Combination RNA therapies

Abstract

Aspects of the technology relate to a composition comprising: (i) a first RNAi agent that targets Proprotein convertase subtilisin / kexin type 9 (PCSK9) and a second RNAi agent that targets angiotensinogen (AGT); (ii) a first RNAi agent that targets PCSK9 and a third RNAi agent that targets Apolipoprotein A (LPA); (iii) a second RNAi agent that targets AGT and a third RNAI agent that targets LPA, or (iv) a first RNAi agent that targets PCSK9, a second RNAi agent that targets AGT, and the third RNAi agent that targets LPA.

Description

[0001] COMBINATION RNA THERAPIES

[0002] RELATED APPLICATIONS

[0003] This Application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 721279, filed on November 15, 2024, and U.S. Provisional Application No. 63 / 866506, filed on August 19, 2025, the entire contents of each of which are incorporated herein by reference.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (C175370002WO00-SEQ-HJD.xml; Size: 72,078 bytes; and Date of Creation: November 4, 2025) is herein incorporated by reference in their entirety.

[0006] BACKGROUND

[0007] Long-acting siRNAs (small interfering RNAs) are a promising development in RNA interference (RNAi)-based therapeutics. Traditional siRNAs typically have a short duration of action due to degradation by nucleases, rapid clearance, and dilution by cell division, limiting their therapeutic efficacy. Long-acting siRNAs are engineered to overcome these challenges and provide prolonged therapeutic effects.

[0008] SUMMARY

[0009] Aspects of the technology provided herein relate to siRNA therapies for treating and preventing cardiovascular disease, including atherosclerotic cardiovascular disease (ASVCD). The siRNA therapies of the disclosure are designed to recapitulate the biological effects of partial loss-of-function variants in the genes that encode these proteins, which are associated with lifetime reductions in exposure to risk factors for cardiovascular disease. The siRNA therapies described herein are engineered, in some embodiments, for once-yearly administration - alone or in combination - to result in sustained reductions in risk factors associated with cardiovascular disease, thereby reducing the risk of atherosclerotic cardiovascular events.

[0010] Some aspects of the technology relate to a composition comprising at least two RNAi agents selected from a first RNAi agent that targets Proprotein convertase subtilisin / kexin type 9 (PCSK9), a second RNAi agent that targets angiotensinogen (AGT), and a third RNAi agent that targets Apolipoprotein A (LPA). In some embodiments, a composition comprises a first RNAi agent that targets PCSK9, a second RNAi agent that targets AGT, and a third RNAi agent that targets LPA.

[0011] Some aspects of the technology relate to a composition comprising: (i) a first RNAi agent that targets PCSK9 and a second RNAi agent that targets AGT; (ii) a first RNAi agent that targets PCSK9 and a third RNAi agent that targets LPA; (iii) a first RNAi agent that targets PCSK9, a second RNAi agent that targets AGT, and the third RNAi agent that targets LPA; or (iv) a second RNAi agent that targets AGT and a third RNAi agent that targets LPA.

[0012] In some embodiments, the first RNAi agent, the second RNAi agent, and / or the third RNAi agent is an siRNA.

[0013] In some embodiments, the siRNA is double stranded.

[0014] In some embodiments, the first RNAi agent comprises an antisense strand comprising a region of complementarity to a PCSK9 RNA, and a sense strand that is complementary to the antisense strand.

[0015] In some embodiments, the second RNAi agent comprises an antisense strand comprising a region of complementarity to an AGT RNA, and a sense strand that is complementary to the antisense strand.

[0016] In some embodiments, the third RNAi agent comprises an antisense strand comprising a region of complementarity to an LPA RNA, and a sense strand that is complementary to the antisense strand.

[0017] In some embodiments, the region of complementarity is at least 8 nucleosides in length.

[0018] In some embodiments, the antisense strand is 15-35 nucleosides in length and / or the sense strand is 15-35 nucleosides in length.

[0019] In some embodiments, the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises an overhang of 0, 1, 2, 3, 4 or 5 nucleosides at the 3’ and / or 5’ end of the sense strand and / or antisense strand.

[0020] In some embodiments, the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one modified nucleoside comprising a chemical modification in the base and / or the sugar moiety of the nucleoside.

[0021] In some embodiments, at least one modified nucleoside comprises a 2’ -modified nucleoside.

[0022] In some embodiments, the 2’ modified nucleoside is a 2’-O-methyl modified nucleoside or 2’-F modified nucleoside. In some embodiments, the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one modified internucleoside linkage.

[0023] In some embodiments, at least one modified internucleoside linkage comprises a phosphorothioate internucleoside linkage.

[0024] In some embodiments, the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises a biostable phosphate mimic. In some embodiments, the biostable phosphate mimic is a vinylpho sphonate moiety. In some embodiments, the biostable phosphate mimic is at the 5’ end of the antisense strand.

[0025] In some embodiments, the first RNAi agent, the second RNAi agent, and / or the third RNAi agent is conjugated to a targeting moiety.

[0026] In some embodiments, the composition comprises the first RNAi agent and the second RNAi agent, wherein the first RNAi agent and the second RNAi agent are not linked.

[0027] In some embodiments, the composition comprises the first RNAi agent and the second RNAi agent, wherein the first RNAi agent and the second RNAi agent are covalently linked (e.g., to form a dimer).

[0028] In some embodiments, the first RNAi agent and the second RNAi agent are covalently linked via a linker, optionally wherein the linker comprises one or more nucleosides, further optionally wherein the linker is a cleavable linker.

[0029] In some embodiments, the composition comprises the first RNAi agent and the third RNAi agent, wherein the first RNAi agent and the third RNAi agent are not linked.

[0030] In some embodiments, the composition comprises the first RNAi agent and the third RNAi agent, wherein the first RNAi agent and the third RNAi agent are covalently linked.

[0031] In some embodiments, the first RNAi agent and the third RNAi agent are covalently linked via a linker, optionally wherein the linker comprises one or more nucleosides, further optionally wherein the linker is a cleavable linker.

[0032] In some embodiments, the composition comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the first RNAi agent, the second RNAi agent, and the third RNAi agent are not linked to each other.

[0033] In some embodiments, the composition comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the first RNAi agent is covalently linked to the second RNAi agent, and the third RNAi agent is not linked to the first RNAi agent or to the second RNAi agent. In some embodiments, the first RNAi agent and the second RNAi agent are covalently linked via a linker, optionally wherein the linker comprises one or more nucleosides, further optionally wherein the linker is a cleavable linker.

[0034] In some embodiments, the composition comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the first RNAi agent is covalently linked to the third RNAi agent, and the second RNAi agent is not linked to the first RNAi agent or to the third RNAi agent.

[0035] In some embodiments, the first RNAi agent and the third RNAi agent are covalently linked via a linker, optionally wherein the linker comprises one or more nucleosides, further optionally wherein the linker is a cleavable linker.

[0036] In some embodiments, the composition comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the second RNAi agent is covalently linked to the third RNAi agent, and the first RNAi agent is not linked to the second RNAi agent or to the third RNAi agent.

[0037] In some embodiments, the second RNAi agent and the third RNAi agent are covalently linked via a linker, optionally wherein the linker comprises one or more nucleosides, further optionally wherein the linker is a cleavable linker.

[0038] In some embodiments, the composition comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the first RNAi agent, the second RNAi agent, and the third RNAi agent are covalently linked to each other.

[0039] In some embodiments, the first RNAi agent, the second RNAi agent, and the third RNAi agent are covalently linked to each other via linkers, optionally wherein each linker comprises one or more nucleosides, further optionally wherein each linker is a cleavable linker.

[0040] In some embodiments, the second RNAi agent and the third RNAi agent are not linked. In some embodiments, the second RNAi agent and the third RNAi agent are covalently linked. In some embodiments, the second RNAi agent and the third RNAi agent are covalently linked via a linker. In some embodiments, the linker comprises one or more nucleosides, further optionally wherein the linker is a cleavable linker.

[0041] In some embodiments, the composition reduces levels of low-density lipoprotein (LDL), systolic blood pressure (SBP), and / or Lipoprotein(a) (Lp(a)) in a subject with high baseline levels of LDL (e.g., LDL-C), SBP, and / or Lp(a). In some embodiments, an elevated baseline level of LDL (e.g., LDL-C) is >130 mg / dL, an elevated base line level of SPB is >130 mmHg, and / or an elevated baseline level of Lp(a) is >50 mg / dL. In some embodiments, the composition reduces the level of LDL (e.g., LDL-C) in a subject with high a baseline level of LDL (e.g., LDL-C) by at least 40%, reduces the level of SBP in a subject with a high baseline level of SBP by at least 5-10 mmHg, and / or reduce the level of Lp(a) in a subject with a high baseline level of Lp(a) by at least 40%. In some embodiments, the composition maintains levels of LDL (e.g., LDL-C), SBP, and / or Lp(a) within approximately 10-15% of baseline levels in subjects with clinically normal profiles (e.g., LDL-C <100 mg / dL, SBP <120 mmHg, Lp(a) <30 mg / dL), thereby preserving biomarker(s) stability and preventing progression into higher-risk thresholds.

[0042] In some embodiments, the composition provides long-lasting reduction of levels of LDL (LDL-C), SBP, and / or Lp(a) in a subject with high baseline levels of LDL (e.g., LDL-C), SBP, and / or Lp(a) (e.g., LDL-C >130 mg / dL, SBP >130 mmHg, or Lp(a) >50 mg / dL). In some embodiments, the composition provides long-lasting maintenance of LDL (e.g., LDL-C), SBP, and / or Lp(a) levels in a subject with clinically normal baseline levels of LDL (e.g., LDL-C), SBP, and / or Lp(a) (e.g., LDL-C <100 mg / dL, SBP <120 mmHg, Lp(a) <30 mg / dL).

[0043] Some aspects of the technology relate to a method of reducing levels of LDL, SBP, and / or Lp(a) in a subject, the method comprising administering to the subject the composition of any one of the preceding paragraphs.

[0044] Other aspects of the technology relate to a method of maintaining LDL, SBP, and / or Lp(a) levels in a subject, the method comprising administering to the subject the composition of any one of the preceding paragraphs.

[0045] Yet aspects of the technology relate to a method of treating cardiovascular disease in a subject, the method the comprising administering to the subject the composition of any one of the preceding paragraphs.

[0046] In some embodiments, the subject is human.

[0047] In some embodiments, the human subject is less than 40 years old.

[0048] In some embodiments, the human subject is more than 40 years old.

[0049] In some embodiments, the subject has a cardiovascular disease. In some embodiments, the subject is at risk of having a cardiovascular disease.

[0050] In some embodiments, the administration begins before the subject develops symptoms of the cardiovascular disease. In some embodiments, the administration begins before the subject has a cardiovascular event.

[0051] In some embodiments, the subject is administered the composition no more than once a year. In some embodiments, the subject is administered the composition no more than twice a year. In some embodiments, the subject is administered the composition once a year.

[0052] In some embodiments, the composition is administered to the subject for a period of time.

[0053] In some embodiments, the period of time is the remainder of the subject’s lifetime. In some embodiments, the method reduces levels of LDL, SBP, and / or Lp(a) in a subject with high baseline levels of LDL, SBP, and / or Lp(a), and / or maintains LDL, SBP, and / or Lp(a) levels in the subject. In some embodiments, the composition maintains LDL, SBP, and / or Lp(a) levels in a subject with normal baseline levels of LDL, SBP, and / or Lp(a).

[0054] In some embodiments, wherein the method provides long-lasting reduction of levels of LDL, SBP, and / or Lp(a) in a subject with high baseline levels of LDL, SBP, and / or Lp(a), and / or provides long-lasting maintenance of LDL, SBP, and / or Lp(a) levels in the subject. In some embodiments, the composition provides long-lasting maintenance of LDL, SBP, and / or Lp(a) levels in a subject with normal baseline levels of LDL, SBP, and / or Lp(a).

[0055] In some embodiments, the cardiovascular disease is atherosclerotic cardiovascular disease and / or hypertension.

[0056] In some embodiments, the method further comprises measuring the levels of LDL, SBP, and / or Lp(a) in the subject before and / or after administration of the composition.

[0057] Other aspects of 6the present disclosure provide compositions comprising two or more RNAi agents, wherein each of at least two of the RNAi agents targets a different gene selected from: proprotein convertase subtilisin / kexin type 9 (PCSK9), angiotensinogen (AGT), apolipoprotein A (LPA). In some embodiments, each of the two or more RNAi agents is an siRNA. In some embodiments, each of the two or more RNAi agents is a double- stranded siRNA. In some embodiments, each of the two or more RNAi agents has a length of 15-35 nucleosides. In some embodiments, each of the two or more RNAi agents is chemically modified. In some embodiments, each of the two or more RNAi agents comprises a 2’ -modified nucleoside. In some embodiments, each of the two or more RNAi agents comprises a 2’-O-methyl modified nucleoside or 2’-F modified nucleoside. In some embodiments, each of the two or more RNAi agents comprises at least one modified intemucleoside linkage. In some embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage. In some embodiments, each of the two or more RNAi agents comprises a biostable phosphate mimic. In some embodiments, the biostable phosphate mimic is a vinylpho sphonate moiety. In some embodiments, each of the two or more RNAi agents is conjugated to a targeting moiety. In some embodiments, two or more RNAi agents are covalently linked to each other, e.g., via a linker. Methods comprising administering to a subject any one of the compositions described herein are provided. In some embodiments, the subject has normal baseline levels of LDL, SBP, and / or Lp(a). In some embodiments, the subject has high baseline levels of LDL, SBP, and / or Lp(a). Methods of treating cardiovascular disease in a subject are also provided, the method comprising administering to the subject a composition described herein. In some embodiments, is older than 40 years old and / or has a cardiovascular disease or is at risk of developing a cardiovascular disease.

