225 mg once every 12 weeks, the LPA RNAi construct comprising a sense strand comprising the sequence of SEQ ID NO:3, an antisense strand comprising the sequence of SEQ ID NO:4, and a targeting moiety comprising an asialoglycoprotein receptor ligand, the targeting moiety being covalently attached to the 5' end of the sense strand; 【Transformation 3】 A pharmaceutical composition having the structure:

3. The pharmaceutical composition of claim 2 , wherein the patient has been diagnosed with or is at risk for cardiovascular disease.

4. 4. The pharmaceutical composition according to claim 3, wherein the cardiovascular disease is coronary artery disease, carotid artery disease, peripheral artery disease, myocardial infarction, cerebrovascular disease, stroke, aortic stenosis, stable or unstable angina, atrial fibrillation, heart failure, hyperlipidemia, heterozygous familial hypercholesterolemia, or homozygous familial hypercholesterolemia.

5. The pharmaceutical composition of claim 2 , wherein the patient has been diagnosed with coronary artery disease.

6. The pharmaceutical composition of claim 2 , wherein the patient has a history of myocardial infarction.

7. The pharmaceutical composition of claim 2 , wherein the patient has been diagnosed with acute coronary syndrome.

8. 1. A pharmaceutical composition for treating, alleviating, or preventing cardiovascular disease in a human patient in need thereof, comprising an LPA RNAi construct, wherein the LPA RNAi construct is administered to the patient at a dose of 10 mg to 225 mg once every 12 weeks, the LPA RNAi construct comprising a sense strand comprising the sequence of SEQ ID NO: 3, an antisense strand comprising the sequence of SEQ ID NO: 4, and a targeting moiety comprising an asialoglycoprotein receptor ligand, the targeting moiety being covalently attached to the 5' end of the sense strand; 【Chemistry 4】 A pharmaceutical composition having the structure:

9. 9. The pharmaceutical composition according to claim 8, wherein the cardiovascular disease is coronary artery disease, carotid artery disease, peripheral artery disease, myocardial infarction, cerebrovascular disease, stroke, aortic stenosis, stable or unstable angina, atrial fibrillation, heart failure, hyperlipidemia, heterozygous familial hypercholesterolemia, or homozygous familial hypercholesterolemia.

10. 1. A pharmaceutical composition for reducing the risk of cardiovascular events in a human patient with atherosclerotic cardiovascular disease, comprising an LPA RNAi construct, wherein the LPA RNAi construct is administered to the patient at a dose of 10 mg to 225 mg once every 12 weeks, the LPA RNAi construct comprising a sense strand comprising the sequence of SEQ ID NO: 3, an antisense strand comprising the sequence of SEQ ID NO: 4, and a targeting moiety comprising an asialoglycoprotein receptor ligand, the targeting moiety being covalently attached to the 5' end of the sense strand; 【Transformation 5】 A pharmaceutical composition having the structure:

11. The pharmaceutical composition of claim 10 , wherein the cardiovascular event is cardiovascular death, myocardial infarction, stroke, and / or coronary revascularization.

12. 11. The pharmaceutical composition of claim 10, wherein the patient has a history of coronary revascularization, a history of coronary artery bypass grafting, a diagnosis of coronary artery disease, a diagnosis of atherosclerotic cerebrovascular disease, a diagnosis of peripheral artery disease, and / or a history of myocardial infarction.

13. The pharmaceutical composition of claim 10, wherein the patient has experienced a myocardial infarction within one year prior to the first administration of the LPA RNAi construct.

14. The pharmaceutical composition of claim 10, wherein the patient is hospitalized for acute coronary syndrome or unstable angina.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the patient has a serum or plasma Lp(a) level of 70 nmol / L or greater before the first administration of the LPA RNAi construct.

16. The pharmaceutical composition of any one of claims 1 to 14, wherein the patient has a serum or plasma Lp(a) level of 100 nmol / L or greater before the first administration of the LPA RNAi construct.

17. The pharmaceutical composition of any one of claims 1 to 14, wherein the patient has a serum or plasma Lp(a) level of 125 nmol / L or greater before the first administration of the LPA RNAi construct.

18. The pharmaceutical composition of any one of claims 1 to 14, wherein the patient has a serum or plasma Lp(a) level of 150 nmol / L or greater before the first administration of the LPA RNAi construct.

19. The pharmaceutical composition of any one of claims 1 to 14, wherein the patient has a serum or plasma Lp(a) level of 175 nmol / L or greater before the first administration of the LPA RNAi construct.

20. The pharmaceutical composition of any one of claims 1 to 14, wherein the patient has a serum or plasma Lp(a) level of 200 nmol / L or greater before the first administration of the LPA RNAi construct.

21. The pharmaceutical composition of any one of claims 1 to 14, wherein the patient has a serum or plasma Lp(a) level of 225 nmol / L or greater before the first administration of the LPA RNAi construct.

22. The pharmaceutical composition of any one of claims 1 to 21, wherein the LPA RNAi construct is administered to the patient at a dose of 50 mg to 100 mg once every 12 weeks.

23. The pharmaceutical composition of any one of claims 1 to 21, wherein the LPA RNAi construct is administered to the patient at a dose of 150 mg to 225 mg once every 12 weeks.

24. The pharmaceutical composition of any one of claims 1 to 21, wherein the LPA RNAi construct is administered to the patient at a dose of 75 mg once every 12 weeks.

25. The pharmaceutical composition according to any one of claims 1 to 21, wherein the LPA RNAi construct is administered to the patient at a dose of 150 mg once every 12 weeks.

26. The pharmaceutical composition of any one of claims 1 to 21, wherein the LPA RNAi construct is administered to the patient at a dose of 225 mg once every 12 weeks.

27. The pharmaceutical composition of any one of claims 1 to 26, wherein the patient is undergoing lipid-lowering therapy.

28. 28. The pharmaceutical composition of claim 27, wherein the lipid-lowering therapy is a statin, ezetimibe, a PCSK9 inhibitor, bempedoic acid, or a combination thereof.

29. 29. The pharmaceutical composition of any one of claims 1 to 28, wherein the patient has a serum low-density lipoprotein cholesterol (LDL-C) level of 100 mg / dL or less prior to the first administration of the LPA RNAi construct.

30. The pharmaceutical composition of any one of claims 1 to 28, wherein the patient has a serum low-density lipoprotein cholesterol (LDL-C) level of 70 mg / dL or less before the first administration of the LPA RNAi construct.

31. The pharmaceutical composition of any one of claims 1 to 30, wherein the sense strand of the LPA RNAi construct comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 5, and the antisense strand of the LPA RNAi construct comprises or consists of a sequence of modified nucleotides according to SEQ ID NO:

6.

32. The pharmaceutical composition of any one of claims 1 to 31, wherein the LPA RNAi construct is olpaciran.

33. The pharmaceutical composition of any one of claims 1 to 32, wherein the composition further comprises potassium phosphate and sodium chloride.

34. The pharmaceutical composition of any one of claims 1 to 33, wherein the LPA RNAi construct is administered to the patient by subcutaneous injection.

35. 35. The pharmaceutical composition of claim 34, wherein the injection volume is 1 mL or less.

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