[0058] The preceding Summary is non-limiting.

[0059] BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Fig. 1 provides an exemplary experimental design to determine the cardiovascular effects of siRNA in hypertensive rats.

[0061] Figs. 2A-2B show relative mRNA expression following administration of siRNAs targeting PCSK9 or AGT, alone or in combination, to hypertensive rats. Valsartan, an angiotensin II receptor blocker, was used as a negative control. Fig. 2A shows relative expression of PCSK9 mRNA. Fig. 2B shows relative expression of AGT mRNA.

[0062] Figs. 3A-3B show relative protein expression following administration of siRNAs targeting PCSK9 or AGT, alone or in combination, to hypertensive rats. Valsartan, an angiotensin II receptor blocker, was used as a negative control. Fig. 3A shows relative expression of PCSK9 protein. Fig. 3B shows relative expression of AGT protein.

[0063] Fig. 4 shows relative cholesterol levels following administration of siRNAs targeting PCSK9 or AGT, alone or in combination, to hypertensive rats. Valsartan, an angiotensin II receptor blocker, was used as a negative control.

[0064] Fig. 5 shows relative change in mean arterial pressure, over time, following administration of siRNAs targeting PCSK9 or AGT, alone or in combination, to hypertensive rats. Valsartan, an angiotensin II receptor blocker, was used as a positive control.

[0065] DEFINITIONS

[0066] In order that the present disclosure may be more readily understood, certain terms are first defined. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also part of this disclosure.

[0067] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. “And” as used herein is interchangeably used with “or” unless expressly stated otherwise. The terms “comprising”, “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value recited or falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited.

[0068] The term “about” or “approximately,” as applied to one or more values provided herein, refers to a value that is similar to a stated reference value. In some embodiments, the term “about” or “approximately” refers to a range of values that fall within and include 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context. In some embodiments, “about” or “approximately” can be understood as about 2 standard deviations from the mean. In some embodiments, “about” or “approximately” means up to and including +10% (e.g., +10%, +9%, +8%, +7%, +6%, +5%, ±4%, +3%, +2%, +1%, or less). In some embodiments, “about” or “approximately” means +5%. When “about” or “approximately” is present before a series of numbers or a range, it is understood that it can modify each of the numbers in the series or range.

[0069] The term “administering” or “administration of,” as used herein, means to provide an agent, e.g., RNAi agent or conjugate to a subject. In some embodiments, “administering” or “administration of ” means to provide an RNAi agent or conjugate to a subject in a manner that is physiologically and / or (e.g., and) pharmacologically useful (e.g., to treat a condition in the subject). Non-limiting examples of routes of administration include intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, or intrathecal routes. In some embodiments, the route of administration is subcutaneous.

[0070] The term “antisense strand” or “guide strand,” as used herein, refers to a single stranded nucleic acid molecule, which is one strand of a double stranded RNAi molecule, and which comprises a region of complementarity to a target sequence (e.g., a target gene sequence, RNA sequence, or mRNA sequence). The antisense strand may contain modified nucleosides with base analogs and is not necessarily 100% complementary over its entire length to the target sequence, but must at least be sufficiently complementary to hybridize with a target RNA and result in RNA interference.

[0071] The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides or nucleosides in a nucleic acid molecule must be an integer. For example, “at least 19 nucleotides of a 21 -nucleotide nucleic acid molecule” means that 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

[0072] The term “biological activity” means any biological property of a molecule, whether present naturally in vivo, or provided or enabled by recombinant means. Biological activities include, but are not limited to, binding to a receptor, inducing cell proliferation, inhibiting cell growth, inducing other cytokines, inducing apoptosis, and modulating enzymatic activity.

[0073] “Blunt” or “blunt end” means that there are no unpaired nucleosides at that end of a double stranded RNAi agent, i.e., no overhang. A “blunt ended” double stranded RNAi agent is double stranded over its entire length, i.e., no overhang at either end of the molecule. The RNAi agents of the disclosure include RNAi agents with overhangs at both ends (i.e., agents with no blunt ends), no overhang at one end (i.e., agents with one overhang and one blunt end), or no overhangs at either end.

[0074] The term “complementary,” as used herein, refers to the capacity for base pairing between two nucleobases or two nucleobase sequences. In particular, complementary is a term that characterizes an extent of hydrogen bond pairing that brings about binding between two nucleobases or two nucleobase sequences. For example, if a base at one position of a nucleobase sequence (e.g., antisense strand of an RNAi agent) is capable of hydrogen bonding with a base at the corresponding position of another nucleobase sequence (e.g., RNAi agent sense strand or target mRNA), then the bases are considered to be complementary to each other at that position. The nucleic acid molecules (e.g., antisense strand and sense strand of an RNAi agent) whose nucleobase sequences are complementary may comprise one or more modified nucleosides and modified internucleoside linkages, which do not affect the capacity of base paring between the nucleobases and do not affect the “complementarity” between two nucleobase sequences. The nucleic acid molecules (e.g., antisense strand and sense strand of an RNAi agent) whose nucleobase sequences are complementary may also comprise nucleobase analogous that result in bases at certain positions not being complementary, but the nucleobase sequences of the two molecules must be sufficiently complementary over the entire length to result in a desired biological activity (e.g., RNA interference).

[0075] Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs (e.g., Wobble base pairs and Hoogsteen base pairs) and may include natural or modified nucleosides or nucleoside mimics. For example, in some embodiments, for complementary base pairings, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guano sine-type bases (G), and that universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.

[0076] Complementarity is independent of modifications in the sugar of a nucleoside. For example, 2 ’-modified A, as defined herein, are complementary to U (or T) and identical to A for the purposes of determining identity or complementarity.

[0077] The term “perfectly complementary” or “fully complementary” means that all (100%) of the nucleobases, nucleosides, or nucleotides in a contiguous sequence of a first nucleotide sequence (e.g., antisense strand of an RNAi agent) will hybridize with the same number of nucleobases, nucleosides, or nucleotides in a contiguous sequence of second nucleotide sequence (e.g., RNAi agent sense strand or target mRNA). The contiguous sequence may comprise all or a part of a first or second nucleotide sequence. The term “partially complementary” means that in a hybridized pair of nucleobase, nucleosides, or nucleotide sequences, at least 70%, but not all, of the bases in a contiguous sequence of a first nucleotide sequence (e.g., antisense strand of an RNAi agent) will hybridize with the same number of bases in a contiguous sequence of a second nucleotide sequence (e.g., RNAi agent sense strand or target mRNA). The term “sufficiently complementary” or “substantially complementary” means that in a hybridized pair of nucleobase, nucleosides, or nucleotide sequences, at least 85%, but not all, of the bases in a contiguous sequence of a first nucleotide sequence (e.g., antisense strand of an RNAi agent) will hybridize with the same number of bases in a contiguous sequence of a second nucleotide sequence (e.g., RNAi agent sense strand or target mRNA). The terms “complementary,” “fully complementary,” “partially complementary,” and “sufficiently / substantially complementary” herein are used with respect to the nucleobase, nucleosides, or nucleotide matching between the sense strand and the antisense strand of an RNAi agent, or between the antisense strand of an RNAi agent and a target mRNA sequence (e.g., PCSK9, AGT, or LPA mRNA). The term “conjugate,” as used herein, refers to an RNAi agent described herein (e.g., an RNAi agent targeting PCSK9, AGT, or LPA RNA, such as mRNA) linked (e.g., covalently linked) to another molecule such as another RNAi agent or a conjugate group (e.g., a targeting moiety). In general, conjugate groups modify one or more properties of the RNAi agent to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and / or clearance properties.

[0078] The term “contiguous” in the context of an oligonucleotide (e.g., RNAi agent) refers to nucleosides, nucleobases, sugar moieties, or intemucleoside linkages that are immediately adjacent to each other. For example, “contiguous nucleobases” means nucleobases that are immediately adjacent to each other in a sequence.

[0079] The term “control” or “reference,” when referring to a substance, means a composition used as a standard or a point of comparison against which other test results are measured. In some embodiments, a “control” or “reference” is a composition known to not contain analyte (“negative control”) or to contain analyte (“positive control”). A positive control can comprise a known concentration of analyte. “Control,” and “positive control,” may be used to refer to a composition comprising a known concentration of analyte. A “positive control” can be used to establish assay performance characteristics and is a useful indicator of the integrity of reagents (e.g., analytes). In some embodiments, an appropriate “control” or “reference” is where only one element is changed in order to determine the effect of the one element. In some embodiments, a control is a level of a target gene (e.g., in a cell or in a subject) before treatment (e.g., with an RNAi agent described herein).

[0080] The term “control” or “reference” also means a baseline level of a measurement depending upon the context, in which the term is used. A baseline level of a measurement is a standard or a point of comparison against which the measurement is compared. In some embodiments, a “control” or a “reference” refers to a level of a measurement for certain biological activity or substance in a cell, a tissue, an organ, or a subject, e.g., the expression level of a gene, copy number of mRNA for such gene, or level of protein encoded by such gene, without treatment of the cell, the tissue, the organ, or the subject, with an agent, e.g., an RNAi agent. In some embodiments, a “control” or a “reference” refers to a level of an average measurement for a certain biological activity or substance in a cell, a tissue, an organ, or a subject, e.g., certain enzyme activity, among a group of healthy subjects, e.g., the general population within in certain geographic or demographic limits or any other limits that may be appropriate for the study of certain disease or disorder, that does not have certain disease or disorder, e.g., cardiovascular disease.

[0081] The term “reference” may also be used in “reference sequence.” The term “reference sequence” refers to a sequence, e.g., a nucleic acid sequence or an amino acid sequence, used as a basis for sequence comparison. In certain embodiments, a reference sequence is the mRNA sequence, e.g., human PCSK9 (NM_174936.4, SEQ ID NO: 2), AGT (NM_001382817.3, SEQ ID NO: 20), or LPA (NM_005577.4, SEQ ID NO: 23) mRNA sequence, upon which the design of the siRNA is based.

[0082] The term “cross-reactive” means the ability of a binding molecule (e.g., an RNAi agent) to bind a target molecule (e.g., an mRNA) other than that against which it was designed or generated. For example, the binding molecule is capable of specifically binding to more than one target molecule of a similar type or class (e.g., mRNA variants or mRNA homologous from closely related species) with similar affinity. Generally, a binding molecule will bind its target molecule with an appropriately high affinity but can bind to the same target molecule of another species or display a low affinity for non-target molecules. In some embodiments, an RNAi agent that is cross-reactive against human and non-human primate PCSK9, AGT, or LPA comprises an antisense strand comprising a region of complementarity to human and non-human primate PCSK9, AGT, or LPA mRNA, respectively, and / or inhibits the expression of human and non-human primate PCSK9, AGT, or LPA. Methods of assessing cross-reactivity are known to one skilled in the art.

[0083] The term “effective amount” or “therapeutically effective amount,” as used herein, refers to that amount of an RNAi agent to produce a molecular (e.g., reduced expression of PCSK9, AGT, or LPA), biological (e.g., reduced SBP and cholesterol), pharmacological, therapeutic (e.g., treatment of a cardiovascular associated disease), or preventive result. The amount administered will likely depend on such variables as the overall health status of the patient, the relative biological efficacy of the compound delivered, the formulation of the drug, the presence and types of excipients in the formulation, and the route of administration. Also, it is to be understood that the initial dosage administered can, in some instances, be increased beyond the above upper level to rapidly achieve the desired blood-level or tissue level, or the initial dosage can, in some instances, be smaller than the optimum.

[0084] The term “GalNAc” refers to N-Acetylgalactosamine (GalNAc), which is a monosaccharide and amino sugar derivative of galactose. GalNAc may also be referred to in the art as 2-(Acetylaniino)-2-deoxy-D-galactopyranose, 2-(Acetylamino)-2-deoxy-D- galactose, N-Acetylchondrosamine, and N-Acetyl-D-galactosamine. Galactose derivatives such as GalNAc have been used to target molecules to hepatocytes in vivo through their binding to the asialoglycoprotein receptor expressed on the surface of hepatocytes. Binding of asialoglycoprotein receptor ligands to the asialoglycoprotein receptor(s) facilitates cell¬ specific targeting to target cells (e.g., hepatocytes) and endocytosis of the molecule into the target cells (e.g., hepatocytes). In some embodiments, any one of the targeting moieties described herein includes an asialoglycoprotein receptor ligand comprising GalNAc. In some embodiments, the asialoglycoprotein receptor ligand comprises a GalNAc trimer.

[0085] Asialoglycoprotein receptor ligands can be monomeric (e.g., having a single GalNAc) or multimeric (e.g., having multiple GalNAcs). The targeting moiety may comprise one or more GalNAcs attached to the 3’ or 5’ end of the sense or antisense strand of the RNAi agent using methods known in the art. In some embodiments, the targeting moiety comprises one or more (e.g., 1, 2, 3, 4, or more) GalNAc, each of which are linked via phosphoro thioate linkages. GalNAc targeting moieties, which comprise one or more GalNAc, have been described, for example, in the following references:

[0086]

[0087] The disclosures in these references related to GalNAc are hereby incorporated herein by reference.

[0088] The terms “hybridize” and “hybridization” refer to the pairing of complementary compounds (e.g., an RNAi agent and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Wobble, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.

[0089] The term “intemucleoside linkage,” as used herein, means a covalent linkage between adjacent nucleosides in an oligonucleotide (e.g., RNAi agent such as a PCSK9, AGT, or LPA RNAi agent described herein). An intemucleoside linkage may be a natural phosphodiester intemucleoside linkage or may be a modified (non-natural) intemucleoside linkage.

[0090] Modified intemucleoside that may be used in an RNAi agent disclosed herein include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3 ’alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 ’-amino phosphoramidate and aminoalkylphosphoramidates, mesyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3’-5’ linkages, 2’-5’ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’; see US patent nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5, 177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799; 5,587,361; and 5,625,050.

[0091] The term, “nucleoside,” as used herein, refers to a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety. The term “nucleoside” encompasses a natural nucleoside and chemically modified nucleosides (e.g., with modifications in the base and / or sugar moiety). As used herein, when referring to the lengths of an oligonucleotide (e.g., the sense or antisense strand of an RNAi agent such as an siRNA) by the number of nucleosides, it is to be understood that such nucleosides (modified or unmodified) linked by intemucleoside linkages (natural or modified) in the oligonucleotide.

[0092] The term “nucleotide,” as used herein, refers to a compound comprising a nucleoside linked to a phosphate group. As used herein, “linked nucleosides” may or may not be linked by phosphate linkages and thus includes but is not limited to “linked nucleotides.” As used herein, “linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e., no additional nucleosides are present between those that are linked). The term “nucleotide” encompasses a natural nucleotide and chemically modified nucleotides (e.g., with modifications in the base, sugar moiety, and / or phosphate group).

[0093] The term “nucleobase,” as used herein, refers to nitrogen-containing compounds that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the compound is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified. As used herein a “naturally occurring nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). The term “nucleobase” encompasses 5 ’-methylated bases (e.g., 5 ’-methyl cytosine or 5 ’-methyl guanine).

[0094] As used herein, a “nucleobase sequence” of an oligonucleotide (e.g., the sense or antisense strand of an RNAi agent such as an siRNA) refers to the sequence of the oligonucleotide represented by the nucleobases (e.g., A, U, T, G, C). It is to be understood that the oligonucleotide comprises nucleosides which include the nucleobases (natural or modified) and sugars (natural or modified), and such nucleosides are linked by intemucleoside linkages (natural or modified) in the oligonucleotide.

[0095] The term “modified internucleoside linkage” refers to a linkage between two nucleosides (e.g., in an oligonucleotide or in a strand of an RNAi agent) that is not the natural phosphodiester linkage. Non-limiting examples of modified intemucleoside linkages include phosphorothioates, phosphorodiamidates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.

[0096] The term “nucleoside modification” or “modified nucleoside” means a nucleoside that has one or more modifications to the nucleoside, including modifications to the nucleobase moiety and / or the sugar moiety. Any of the modified chemistries or formats of nucleosides described herein can be combined with each other. Non-limiting examples of modified nucleosides includes 2’-fluoro (2’-F), 2’-O-methyl (2’-0-Me), 2’-0-methoxyethyl (2’-M0E), 2’-0-aminopropyl (2’-O-AP), 2’-0-dimethylaminoethyl (2’-0-DMA0E), 2’-O-dimethylaminopropyl (2’-0-DMAP), 2’-0-dimethylaminoethyloxyethyl (2’-0-DMAE0E), or 2’-0-N-methylacetamido (2’-0-NMA), locked nucleic acid (LNA, methylene-bridged nucleic acid), unlocked nucleic acid (UNA), ethylene-bridged nucleic acid (ENA), and (S)-constrained ethyl-bridged nucleic acid (cEt) modified nucleosides. Further non-limiting examples of modified nucleosides include a conformationally restricted nucleoside, an abasic nucleoside, a 2’-amino-modified nucleoside, a morpholino nucleoside, a phosphoramidate, a non-natural base comprising nucleoside, a tetrahydropyran modified nucleoside, a 1,5-anhydrohexitol modified nucleoside (HNA), a cyclohexenyl modified nucleoside (CeNA), a nucleoside comprising a phosphorothioate group, a nucleoside comprising a methylphosphonate group, a nucleoside comprising a 5 ’-phosphate, a nucleoside comprising a 5 ’-phosphate mimic, a thermally destabilizing nucleoside, a glycol modified nucleoside (GNA).

[0097] The term “2’ -modified nucleoside” refers to a nucleoside having a sugar moiety modified at the 2’ position, meaning the sugar moiety comprises at least one 2’ -substituent group other than H or OH. Non-limiting examples of 2’ -modified nucleosides include: 2’-fluoro (2’-F), 2’-O-methyl (2’-0-Me), 2’-0-methoxyethyl (2’-M0E), 2’-deoxy, 2’-O-aminopropyl (2’-O-AP), 2’-0-dimethylaminoethyl (2’-0-DMA0E), 2’-O-dimethylaminopropyl (2’-0-DMAP), 2’-0-dimethylaminoethyloxyethyl (2’-0-DMAE0E), or 2’-0-N-methylacetamido (2’-0-NMA) modified nucleosides. In some embodiments, any one of the 2’-modified nucleosides described herein are high-affinity modified nucleosides and a modified RNAi agent has increased affinity to target sequences, relative to an unmodified RNAi agent. In some embodiments, at least one modified nucleoside is a 2’ modified nucleoside. In some embodiments the 2’ modified nucleoside is a 2’-O-methyl (2’-O-Me) modified nucleoside or a 2’-fluoro (2’-F) modified nucleoside or combinations thereof.

[0098] The term “modified oligonucleotide” or “modified RNAi agent” or “modified siRNA” refers to oligonucleotides, modified RNAi agents, or modified siRNAs that comprise one or more modified nucleosides and / or one or more modified internucleoside linkages. In some embodiments, a “modified oligonucleotide” or “modified RNAi agent” or “modified siRNA” comprises a mix of modified nucleosides and unmodified nucleosides and / or a mix of modified intemucleoside linkages and unmodified modified intemucleoside linkages. In some embodiments, each nucleoside of a modified oligonucleotide” or “modified RNAi agent” or “modified siRNA” is a modified nucleoside, and / or each intemucleoside linkage of a modified oligonucleotide” or “modified RNAi agent” or “modified siRNA” is a modified intemucleoside linkage.

[0099] As used herein, the term “overhang” refers to at least one unpaired nucleoside that protrudes from the duplex structure of an RNAi agent. One skilled in the art would understand that the unpaired nucleosides in the overhang are linked via intemucleoside linkages, and are linked to the adjacent nucleoside in the duplex region via an intemucleoside linkage. For example, when a 3 ’-end of one strand of an RNAi agent extends beyond the 5’-end of the other strand, or vice versa, there is an overhang. An RNAi agent can comprise an overhang of at least one nucleoside, at least two nucleosides, at least three nucleosides, at least four nucleosides, or more. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleoside(s) of an overhang can be present on the 5 ’-end, 3 ’-end, or both ends of either an antisense or sense strand of an RNAi agent.

[0100] In some embodiments, at least one strand of an RNAi agent comprises a 3’ overhang of at least 1 nucleoside. In some embodiments, at least one strand comprises a 3’ overhang of at least 2 nucleosides, e.g., 2, 3, 4, 5, etc. nucleosides. In some embodiments, at least one strand of an RNAi agent comprises a 5’ overhang of at least 1 nucleoside. In some embodiments, at least one strand comprises a 5’ overhang of at least 2 nucleosides, e.g., 2, 3, 4, 5, etc. nucleosides. In some embodiments, both the 3’ and the 5’ end of one strand of an RNAi agent comprises an overhang of at least 1 nucleoside.

[0101] In some embodiments, the antisense strand of an RNAi agent has a 1-10 nucleoside, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleoside, overhang at the 3’-end or the 5’-end. In some embodiments, the sense strand of an RNAi agent has a 1-10 nucleoside, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleoside, overhang at the 3’-end or the 5’-end. In another embodiment, one or more of the nucleosides in the overhang is replaced with a nucleoside thiophosphate.

[0102] In some embodiments, the antisense strand of an RNAi agent has a 1-10 nucleosides, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleoside, overhang at the 3’-end or the 5’-end. In some embodiments, the antisense strand of an RNAi agent has a 1-3 nucleosides, e.g., a 1, 2, or 3 nucleoside, overhang at the 3 ’-end.

[0103] In some embodiments, the nucleosides in an overhang that is immediately adjacent to the duplex region is linked to the adjacent nucleoside in the duplex region via a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. In some embodiments, the overhang comprises two or more nucleosides and the nucleosides in an overhang are linked via a phosphorothioate intemucleoside linkage or a phosphodiester intemucleoside linkage.

[0104] The term “passenger strand” or “sense strand,” as used herein, refers to a single stranded nucleic acid molecule which is one strand of a double stranded RNAi molecule, and which has a sequence that is at least substantially complementary (e.g., at least 85% complementary) to that of the guide strand / antisense strand. The sense strand need not be fully complementary over the entire length of the antisense strand but must at least be sufficiently complementary to hybridize with the antisense strand and result in RNA interference.

[0105] The term “region of complementarity,” as used herein, refers to a nucleobase sequence, e.g., of an RNAi agent, that is sufficiently complementary to a cognate nucleobase sequence, e.g., of a target nucleic acid, such that the two nucleobase sequences are capable of annealing to one another under physiological conditions (e.g., in a cell). In some embodiments, a region of complementarity is fully complementary to a cognate nucleobase sequence of target nucleic acid. However, in some embodiments, a region of complementarity is partially complementary to a cognate nucleobase sequence of target nucleic acid (e.g., at least 80%, 90%, 95% or 99% complementarity). In some embodiments, a region of complementarity contains 1, 2, 3, 4, or 5 mismatches compared with a cognate nucleobase sequence of a target nucleic acid.

[0106] The term “RNAi agent,” or “RNA interference agent” means a composition that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner. As used herein, RNAi agents may operate through the RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells), or by any alternative mechanism(s) or pathway(s). While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interference mechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action. An RNAi agent modulates, e.g., inhibits, the expression of a PCSK9, AGT, or LPA in a cell, e.g., a cell within a subject, such as a mammalian subject. RNAi agents include, but are not limited to: single- stranded oligonucleotides, single- stranded antisense oligonucleotides, short interfering RNAs (siRNAs), double-strand RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates. Any one of the RNAi agents described herein comprises a strand that is at least partially complementary to the mRNA being targeted. In some embodiments, an RNAi agent is single stranded (e.g., it can be an antisense oligonucleotide). In some embodiments, an RNAi agent is double stranded. In some embodiments, the double stranded RNAi agent is a double stranded siRNA.

[0107] In some embodiments, an RNAi agent described herein is double-stranded, and comprises an antisense strand and a sense strand, wherein the antisense strand is at least partially complementary to the mRNA being targeted (e.g., PCSK9, AGT, or LPA mRNA), and the sense strand is at least partially complementary to the antisense strand. It is not necessary that there be perfect complementarity between the RNAi agent and the target, but the correspondence is preferably sufficient to enable the RNAi agent to direct sequence specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., PCSK9, AGT, or LPA mRNA. An RNAi agent described herein may comprise one or more modified nucleosides and / or one or more modified (e.g., non-phosphodiester) intemucleoside linkages.

[0108] Modification to stabilize one or more 3’- or 5 ’-terminus of an RNAi agent, e.g., against exonucleases may also be present in an RNAi agent described herein. Other modifications can include C3 (or C6, C7, Cl 2) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis.

[0109] Modifications can also include, e.g., the use of modifications at the 2’ OH group of the ribose sugar, e.g., the use of deoxyribonucleosides, e.g., deoxythymidine, instead of ribonucleosides, and modifications in the intemucleoside linkages, e.g., phosphothioate intemucleoside linkages. In some embodiments, the different strands will include different modifications. In some embodiments, an RNAi agent of the disclosure includes a short interfering RNA (siRNA) that interacts with a target RNA sequence, e.g., a PCSK9, AGT, or LPA target sequence, to direct the cleavage of the target RNA. In some embodiments, an RNAi agent described herein is a small interfering RNA (siRNA).

[0110] The term “siRNA,” as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary (e.g., at least 85% complementary) nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target sequence, i.e., a PCSK9, AGT, or LPA sequence. Each strand of the siRNA may optionally and independently comprise ribonucleosides (RNA), RNA analog(s) (e.g., chemically modified ribonucleosides), and / or deoxyribonucleosides (DNA). In some embodiments, each strand of an siRNA comprises between 15 and 35 nucleosides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleosides). In some embodiments, each strand of an siRNA comprises between 18 and 28 nucleosides (e.g., 18-28, 19-25, 19-23, 19-21). In some embodiments, each strand of an siRNA is 19, 20, 21, 22, or 23 nucleosides in length. In some embodiments of the disclosure, an siRNA induces the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

[0111] In some embodiments, any one of the RNAi agents (e.g., siRNAs) disclosed herein comprises a duplex region of 10-30 base pairs in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length). In some embodiments, an RNAi agent of the present disclosure is blunt ended. In some embodiments, an RNAi agent of the present disclosure has overhangs on one or both strands. The overhang may include 1-10 (e.g., 1-10, 1-8, 1-5, 1-3, 1-2) nucleosides, such that the duplex region in the RNAi agent comprises 17-21 nucleosides, or 19 nucleosides. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. Without wishing to be bound by theory, it is believed that long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3’ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0112] In some embodiments, an RNAi agent described herein is a single stranded RNA generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., PCSK9, AGT, or LPA gene. In some embodiments, an RNAi agent may be a single- stranded RNA (ssRNAi) that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single- stranded RNAi agents are generally 15-30 nucleosides and may be chemically modified. The design and testing of single- stranded RNAi agents are described in U.S. Patent No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference.

[0113] The term “vinylphosphonate” or “5’-vinylphosphonate” in the context of a 5’-phosphate of a nucleotide refers to a structure in which the bridging 5 '-oxygen atom is replaced with a methylidene ( — CH=) group (e.g., as illustrated below): << "

[0114]

[0115] < <

[0116] b- <

[0117] A terminal 5’-vinylphosphonate nucleotide is a nucleotide in which the natural phosphate group at the 5’ end has been replaced with a vinylphosphonate. A 5’-vinylphosphonate is a 5’ biological mimic of a phosphate that is biostable (also referred to herein as a “biostable phosphate mimic.” A biological mimic is a molecule that is capable of carrying out the same function as and is structurally very similar to the original molecule that is being mimicked. In the context of the present disclosure, 5 ’-vinylphosphonate mimics the function of a normal 5’ phosphate, e.g., enabling efficient RISC loading, while being capable of stabilizing the 5’ end nucleotide by protecting it from dephosphorylation by enzymes such as phosphatases. RNAi agents (e.g., siRNAs) comprising a vinylphosphonate moiety or a 5’-vinylphosphonate nucleotide has been described, e.g., in US Patent No. 11560563 and US Patent No. 10233448.

[0118] Other biostable phosphate mimics may also be used in RNAi agents described herein. Non-limiting examples of other biostable phosphate mimics are described in, e.g., US Patent No. 8927513, US Patent No. 11078227, US Patent Application Publication No.

[0119] US20190177729m and Varley et al., RSC Adv., 2021, 11, 2415-2426, each of which is incorporated herein by reference in their entirety. In some embodiments, other biostable phosphate mimics that may be used in an RNAi agent described herein (e.g., at the 5’ end of the antisense strand) may be a 5 ’-cyclopropyl phosphonate, or an oxymethylphosphonate (e.g., where the oxygen atom of the oxymethyl group is bound to the 4’-carbon of the sugar moiety or analog thereof). In some embodiments, in an RNAi agent described herein, the 5 ’-terminal nucleotide of the antisense strand is a 5’-vinylphosphonate 2’-0-Me-U. In some embodiments, in an RNAi agent described herein, the 5 ’-terminal nucleotide of the antisense strand is a 5’-cyclopropyl phosphonate 2’-0-Me-U. In some embodiments, in an RNAi agent described herein, the 5’-terminal nucleotide of the antisense strand is an oxymethylphosphonate 2’-O-Me-U, wherein the oxygen atom of the oxymethyl group is bound to the 4’ -carbon of the sugar moiety or analog thereof.

[0120] The term “sequence identity,” as used herein, refers to the extent that sequences are identical (independent of chemical modification) on a nucleobase-by- nucleobase basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0121] The terms “silence,” “reduce,” “inhibit,” “down-regulate,” or “knockdown” when referring to expression of a given gene (e.g., PCSK9, AGT, or LPA), mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with an RNAi agent or conjugate described herein as compared to a control or reference cell, group of cells, tissue, organ or a subject, e.g., a second cell, group of cells, tissue, organ, or subject that has not or have not been so treated. In some embodiments, when a cell, group of cells, tissue, organ, or subject is treated with an RNAi agent or conjugate described herein, expression of a target gene (e.g., PCSK9, AGT, or LPA) is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% relative to a control, e.g., baseline level of gene expression prior to treatment.

[0122] The term “subject,” as used herein, refers to a mammal. In some embodiments, a subject is non-human primate, or rodent. In some embodiments, a subject is a human. In some embodiments, a subject is a patient, e.g., a human patient that has or is suspected of having a disease. In some embodiments, the subject is a human patient who has or is suspected of having a cardiovascular disease or is at risk of developing cardiovascular disease in the future (e.g., atherosclerotic cardiovascular disease, heart attack, stroke, or hypertension.

[0123] Reference is made herein to “a subject with high baseline levels of LDL, SBP, and / or Lp(a).” A “baseline level” of a biomarker (such as LDL, SBP, and / or Lp(a)) refers to the level of the biomarker or clinical measure at the time of administration of a composition of the disclosure (e.g., RNAi agents targeting PCSK9, AGT, and / or LPA) — that is, before a subject is initially treated with, or before a subject is treated with a maintenance dose of, an RNAi agent intended to lower the expression and / or function of PCSK9, AGT, and / or LPA). A “high baseline level” of a biomarker is any level above a normal level, which is typically determined by a healthcare provider (e.g., a primary care physician, cardiologist, or lipid specialist) using guidelines established by national and international health organizations, for example. Such organizations include American College of Cardiology (ACC), American Heart Association (AHA), National Cholesterol Education Program (NCEP), U.S. Preventive Services Task Force (USPSTF), and European Society of Cardiology (ESC). For example, “normal” with respect to cholesterol levels, can be individualized based on a patient's overall cardiovascular risk, which can include factors such as age, family history, diabetes, smoking, and blood pressure. A clinician uses these factors — often via risk calculators like the ASCVD Risk Estimator — to determine target cholesterol levels and the need for treatment. In some embodiments, a composition reduces levels of LDL, SBP, and / or Lp(a) to, or maintains levels of LDL, SBP, and / or Lp(a) at normal levels. A normal level of LDL, in some embodiments, is less than 100 mg / dL. A normal level of SBP, in some embodiments, is less than 100 mm Hg. A normal level of Lp(a), in some embodiments, is less than 30 mg / dL.

[0124] The term “specificity” means the ability to inhibit the target RNA without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS).

[0125] The term “symptom” as used herein, refers to any manifestation or indication of an underlying disease. A symptom can be any biochemical, cellular, genetic, histological, and / or physiological observation, measurement, and / or test result in a subject that deviate from those of a control or reference. For example, a symptom may be an elevated level of an enzyme as compared to a normal reference range.

[0126] The term “target sequence,” as used herein, refers to a nucleotide sequence whose expression or activity is to be modulated. In some embodiments, the target sequence is a contiguous portion of the nucleotide sequence of a gene, a cDNA, or an mRNA molecule formed during the transcription of a target gene, e.g., PCSK9, AGT, or LPA gene, including an unprocessed pre-mRNA transcript and mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the target gene, e.g., PCSK9, AGT, or LPA gene. In some embodiments, the target sequence is within the protein coding region of the target gene, e.g., PCSK9, AGT, or LPA.

[0127] As used herein, the term “cardiovascular disease” refers to disorders caused by, or associated with, PCSK9, AGT, and / or LPA expression and / or activity, and / or cumulative exposure to cholesterol and high blood pressure, including those that would benefit from a reduction in PCSK9, AGT, and / or LPA gene expression, replication, or protein activity. In some embodiments, a subject having a cardiovascular disease may benefit from the reduction in expression of PCSK9, AGT, and / or LPA. Cardiovascular diseases typically affect the heart and / or blood vessels. In some embodiments, a cardiovascular disease is atherosclerotic cardiovascular disease. In some embodiments, a cardiovascular disease is hypertension.

[0128] Other non-limiting examples of cardiovascular diseases include coronary artery diseases, cerebrovascular disease, peripheral artery disease, and hypertension. A “cardiovascular event” refers to any incident that disrupts normal heart function. While a cardiovascular event may indicate or result from cardiovascular disease, it should be understood that the term “cardiovascular disease,” as used herein, encompasses “cardiovascular events”. Non-limiting examples of cardiovascular events include myocardial infarction (heart attack), cardiac arrest, stroke, angina, heart failure, and arrhythmia. In some embodiments, a cardiovascular disease is a cardiovascular event. In some embodiments, a cardiovascular event is a heart attack. The terms “treatment” or “treat,” as used herein, refer to any action taken (e.g., administration of an RNAi agent to a subject) to prevent, alleviate, or otherwise manage a symptom of a cardiovascular disease (including a cardiovascular event). “Treatment” includes delaying the onset of a cardiovascular disease as well as delaying the progression of a cardiovascular disease, for example, in a subject who has a cardiovascular disease or who is at risk but does not yet exhibit symptoms of a cardiovascular disease. As an example, statins, such as atorvastatin or simvastatin, are lipid-lowering agents that reduce levels of low-density lipoprotein cholesterol in the blood. By inhibiting HMG-CoA reductase, a key enzyme in cholesterol biosynthesis, statins decrease the accumulation of cholesterol in arterial walls, thereby slowing the development of atherosclerotic plaques. In individuals at high risk for cardiovascular events — such as those with diabetes, hypertension, or a family history of heart disease — statin therapy has been shown to delay the onset of cardiovascular diseases and cardiovascular events, including coronary artery disease and myocardial infarction. Similarly, ACE inhibitors, such as enalapril or lisinopril, block the conversion of angiotensin I to angiotensin II, a potent vasoconstrictor. This leads to reduced blood pressure, decreased afterload, and diminished strain on the heart. In patients with heart failure with reduced ejection fraction (HFrEF), ACE inhibitors have been shown to delay the progression of disease by preventing or slowing left ventricular remodeling, reducing hospitalizations, and prolonging survival. Thus, in some embodiments, treatment of a subject (e.g., at risk of a cardiovascular disease or a cardiovascular event) includes administration (e.g., once-yearly administration) of a composition comprising two or more RNAi agents selected from an RNAi agent that targets PCKS9, an RNAi agent that targets AGT, and an RNAi agent that targets LPA, resulting in the delayed onset of a cardiovascular disease. In some embodiments, administration of the composition results in avoidance of cardiovascular disease altogether. In other embodiments, treatment of a subject (e.g., a subject already diagnosed with a cardiac disease) includes administration (e.g., once-yearly administration) of a composition comprising two or more RNAi agents selected from an RNAi agent that targets PCKS9, an RNAi agent that targets AGT, and an RNAi agent that targets LPA, resulting in the delayed progression of a cardiovascular disease. Such delayed progression could add years and / or improve quality of life in the subject, for example.

[0129] The term “variant” means a molecule (e.g., nucleic acid or polypeptide) that differs from a given molecule (e.g., a reference nucleic acid or polypeptide) in sequence (nucleic acid or amino acid respectively) by the addition (e.g., insertion), deletion, or conservative substitution of nucleic acids or amino acids, respectively, and / or in chemical modifications (e.g., to nucleosides, internucleoside linkages, terminal modifications, amino acids, and / or targeting ligands), but that retains the biological activity of the given molecule. Nucleic acid variants are closely related overall and, in many regions, identical. Changes in the reference nucleic acid sequence of the variant may be silent. That is, they may not alter the amino acid sequence encoded by the nucleic acid. Alternatively, changes in the nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleoside changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. The term “variant” encompasses fragments of a variant unless otherwise defined. A variant may be 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, or 75% identical to the reference sequence. The degree of homology (percent identity) between a native and a variant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g., BLASTn with default settings).

[0130] As used herein, the term “salt” refers to any and all salts, and encompasses pharmaceutically acceptable salts.

[0131] The term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and / or animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.

[0132] Pharmaceutically acceptable salts of the compounds of this disclosure include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N+(CI-4 alkyl)4~ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0133] It is also to be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers”. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”.

[0134] Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”. When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture”.

[0135] The term “proprotein convertase subtilisin / kexin type 9 (PCSK9)” is used interchangeably in the art or herein with the term “FH3,” “PC9,” “FHCL3,” “NARC1,” “LDLCQ1,” “NARC-1,” and “HCHOLA3.” PCSK9 binds to low density lipoprotein (“LDL”) receptors, reducing their expression, and thereby regulates cholesterol metabolism. Inhibition of PCSK9 can therefore help lower cholesterol levels, reducing the risk of cardiovascular-related events. Consistent with this, gain-of-function mutations in PCSK9 reduce LDL receptor levels in the liver, resulting in high levels of LDL cholesterol in the plasma and increased susceptibility to coronary heart disease. Conversely, loss-of-function mutations lead to higher levels of the LDL receptor, lower LDL cholesterol levels, and protection from coronary heart disease. Consequently, there is considerable interest in developing antibody therapeutics to inhibit PCSK9-LDL receptor interactions (Peterson et al., (2008) J Lipid Res 49: 1152-1156, the entire contents of which are hereby incorporated by reference).

[0136] The term “PCSK9” includes human (Homo sapiens) PCSK9, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI:

[0137] 255738, NCBI Accession Nos. NP_777596.2 and NM_174936.4, NP_ 001394169.1 and NM_001407240.1, NP_001394170.1 and NM_001407241.1, NP_001394171.1 and NM_001407242.1, NP_001394172.1 and NM_001407243.1, NP_001394173.1 and NM_001407244.1, NP_001394174.1 and NM_001407245.1, NP_001394175.1 and NM_001407246.1, and NP_001394176.1 and NM_001407247.1, UniProt ID: Q8NBP7.3; mouse (Mus musculus) PCSK9, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 100102, NCBI Accession Nos.

[0138] NP_705793.1 and NM_153565.2, UniProt ID: Q80W65.2; rat (Rattus norvegicus) PCSK9, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 298296, NCBI Accession Nos. NP_954862.2 and NM_199253.2, UniProt ID: P59996.1; and chimpanzee (Pan troglodytes') PCSK9, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 456880, NCBI Accession Nos. NP_001104592.1 and NM_001111122.1, UniProt ID: A8T644.1. The term “PCSK9” also includes rhesus monkey (Macaca mulatto) AGT, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI:

[0139] 717147, NCBI Accession Nos. NP_001106130.1 and NM_001112660.1, UniProt ID:

[0140] A8T666.1. Additional examples of PCSK9 mRNA sequences are readily available using, e.g., GenBank, UniProt, OMIM, and the Macaca genome project web site. Exemplary PCSK9 nucleotide and amino acid sequences may also be found in Table 1, SEQ ID NOs: 1-18.

[0141] The term “PCSK9,” as used herein, also refers to naturally occurring DNA sequence variations of the PCSK9 gene. Numerous sequence variations within the PCSK9 gene have been identified and may be found at, for example, NCBI dbSNP and UniProt (see, e.g., ncbi.nlm.nih.gov / snp).

[0142] Further information on PCSK9 can be found, for example, at ncbi.nlm.nih.gov / gene / 255738. The entire contents of each of the foregoing GenBank Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of filing this application.

[0143] Table 1 below summarizes exemplary amino acid sequences of PCSK9 proteins and nucleic acid (e.g., DNA or mRNA) sequence of PCSK9 genes of humans. able 1: Exemplary Amino Acid and Nucleic Acid Sequences of PCSK9.

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] The term “angiotensinogen (AGT)” is used interchangeably in the art or herein with the term “ANHU,” “hFLTl,” and “SERPINA8.” The protein encoded by the AGT gene, pre- angiotensinogen, is cleaved by renin, in response to a drop in blood pressure, producing angiotensin I. Angiotensin I is subsequently cleaved by angiotensin converting enzyme (“ACE”) to generate angiotensin II, which acts on its cognate receptors to induce vasoconstriction, stimulate production of aldosterone, thereby promoting sodium and water retention, and increase glomerular filtration rate and sodium reabsorption (Cruz-Lopez et al., (2022) Hypertension 79:2115-2126, the entire contents of which are hereby incorporated by reference). Collectively, these events increase blood pressure and regulate fluid balance.

[0158] The term “AGT” includes human (Homo sapiens) AGT, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 183, NCBI Accession Nos. NP_001369746.2 and NM_001382817.3, and NP_001371408.1 and NM_001384479.1, UniProt ID: P01019.4; mouse (Mus musculus) AGT, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 11606, NCBI Accession Nos. NP_001403241.1 and NM_001416312.1, and NP_031454.4 and NM_007428.4, UniProt ID: Pl 1859.1 ; rat (Rattus norvegicus) AGT, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession Nos. GI: 24179, NCBI Accession No. NP_602308.1 and NM_134432.2, UniProt ID: P01015.1; and chimpanzee (Pan troglodytes) AGT, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession Nos. GI: 450104, NCBI Accession No.

[0159] NP_001009032.1 and NM_001009032.1, UniProt ID: Q9GLN8.2. The term “AGT” also includes cynomolgus monkey Macacafascicularis) AGT, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 101926221, NCBI Accession Nos. XP_015299181.3 and XM_015443695.3, XP_065403648.1 and XM_065547576.1, and XP_045247767.2 and XM.045391832.2, UniProt IDs:

[0160] A0A2K5UDY1 and G8F2E1. Additional examples of AGT mRNA sequences are readily available using, e.g., GenBank, UniProt, OMIM, and the Macaca genome project web site. Exemplary AGT nucleotide and amino acid sequences may also be found in Table 2, SEQ ID NOs: 19-21.

[0161] The term “AGT,” as used herein, also refers to naturally occurring DNA sequence variations of the AGT gene. Numerous sequence variations within the AGT gene have been identified and may be found at, for example, NCBI dbSNP and UniProt (see, e.g., ncbi.nlm.nih.gov / snp).

[0162] Further information on AGT can be found, for example, at ncbi.nlm.nih.gov / gene / 183. The entire contents of each of the foregoing GenBank Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of filing this application.

[0163] Table 2 below summarizes exemplary amino acid sequences of AGT proteins and nucleic acid (e.g., DNA or mRNA) sequence of AGT genes of humans.

[0164] Table 2: Exemplary Amino Acid and Nucleic Acid Sequences of AGT.

[0165]

[0166]

[0167]

[0168] The term “apolipoprotein(a) (LPA)” is used interchangeably in the art or herein with the term “apoA,” “Lp(a),” “LP,” and “AK38.” Apolipoprotein(a) inhibits the activity of tissue-type plasminogen activator I, a serine protease produced primarily by vascular endothelial cells that converts plasminogen to plasmin, thereby increasing risk of atherosclerosis. Consequently, increased LPA levels have been linked to increased risk of cardiovascular disease and stroke, and cannot be lowered through lifestyle changes (e.g., diet, exercise). Approximately 70%-90% of inter-individual LPA levels are genetically determined. Novel therapies for elevated LPA are in various phases of clinical development (Reyes-Soffer et al., (2021) Arterioscler Thromb Vase Biol 42:e48-e60, the entire contents of which are hereby incorporated by reference).

[0169] The term “LPA” includes human (Homo sapiens) LPA, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 4018, NCBI Accession Nos. NP_005568.2 and NM_005577.4, UniProt ID: P08519.2; and chimpanzee (Pan troglodytes) LPA, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 463116, NCBI Accession Nos. XP_054542942.1 and XM_054686967.2, XP_063669709.1 and XM_063813639.1, XP_054542943.1 and XM_054686968.2, XP_054542944.1 and XM_054686969.2, and XP.063669710.1 and XM_063813640.1, UniProt ID: H2QU05. The term “LPA” also includes rhesus monkey (Macaco mulatto) LPA, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 709528, NCBI Accession Nos. XP_028702834.1 and XM_028847001.1, UniProt ID: P14417.1. Additional examples of LPA mRNA sequences are readily available using, e.g., GenBank, UniProt, OMIM, and the Macaca genome project web site. Exemplary LPA nucleotide and amino acid sequences may also be found in Table 3, SEQ ID NOs: 22 and 23.

[0170] The term “LPA,” as used herein, also refers to naturally occurring DNA sequence variations of the LPA gene. Numerous sequence variations within the LPA gene have been identified and may be found at, for example, NCBI dbSNP and UniProt (see, e.g., ncbi.nlm.nih.gov / snp).

[0171] Further information on LPA can be found, for example, at ncbi.nlm.nih.gov / gene / 4018. The entire contents of each of the foregoing GenBank Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of filing this application.

[0172] Table 3 below summarizes exemplary amino acid sequences of LPA proteins and nucleic acid (e.g., DNA and mRNA) sequence of LPA genes of humans.

[0173] Table 3: Exemplary Amino Acid and Nucleic Acid Sequences of LPA.

[0174]

[0175]

[0176]

[0177]

[0178] * The nucleic acid sequence Access Numbers listed in Table 3 provide the DNA sequences of LPA (i.e., with T’s). The mRNA sequences provided in Table 3 represent the DNA sequences associated with the respective Accession Numbers, with all the T’s replaced with U’s.

[0179] DETAILED DESCRIPTION

[0180] Atherosclerotic cardiovascular disease and hypertension are by far the leading causes of morbidity, mortality, and healthcare costs around the world. Atherosclerosis develops over several decades before the accumulated plaque burden becomes large enough to increase the risk of having a heart or stroke, and systolic blood pressure (SBP) begins to rise linearly with age several decades before the development of hypertension. Thus, it is possible to precisely predict who is developing these diseases, and then intervene to lower LDL, Lp(a), and SBP prior to disease initiation or early in the disease process to slow the trajectory of atherosclerosis and rising SBP enough to largely eliminate the lifetime risk of heart attack, stroke, and hypertension, extending the average healthy lifespan.

[0181] To achieve this goal, many people will likely require modest sustained reductions in LDL and SBP over several decades to slow the trajectory of atherosclerosis and rising SBP enough to largely eliminate their lifetime risk of developing heart attack, stroke, and hypertension. However, long-term compliance with the therapies needed to modulate LDL and SBP enough to accomplish this goal is likely to be very poor, thus substantially undermining the potential clinical and economic benefits that can be achieved through early intervention to prevent cardiometabolic disease. This problem can be addressed, at least in part, with long-acting siRNA that can be administered yearly to ensure that the sustained reductions to the increases LDL, Lp(a), and SBP are being achieved.

[0182] Treating and / or Preventing Atherosclerotic Cardiovascular Disease Using Long-Acting siRNA

[0183] Atherosclerosis is caused by the progressive trapping of LDL and other apoB-containing lipoproteins within the artery wall. As more LDL particles become trapped within the artery wall over time, the size of the accumulating atherosclerotic plaque burden gradually enlarges and risk of having an acute cardiovascular event increases directly proportional to the size of the accumulated plaque. Therefore, the biological effect of LDL on the risk of atherosclerotic cardiovascular disease depends on both the magnitude and duration of exposure.

[0184] Summing LDL-C levels measured over time to calculate the cumulative exposure to LDL creates a unique biomarker that captures the biological effect of both the magnitude and duration of exposure to LDL. The magnitude of plasma LDL determines the rate of plaque progression, while the duration of exposure to LDL determines the lipoproteins that become trapped within the artery wall and therefore determines the size of the accumulated plaque and corresponding risk of having an acute cardiovascular event. As a result, cumulative exposure to LDL is a biomarker that can be used to estimate the size of a person’s accumulated plaque burden, track the rate of their plaque progression, and predict their risk of having an acute atherosclerotic cardiovascular event at any point in time.

[0185] Titrating LDL-C lowering to keep cumulative exposure to LDL and the resulting size of the accumulated plaque burden below the threshold at which acute cardiovascular events occur can dramatically reduce the lifetime risk of atherosclerotic cardiovascular events including heart attack and stroke. A long-acting siRNA directed against PCSK9, for example, administered once yearly over several decades provides a way to maintain the long-term sustained reductions in LDL needed to slow the trajectory of atherosclerosis enough to keep the accumulated plaque burden below the threshold at which cardiovascular events occur. However, there is no single threshold for cumulative exposure to LDL and corresponding size of accumulated plaque burden above which atherosclerotic cardiovascular events occur. Indeed, the risk of having an acute cardiovascular event is increased at all levels of plaque burden by other exposures that cause accumulating irreversible injury to artery wall - including elevated SBP. This arterial wall injury decreases the capacity of the artery to tolerate the accumulated plaque burden.

[0186] Elevated SBP is the most common cause of accumulating irreversible structural injury to the artery wall. Over time, non-laminar flow caused by elevated SBP increases the transcytosis of LDL particles into the artery wall and promotes the proliferation of vascular smooth muscle cells, thus leading to increased proteoglycan synthesis and the trapping of more LDL particles within the artery wall at vulnerable branch points. In addition, elevated SBP increases shear stress across atherosclerotic plaques, thus increasing the risk of having an acute atherosclerotic event by increasing the likelihood of plaque erosion and rupture. As a result, persons with elevated SBP have a higher risk of experiencing an acute cardiovascular event at the same level of cumulative exposure to LDL and resulting accumulated plaque burden and begin to experience acute cardiovascular events at a lower plaque burden, as compared to persons with lower SBP.

[0187] Indeed, LDL and SBP have independent, additive, causal, and cumulative effects on the risk of major cardiovascular events. Mendelian randomization studies - often referred to as ‘nature’s randomized trials’ - demonstrate that reducing long-term exposure to LDL by 1 mmol / L and SBP by 10 mmHg can reduce the lifetime risk of major cardiovascular events by 80%, cardiovascular mortality by 67%, and all-cause mortality by 33%, which is nearly twice the benefit that can be achieved by lowering LDL alone.

[0188] Therefore, an effective way to reduce the lifetime risk of atherosclerotic cardiovascular events is to maintain normal levels of LDL to slow the progression of atherosclerotic plaque while also maintaining normal levels of SBP to protect the artery wall from accumulating irreversible structural injury to maximize the capacity of the artery to tolerate the accumulated plaque burden. A combination of long-acting siRNAs directed against PCSK9 and AG7', for example, administered once yearly over several decades would provide a way to maintain the long-term sustained reductions in both LDL and SBP needed to slow the trajectory of atherosclerosis and protect the artery wall from accumulating vascular injury enough to prevent the developing of heart attacks and stroke.

[0189] Indeed, because the clinical benefits of lowering LDL and SBP are independent and additive, a long-acting siRNA, or a combination of long-acting siRNAs, designed to lower both LDL and SBP has the potential reduce almost twice as many events for the same reduction in LDL as compared to any other therapy that only lowers LDL. This includes other therapies that are being developed to produce sustained long-term reductions in LDL, including base-editing therapies, and potential ‘vaccines’ directed against LDL, apoB, or PCSK9 (which have the potential risk of provoking ‘immunologic switch’ thus diminishing their effectiveness to lower LDL over time; or an ‘auto-immune’ like condition provoked by immunologically targeting a constitutive highly expressed protein like PCSK9).

[0190] In addition, Lp(a) is another circulating apoB-containing lipoprotein that can become trapped within the artery wall over time to increase the rate of plaque progression and thus increase the corresponding risk of having an acute atherosclerotic cardiovascular event. Like LDL and SBP, the biological effect of Lp(a) on the risk of atherosclerotic cardiovascular disease accumulates over time and thus depends on both the magnitude and duration of exposure. However, unlike LDL and SBP, circulating Lp(a) levels are almost entirely inherited. As a result, plasma Lp(a) cannot be lowered with diet or exercise but instead is lowered therapeutically. Furthermore, unlike LDL and SBP, the distribution of plasma Lp(a) levels in the population is highly skewed. As a result, about 20% of the population have a substantially increased lifetime risk of cardiovascular disease caused by markedly elevated circulating Lp(a).

[0191] Because Lp(a) levels are inherited and because the biological effect of Lp(a) increases over time, a long acting siRNA designed to lower Lp(a) (e.g., by targeting LPA) that can be administered once yearly beginning early in life will be an essential therapy to reduce the very high inherited lifetime risk of atherosclerotic cardiovascular events among persons with very high Lp(a) levels. For persons with more moderately elevated circulating Lp(a) levels, a long-acting siRNA to lower Lp(a) (e.g., targeting LPA) can be used to provide additional modest risk reduction when treatment with a long-acting siRNA designed to lower LDL (or LDL and SBP) does not produce the desired level of risk reduction.

[0192] Treating and / or Preventing Hypertension & Hypertensive Cardiovascular Disease Using Long-Acting siRNA

[0193] Hypertension is caused by a cycle of increasing vascular injury that leads to progressively rising SBP over time. Beginning in early adulthood, SBP starts to slowly rise in most people as they become less active and gain weight. However, once SBP crosses a certain threshold, the increased blood pressure injures the artery wall causing it to become inflamed and stiffen. In response, the heart must pump harder to generate a higher blood pressure to force blood through the stiffened vessel. The higher blood pressure generated by the heart, in turn, leads to more vascular injury and initiates a vicious cycle of accumulating vascular injury that causes SBP to rise linearly with age. The blood pressure inflection point at which SBP begins to rise linearly with age, and the rate at which SBP rises beyond this inflection point, varies substantially from person to person.

[0194] Lowering SBP once it begins to rise linearly with age should interrupt this cycle of accumulating vascular injury and prevent further rises in SBP and thus prevent the development of hypertension. A long-acting siRNA directed against AGT, for example, administered once yearly to persons whose SBP begins to rise linearly with age, and therefore will therefore eventually develop hypertension, would lower their SBP and prevent further rises in SBP, thus personalizing the prevention of hypertension. Importantly, preventing hypertension would also prevent all pressure-related cardiovascular morbidity; including myocardial hypertrophy, heart failure with a preserved ejection fraction, atrial fibrillation, non-ischemic strokes, and vascular dementia.

[0195] Furthermore, clinical practice guidelines for the treatment of elevated blood pressure and hypertension now increasingly recommend that the decision to initiate treatment to lower SBP among persons with ‘pre-hypertension’ and Stage 1 hypertension should be based on the estimated risk of developing atherosclerotic cardiovascular disease. This is a compelling rationale to use a long-acting siRNA designed to lower both LDL and SBP because it creates a unique asset that can reduce the risk of atherosclerotic cardiovascular disease more effectively than lowering SBP alone, while at the same time preventing hypertension and all related hypertensive heart disease.

[0196] Accordingly, the present disclosure, in some aspects, provide compositions comprising RNAi agents that target Proprotein convertase subtilisin / kexin type 9 (PCSK9), angiotensinogen (AGT), and / or Apolipoprotein A (LPA). In some embodiments, each of the RNAi agents that target Proprotein convertase subtilisin / kexin type 9 (PCSK9), angiotensinogen (AGT), and / or Apolipoprotein A (LPA) is a long-acting RNAi agent.

[0197] The term “long-acting RNAi agent,” as used herein, refers to RNAi agents that, once administered to a subject, can reduce the expression and / or activity of a target gene (e.g., PCSK9, AGT, or LPA) for an extended period of time (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months, or longer). In some embodiments, a subject treated by a long-acting RNAi agent is administered the long-acting RNAi agent no more than once every 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months, or even less frequently). Thus, the term “long-lasting,” as used herein, refers to an extended period of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months, or longer. In some embodiments, a composition described herein comprises a first RNAi agent that targets PCSK9 and a second RNAi agent that targets AGT. In some embodiments, a composition described herein comprises a first RNAi agent that targets PCSK9 and a third RNAi agent that targets LPA. In some embodiments, a composition described herein comprises a first RNAi agent that targets PCSK9, a second RNAi agent that targets AGT, and a third RNAi agent that targets LPA. In some embodiments, a composition described herein comprises a second RNAi agent that targets AGT and a third RNAi agent that targets LPA. In some embodiments, in a composition described herein, each of the first, second, and third RNAi agent is an siRNA (e.g., a double stranded siRNA).

[0198] In some embodiments, in any one of the compositions described herein, the first RNAi agent comprises an antisense strand comprising a region of complementarity to a PCSK9 RNA (e.g., a PCSK9 RNA as set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, or 18), and a sense strand that is complementary (e.g., at least substantially complementary) to the antisense strand. In some embodiments, the region of complementarity of the antisense strand of the first RNAi agent is at least 8 nucleosides (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleosides) in length. In some embodiments, the antisense strand of the first RNAi agent is 15-35 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleosides in length, and / or the sense strand of the first RNAi agent is 15-35 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleosides in length. In some embodiments, the first RNAi agent comprises an overhang of 0, 1, 2, 3, 4 or 5 nucleosides at the 3’ and / or 5’ end of the sense strand and / or antisense strand. For example, in some embodiments, the first RNAi agent comprises no overhang at either end of the sense strand or antisense strand. In some embodiments, the first RNAi agent comprises an overhang of 1 or 2 nucleosides at the 3’ end of the sense strand and / or antisense strand. In some embodiments, the first RNAi agent comprises no overhang at the 3’ end of the sense strand and the 5’ end of the antisense strand and comprises an overhang of 2 nucleosides at the 3’ end of the antisense strand. In some embodiments, the sense strand and the antisense strand of the first RNAi agent form a duplex region of 12-30 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26, 27, 28, 29, or 30) base pairs in length. In some embodiments, in the duplex region, the sense strand and the antisense strand are fully complementary.

[0199] In some embodiments, in any one of the compositions described herein, the second RNAi agent comprises an antisense strand comprising a region of complementarity to an AGT RNA (e.g., an AGT RNA as set forth in any one of SEQ ID NOs: 20 and 21), and a sense strand that is complementary (e.g., at least substantially complementary) to the antisense strand. In some embodiments, the region of complementarity of the antisense strand of the second RNAi agent is at least 8 nucleosides (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleosides) in length. In some embodiments, the antisense strand of the second RNAi agent is 15-35 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleosides in length, and / or the sense strand of the second RNAi agent is 15-35 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleosides in length. In some embodiments, the second RNAi agent comprises an overhang of 0, 1, 2, 3, 4 or 5 nucleosides at the 3’ and / or 5’ end of the sense strand and / or antisense strand. For example, in some embodiments, the second RNAi agent comprises no overhang at either end of the sense strand or antisense strand. In some embodiments, the second RNAi agent comprises an overhang of 1 or 2 nucleosides at the 3’ end of the sense strand and / or antisense strand. In some embodiments, the second RNAi agent comprises no overhang at the 3’ end of the sense strand and the 5’ end of the antisense strand and comprises an overhang of 2 nucleosides at the 3’ end of the antisense strand. In some embodiments, the sense strand and the antisense strand of the second RNAi agent form a duplex region of 12-30 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26, 27, 28, 29, or 30) base pairs in length. In some embodiments, in the duplex region, the sense strand and the antisense strand are fully complementary.

[0200] In some embodiments, in any one of the compositions described herein, the third RNAi agent comprises an antisense strand comprising a region of complementarity to an LPA RNA (e.g., an LPA RNA as set forth in any one of SEQ ID NO: 23), and a sense strand that is complementary (e.g., at least substantially complementary) to the antisense strand. In some embodiments, the region of complementarity of the antisense strand of the third RNAi agent is at least 8 nucleosides (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleosides) in length. In some embodiments, the antisense strand of the third RNAi agent is 15-35 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleosides in length, and / or the sense strand of the third RNAi agent is 15-35 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleosides in length. In some embodiments, the third RNAi agent comprises an overhang of 0, 1, 2, 3, 4 or 5 nucleosides at the 3’ and / or 5’ end of the sense strand and / or antisense strand. For example, in some embodiments, the third RNAi agent comprises no overhang at either end of the sense strand or antisense strand. In some embodiments, the third RNAi agent comprises an overhang of 1 or 2 nucleosides at the 3’ end of the sense strand and / or antisense strand. In some embodiments, the third RNAi agent comprises no overhang at the 3’ end of the sense strand and the 5’ end of the antisense strand and comprises an overhang of 2 nucleosides at the 3’ end of the antisense strand. In some embodiments, the sense strand and the antisense strand of the third RNAi agent form a duplex region of 12-30 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26, 27, 28, 29, or 30) base pairs in length. In some embodiments, in the duplex region, the sense strand and the antisense strand are fully complementary.

[0201] In some embodiments, in a composition described herein, the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26. 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more) modified nucleoside. In some embodiments, the antisense strand of the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) modified nucleoside. In some embodiments, the sense strand of the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) modified nucleoside. In some embodiments, the antisense strand of the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) modified nucleoside, and the sense strand of the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) modified nucleoside. In some embodiments, each nucleoside of the first RNAi agent is a modified nucleoside. In some embodiments, each nucleoside of the second RNAi agent is a modified nucleoside. In some embodiments, each nucleoside of the third RNAi agent is a modified nucleoside. In some embodiments, at least one modified nucleoside comprises a chemical modification in the base of the nucleoside. In some embodiments, at least one modified nucleoside comprises a chemical modification in the sugar moiety of the nucleoside. In some embodiments, at least one modified nucleoside comprises a chemical modification in the base of the nucleoside and a chemical modification in the sugar moiety of the modified nucleoside. In some embodiments, a modified nucleoside in each of the first, second, and third RNAi agent in any one of the compositions described herein is a 2 ’-modified nucleoside (e.g., any one of the 2’ -modified nucleoside known in the art or described herein). In some embodiments, the 2’-modified nucleoside is selected from 2’-fluoro (2’-F), 2’-O-methyl (2’-O-Me), 2’-0-methoxyethyl (2’-M0E), 2’-0-aminopropyl (2’-O-AP), 2’-O-dimethylaminoethyl (2’-0-DMA0E), 2’-0-dimethylaminopropyl (2’-O-DMAP), 2’-O-dimethylaminoethyloxyethyl (2’-O-DMAEOE), or 2’-0-N-methylacetamido (2’-0-NMA) modified nucleoside and combinations thereof, optionally wherein the 2’-modified nucleoside is selected from a 2’-O-methyl (2’-0-Me) modified nucleoside, a 2’-fluoro (2’-F) modified nucleoside, and combinations thereof. In some embodiments, the 2’ -modified nucleoside is a 2’-O-methyl (2’-0-Me) modified nucleoside, a 2’-fluoro (2’-F) modified nucleoside, or a 2’ -deoxyribonucleotide (DNA).

[0202] In some embodiments, a composition described herein comprises the first RNAi agent and the second RNAi agent, wherein each nucleoside of the first RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, and each nucleoside of the second RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside.

[0203] In some embodiments, a composition described herein comprises the first RNAi agent and the third RNAi agent, wherein each nucleoside of the first RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, and each nucleoside of the third RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside.

[0204] In some embodiments, a composition described herein comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein each nucleoside of the first RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, each nucleoside of the second RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, and each nucleoside of the third RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside.

[0205] In some embodiments, in a composition described herein, the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) modified internucleoside linkages. In some embodiments, the antisense strand of the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one (e.g., at least 1, 2, 3, 4, 5, or more) modified intemucleoside linkages. In some embodiments, the sense strand of the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one (e.g., at least 1, 2, 3, 4, 5, or more) modified intemucleoside linkages. In some embodiments, the antisense strand of the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one (e.g., at least 1, 2, 3, 4, 5, or more) modified intemucleoside linkages, and the sense strand of the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one (e.g., at least 1, 2, 3, 4, 5, or more) modified internucleoside linkages. In some embodiments, the modified intemucleoside linkage is a phosphorothioate intemucleoside linkage.

[0206] In some embodiments, the first RNAi agent comprises a block of two phosphorothioate intemucleoside linkages at the 3’ end or 5’ end of the sense strand. In some embodiments, the first RNAi agent comprises a block of two phosphorothioate intemucleoside linkages at the 3’ end or 5’ end of the antisense strand. In some embodiments, the first RNAi agent comprises a block of two phosphorothioate intemucleoside linkages at the 3’ end and / or 5’ end of the sense strand, and comprises a block of two phosphorothioate intemucleoside linkages at the 3’ end and / or 5’ end of the antisense strand.

[0207] In some embodiments, the second RNAi agent comprises a block of two phosphorothioate intemucleoside linkages at the 3’ end or 5’ end of the sense strand. In some embodiments, the first RNAi agent comprises a block of two phosphorothioate intemucleoside linkages at the 3’ end or 5’ end of the antisense strand. In some embodiments, the second RNAi agent comprises a block of two phosphorothioate intemucleoside linkages at the 3’ end and / or 5’ end of the sense strand, and comprises a block of two phosphorothioate intemucleoside linkages at the 3’ end and / or 5’ end of the antisense strand.

[0208] In some embodiments, the third RNAi agent comprises a block of two phosphorothioate intemucleoside linkages at the 3’ end or 5’ end of the sense strand. In some embodiments, the first RNAi agent comprises a block of two phosphorothioate intemucleoside linkages at the 3’ end or 5’ end of the antisense strand. In some embodiments, the third RNAi agent comprises a block of two phosphorothioate intemucleoside linkages at the 3’ end and / or 5’ end of the sense strand, and comprises a block of two phosphorothioate intemucleoside linkages at the 3’ end and / or 5’ end of the antisense strand.

[0209] In some embodiments, a composition described herein comprises the first RNAi agent and the second RNAi agent, wherein each nucleoside of the first RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, and each nucleoside of the second RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, wherein each of the first RNAi agent and the second RNAi agent comprises least one (e.g., at least 1, 2, 3, 4, 5, or more) phosphorothioate internucleoside linkages.

[0210] In some embodiments, a composition described herein comprises the first RNAi agent and the third RNAi agent, wherein each nucleoside of the first RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, and each nucleoside of the third RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, wherein each of the first RNAi agent and the third RNAi agent comprises least one (e.g., at least 1, 2, 3, 4, 5, or more) phosphorothioate internucleoside linkages.

[0211] In some embodiments, a composition described herein comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein each nucleoside of the first RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, each nucleoside of the second RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, and each nucleoside of the third RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, wherein each of the first RNAi agent, the second RNAi agent, and the third RNAi agent comprises least one (e.g., at least 1, 2, 3, 4, 5, or more) phosphorothioate intemucleoside linkages.

[0212] In some embodiments, a composition described herein comprises the second RNAi agent and the third RNAi agent, wherein each nucleoside of the second RNAi agent is a 2’-O-Me modified nucleoside or a 2’-F modified nucleoside, and each nucleoside of the third RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, wherein each of the second RNAi agent and the third RNAi agent comprises least one (e.g., at least 1, 2, 3, 4, 5, or more) phosphorothioate internucleoside linkages.

[0213] In some embodiments, in any one of the compositions described herein, the first RNAi agent, the second RNAi agent, and the third RNAi agent comprises a phosphorylated 5’ terminus, or includes a phosphoryl analog at the 5’ terminus. In some embodiments, 5’-phosphoryl analogs include those which are compatible with RISC mediated gene silencing. In some embodiments, a 5’- phosphoryl analog is a biostable phosphate mimic (e.g., any one of the biostable phosphate mimic described herein or known in the art). Suitable 5’-phosphoryl analogs include, without limitation: 5'-monophosphate ((HO)2(O)P-O-5’); 5’-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5’); 5 ’-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5’); 5’-guanosine cap (7-methylated or non-methylated) (7m-G-O-5’-(HO)(O)P-O-HO)(O)P-O-P(HO)(O)-O-5’); 5’-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5’-(HO)(O)P-O-HO)(O)P-O-P(HO)(O)-O-5’); 5’-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5’); 5 ’-monodithiophosphate (phosphorodithioate; (H0)(HS)(S)P-0-5’), 5’-phosphorothiolate ((HO)2(O)P-S-5’); any additional combination of oxygen / sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5 ’-alpha-thiotriphosphate, 5 ’-gamma-thiotriphosphate, etc.), 5’-phosphoramidates ((HO)2(O)P-NH-5’, (HO)(NH2)(O)P-O-5’), 5’-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(0H)(0)-0-5’, 5’-alkenylphosphonates (i.e. vinyl, substituted vinyl), (OH)2(O)P-5’-CH2-), 5’-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(0H)(0)-0-5’-).

[0214] In some embodiments, in any one of the composition described herein, the first RNAi agent, the second RNAi agent, and the third RNAi agent comprises a 5’-vinalphosphonate (e.g., at the 5’ terminus of the antisense strand).

[0215] In some embodiments, in any one of the compositions described herein, the first RNAi agent, the second RNAi agent, and / or the third RNAi agent is conjugated (e.g., covalently linked) to a targeting moiety (e.g., for improved delivery, cellular uptake, and distribution). Any suitable targeting moiety in the field of RNA interference may be conjugated (e.g., covalently linked) to any one of the RNAi agents described herein. Nonlimiting examples of targeting moieties include carbohydrates (e.g., monosaccharides (such as GalNAc)), disaccharides, trisaccharides, tetrasaccharides, polysaccharides), folate, mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster, galactose cluster, an aptamer, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, asialoglycoprotein receptor ligands, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands.

[0216] In some embodiments, the targeting moiety is an antibody or antigen binding fragment thereof that specifically binds to a cell surface protein, e.g., a cell surface receptor that promotes receptor mediated endocytosis and internalization of the RNAi agents described herein. Cell surface proteins that may be targeted by the targeting moiety include, without limitation: asialoglycoprotein receptors, CD63, MHC-I, Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL-receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein-2 (APLP2), apelin receptor (APL R), PRLR (prolactin receptor), MAL (Myelin And Lymphocyte protein, a.k.a. VIP 17), IGF2R, vacuolar-type H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptors, glutathione receptor, leptin receptors, scavenger receptors, SCARA1-5, SCARB1-3, CD36, CDH16 (Cadheri-16), CLDN16 (Claudn-16), KL (Klotho), PTH1R (parathyroid hormone receptor), SLC22A13 (Solute carrier family 22 member 13), SLC5A2 (Sodium / glucose cotransporter 2), UMOD (Uromodulin), BMPRIA (Bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4 / GPR48 (G protein-coupled receptor 48), cholinergic receptor (nicotinic) alpha 1, CDH15 (Cadheri-15), ITGA7 (Integrin alpha-7), CACNG1 (L-type calcium channel subunit gamma- 1), CACNAls (L-type calcium channel subunit alpha- 15), CACNG6 (L-type calcium channel subunit gamma-6), SCN1B (Sodium channel subunit beta-1), CHRNA1 (ACh receptor subunit alpha), CHRND (ACh receptor subunit delta), LRRC14B (Leucine-rich repeat-containing protein 14B), and POPDC3 (Popeye domain-containing protein 3). In some embodiments, antibodies or antigen binding fragments that may be used as a targeting moiety according to the present disclosure is a Fab fragment, a Fab’, a F(ab’)2 fragment, a Fv fragment, a scFv, a VHH, or a diabody. In some embodiments, the antibody or antigen binding fragment thereof is covalently linked directly or indirectly (e.g., via a linker) to an RNAi agent described herein. In some embodiments, the targeting moiety is a hepatospecific targeting moiety (i.e., it directs the RNAi agent composition to liver cells). In some embodiments, the targeting moiety comprises a monosaccharide. In some embodiments, the monosaccharide is an N-acetylgalactosamine (GalNAc).

[0217] In some embodiments, in any one of the compositions described herein, the first RNAi agent, the second RNAi agent, and / or the third RNAi agent is conjugated (e.g., covalently linked) to a targeting moiety, wherein the targeting moiety comprises one or more (e.g., 1, 2, 3, 4, or more) N-acetyl-galactosamine (GalNAc) moieties (e.g., a tri-antennary GalNac moiety, or any one of the GalNAc moieties known in the art or described herein). In some embodiments, the targeting moiety (e.g., one or more GalNac moieties) is covalently linked to any one of the RNAi agent described herein via a linker. In some embodiments, the linker may be a bond (e.g., covalent bond) or chemical moiety between two components of the conjugate, e.g., between an RNAi agent and a targeting moiety. In some embodiments, the linker facilitates covalent linkage of an RNAi agent to a targeting moiety. In some embodiments, the linker is conjugated (e.g., covalently linked) to the 5’ or 3 ’end of the sense strand or antisense strand of an RNAi agent. In some embodiments, the linker is conjugated (e.g., covalently linked) to the 5’ or 3’ end of the sense strand of an RNAi agent. In some embodiments, a linker is conjugated (e.g., covalently linked) to the 5’ end of the sense strand of an RNAi agent. In some embodiments, a linker is conjugated (e.g., covalently linked) to the 3’ end of the sense strand of an RNAi agent. The linker can be any suitable group for coupling the RNAi agent to the targeting moiety. In some embodiments, the linker is a monovalent, bivalent, trivalent, or tetravalent branched linker. In some embodiments, the linker is a phosphorus-containing linker (e.g., phosphate, phosphodiester, phosphorothioate, phosphorodithioate, phosphoroamidate, etc.), or other linker. Non-limiting examples of other linkers can include, but are not limited to: reactive groups such a primary amines and alkynes, alkyl groups, abasic nucleotides, ribitol (abasic ribose), and / or PEG groups. In some embodiments, the linker comprises a phosphodiester linkage or a phosphorothioate linkage.

[0218] In some embodiments, in any one of the compositions described herein, one or more of the first RNAi agent, the second RNAi agent, and the third RNAi agent is covalently linked (e.g., via the 5’ end of the 3’ end of the sense strand) to one or more (e.g., 1, 2, 3, 4, or more) GalNAc moieties via a trivalent branched linker. In some embodiments, in any one of the compositions described herein, one or more of the first RNAi agent, the second RNAi agent, and the third RNAi agent is covalently linked (e.g., via the 5’ end of the 3’ end of the sense strand) to a tri-antennary GalNAc moiety via a trivalent branched linker.

[0219] In some embodiments, a composition described herein comprises the first RNAi agent and the second RNAi agent, wherein each nucleoside of the first RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, and each nucleoside of the second RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, wherein each of the first RNAi agent and the second RNAi agent comprises least one (e.g., at least 1, 2, 3, 4, 5, or more) phosphorothioate internucleoside linkages, and wherein the first RNAi agent and / or (e.g., and) the second RNAi agent is covalently linked (e.g., via the 5’ end of the 3’ end of the sense strand) to a tri-antennary GalNac moiety via a trivalent branched linker.

[0220] In some embodiments, a composition described herein comprises the first RNAi agent and the third RNAi agent, wherein each nucleoside of the first RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, and each nucleoside of the third RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, wherein each of the first RNAi agent and the third RNAi agent comprises least one (e.g., at least 1, 2, 3, 4, 5, or more) phosphorothioate internucleoside linkages, and wherein the first RNAi agent and / or (e.g., and) the third RNAi agent is covalently linked (e.g., via the 5’ end of the 3’ end of the sense strand) to a tri-antennary GalNac moiety via a trivalent branched linker.

[0221] In some embodiments, a composition described herein comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein each nucleoside of the first RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, each nucleoside of the second RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, and each nucleoside of the third RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, wherein each of the first RNAi agent, the second RNAi agent, and the third RNAi agent comprises least one (e.g., at least 1, 2, 3, 4, 5, or more) phosphorothioate intemucleoside linkages, and wherein the first RNAi agent, the second RNAi agent, and / or (e.g., and) the third RNAi agent is covalently linked (e.g., via the 5’ end of the 3’ end of the sense strand) to a tri-antennary GalNac moiety via a trivalent branched linker.

[0222] In some embodiments, a composition described herein comprises the second RNAi agent, and the third RNAi agent, wherein each nucleoside of the second RNAi agent is a 2’-O-Me modified nucleoside or a 2’-F modified nucleoside, and each nucleoside of the third RNAi agent is a 2’-0-Me modified nucleoside or a 2’-F modified nucleoside, wherein each of the second RNAi agent and the third RNAi agent comprises least one (e.g., at least 1, 2, 3, 4, 5, or more) phosphorothioate internucleoside linkages, and wherein the second RNAi agent and / or (e.g., and) the third RNAi agent is covalently linked (e.g., via the 5’ end of the 3’ end of the sense strand) to a tri-antennary GalNac moiety via a trivalent branched linker.

[0223] In some embodiments, a composition described herein comprises a first RNAi agent (targeting PCSK9) and a second RNAi agent (targeting AGT), wherein the first RNAi agent is any one of the PCSK9 RNAi agents (e.g., siRNAs) known in the art (e.g., Inclisiran) or described herein, and wherein the second RNAi agent is any one of the AGT RNAi agents (e.g., siRNAs) known in the art (e.g., zilebesiran or SGB-3908 (IBI3016)) or described herein. Inclisiran has a has a CAS Registration No. of 1639324-58-5. Zilebesiran has a has a CAS Registration No. of 2380166-33-4.

[0224] In some embodiments, a composition described herein comprises a first RNAi agent (targeting PCSK9) and a third RNAi agent (targeting LPA), wherein the first RNAi agent is any one of the PCSK9 RNAi agents (e.g., siRNAs) known in the art (e.g., Inclisiran) or described herein, and wherein the third RNAi agent is any one of the LPA RNAi agents (e.g., siRNAs) known in the art (e.g., SLN360) or described herein. SLN360 is described in, e.g., Nissen et al., JAMA. 2022 May 3;327(17): 1679-1687, the entire contents of which are incorporated herein by reference.

[0225] In some embodiments, a composition described herein comprises a first RNAi agent (targeting PCSK9), a second RNAi agent (targeting AGT), and a third RNAi agent (targeting LPA), wherein the first RNAi agent is any one of the PCSK9 RNAi agents (e.g., siRNAs) known in the art (e.g., Inclisiran) or described herein, wherein the second RNAi agent is any one of the AGT RNAi agents (e.g., siRNAs) known in the art (e.g., zilebesiran or SGB-3908 (IBI3016)) or described herein, and wherein the third RNAi agent is any one of the LPA RNAi agents (e.g., siRNAs) known in the art (e.g., SLN360) or described herein. SLN360 is described in, e.g., Nissen et al., JAMA. 2022 May 3;327(17): 1679-1687, the entire contents of which are incorporated herein by reference.

[0226] In some embodiments, a composition described herein comprises a second RNAi agent (targeting AGT) and a third RNAi agent (targeting LPA), wherein the second RNAi agent is any one of the AGT RNAi agents (e.g., siRNAs) known in the art (e.g., zilebesiran or SGB-3908 (IB 13016)) or described herein, and wherein the third RNAi agent is any one of the LPA RNAi agents (e.g., siRNAs) known in the art (e.g., SLN360) or described herein. SLN360 is described in, e.g., Nissen et al., JAMA. 2022 May 3;327(17): 1679-1687, the entire contents of which are incorporated herein by reference.

[0227] Additional PCSK9 RNAi agents (e.g., siRNAs) that may be used in a composition described herein are provided in, e.g., US Patent Nos.: US8222222, US8809292, US8828956, US8106022, US10131907, US10125369, US10851377, US10668170, US10167471, US8598139, US76052512, US9493774, US9187746, US8431544, US8273869, and

[0228] US 10633654, the entire contents of each of which are incorporated herein by reference.

[0229] Additional AGT RNAi agents (e.g., siRNAs) that may be used in a composition described herein are provided in, e.g., US Patent Nos. US11834661, US10238749, US12247204, US11419942, US11015201, and US10814007, the entire contents of each of which are incorporated herein by reference.

[0230] Additional LPA RNAi agents (e.g., siRNAs) that may be used in a composition described herein are provided in, e.g., US Patent Nos.: US9932586, US7741305, US10662427, US12054717, US11499153, US10472629, US11319536 and US12252691, US Patent Application Publication Nos.: US2023405040, US2024035029, and US2024271140, International Patent Application Publication Nos.: WO2023152194, WO2024222686, WO2023169548, W02023017004, WO2022121959, W02023109940, WO2023138689, WO2024035898, W02024088190, W02024210530, WO2024222686, WO2024222686, and WO2024212954, the entire contents of each of which are incorporated herein by reference.

[0231] The RNAi agents in any one of the compositions described herein may be separate molecular entities (e.g., not covalently linked), or may be linked (e.g., covalently linked) via a linker or a chemical moiety.

[0232] In some embodiments, a composition described herein comprises the first RNAi agent and the second RNAi agent, wherein the first RNAi agent and the second RNAi agent are not covalently linked. In some embodiments, a composition described herein comprises the first RNAi agent and the second RNAi agent, wherein the first RNAi agent and the second RNAi agent are covalently linked (e.g., via a covalent bond or a linker). In some embodiments, a composition described herein comprises the first RNAi agent and the third RNAi agent, wherein the first RNAi agent and the third RNAi agent are not covalently linked. In some embodiments, a composition described herein comprises the first RNAi agent and the third RNAi agent, wherein the first RNAi agent and the third RNAi agent are covalently linked (e.g., via a covalent bond or a linker).

[0233] In some embodiments, a composition described herein comprises the second RNAi agent and the third RNAi agent, wherein the second RNAi agent and the third RNAi agent are not covalently linked. In some embodiments, a composition described herein comprises the second RNAi agent and the third RNAi agent, wherein the second RNAi agent and the third RNAi agent are covalently linked (e.g., via a covalent bond or a linker).

[0234] In some embodiments, a composition described herein comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the first RNAi agent, the second RNAi agent, and the third RNAi agent are not covalently linked to each other.

[0235] In some embodiments, a composition described herein comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the first RNAi agent is covalently linked to the second RNAi agent (e.g., via a covalent bond or a linker), and the third RNAi agent is not linked to the first RNAi agent or to the second RNAi agent.

[0236] In some embodiments, a composition described herein comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the first RNAi agent is covalently linked (e.g., via a covalent bond or a linker) to the third RNAi agent, and the second RNAi agent is not linked to the first RNAi agent or to the third RNAi agent.

[0237] In some embodiments, a composition described herein comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the second RNAi agent is covalently linked (e.g., via a covalent bond or a linker) to the third RNAi agent, and the first RNAi agent is not linked to the second RNAi agent or to the third RNAi agent.

[0238] In some embodiments, a composition described herein comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the first RNAi agent, the second RNAi agent, and the third RNAi agent are covalently linked (e.g., via a covalent bond or a linker) to each other.

[0239] Any suitable covalent bond or linker known in the art may be used to link the RNAi agents in any one of the compositions provided herein. Suitable linkers that may be used include, without limitation, chemical linkers, peptide linkers, and nucleotide linkers. In some embodiments, the linker comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleosides. In some embodiments, the linker is a cleavable linker (e.g., a disulfide bond, a protease cleavable linker, or a nucleotide linker containing natural ribonucleotides and / or deoxyribonucleotides). In some embodiments, a protease cleavable linker is cleavable by a lysosomal protease and / or endosome proteas (e.g., cathepsin). In some embodiments, a protease cleavable linker comprises a valine-citrulline sequence. In some embodiments, the linker is a noncleavable linker (e.g., chemical linkers that are not cleavable, nucleotide linkers with modified nucleosides that are resistant to cleavage, e.g., by a nuclease).

[0240] In some embodiments, in a composition described herein where the siRNAs are covalently linked to each other, a nucleotide linker is used to covalently link the one strand from each siRNA to form a dimer, and the second strand of each siRNA anneals to the dimer. In some embodiments, the dimer is formed by covalently linking two sense strands via a nucleotide linker. In some embodiments, the dimer is formed by covalently linking the sense strand from one siRNA, and the antisense strand from another siRNA via a nucleotide linker. In some embodiments, the nucleotide linker contains only modified nucleotides and is noncleavable. In some embodiments, the nucleoside linkers an unmodified ribonucleotide (RNA) and / or a deoxyribonucleotide (DNA), and is cleavable (e.g., by a nuclease).

[0241] In some embodiments, any one of the compositions described herein further comprises a pharmaceutically acceptable carrier. A composition provided herein is for use in, but not limited to, preventing, treating, managing, or ameliorating a disease or one or more symptoms thereof, and / or in research. In some embodiments, a composition may further comprise any other suitable therapeutic agent for treatment of a subject, e.g., a human subject having a cardiovascular disease. In some embodiments, the other therapeutic agents may enhance or supplement the effectiveness of the compositions described herein.

[0242] In some embodiments, a composition described herein is an unbuffered solution. In some embodiments, a composition described herein is formulated in water or in an aqueous solution (e.g., water with pH adjustments). In some embodiments, the unbuffered solution is saline. In some embodiments, a composition described herein is formulated in a buffer solution such as a phosphate-buffered saline solution, liposome, micellar structure, and capsid. In some embodiments, a composition described herein formulated in a basic buffered aqueous solution (e.g., PBS). In some embodiments, formulations as disclosed herein comprise an excipient. In some embodiments, an excipient confers to a composition improved stability, improved absorption, improved solubility and / or (e.g., and) therapeutic enhancement of the active ingredient. In some embodiments, an excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, any one of compositions described herein can be packaged into kits, containers, packs, or dispensers. In some embodiments, any one of the compositions described herein can be packaged in pre-filled syringes or vials.

[0243] In some embodiments, any one of the compositions described herein is formulated to be compatible with its intended route of administration. Non-limiting examples of routes of administration include intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, or intrathecal routes. In some embodiments, the route of administration is subcutaneous.

[0244] Some aspects of the present disclosure provide methods of reducing or maintaining levels of low-density lipoprotein (LDL) and / or cholesterol, arterial pressure and / or systolic blood pressure (SBP), and / or Lipoprotein(a) (Lp(a)) in a subject (e.g., human subject) by administering to the subject any one of the compositions described herein. In some embodiments, the subject has high (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, 2-fold, 5-fold, or higher) levels of LDL, SBP, and / or Lp(a), relative to normal levels of LDL, SBP, and / or Lp(a). In some embodiments, the subject has normal (e.g., considered normal by a clinician in the medical field) levels of LDL, SBP and / or Lp(a). In some embodiments, the subject has an elevated life-time risk of developing cardiovascular disease. In some embodiments, the subject has symptoms of a cardiovascular disease. In some embodiments, the subject is diagnosed with a cardiovascular disease. In some embodiments, the administration is via subcutaneous injection.

[0245] Some aspects of the present disclosure provide methods of maintaining levels LDL and / or cholesterol, arterial pressure and / or SBP, and / or Lp(a) in a subject (e.g., human subject) by administering to the subject any one of the compositions described herein. For example, the compositions herein, in some embodiments, are formulated for maintenance dosing (repeat administration), such as on a yearly basis. Thus, following an initial dose, the compositions can be administered, for example, once or twice a year, to maintain normal levels of LDL and / or cholesterol, arterial pressure and / or SBP, and / or Lp(a) in a subject. In some embodiments, the composition can be administered to a subject that has normal (e.g., considered normal by a clinician in the medical field) levels of LDL, SBP and / or Lp(a). In some embodiments, such a subject is at risk of developing a cardiovascular disease. In some embodiments, a composition is administered (e.g., once or twice) a year to maintain normal levels of LDL and SBP. In some embodiments, a composition is administered (e.g., once or twice) a year to maintain normal levels of LDL and Lp(a). In some embodiments, a composition is administered (e.g., once or twice) a year to maintain normal levels of Lp(a) and SBP. In some embodiments, a composition is administered (e.g., once or twice) a year to maintain normal levels of LDL, SBP, and Lp(a). In some embodiments, the compositions can be used in a subject to maintain levels LDL and / or cholesterol, arterial pressure and / or SBP, and / or Lp(a) at or below a threshold level for the biomarker (e.g. LDL, SBP, and / or Lp(a)). In some embodiments, a threshold level is a normal level for the biomarker, as discussed elsewhere herein.

[0246] In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a first RNAi agent and a second RNAi agent, wherein the method reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more) the expression level (e.g., mRNA and / or protein level) of PCSK9 and AGT in the subject, compared to before the subject is administered the composition. In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a first RNAi agent and a second RNAi agent, wherein the method reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or more) the levels of LDL and / or cholesterol in the subject, and reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or more) arterial pressure and / or systolic blood pressure (SBP) in the subject, compared to before the subject is administered the composition. In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a first RNAi agent and a second RNAi agent, wherein the method restores the levels of LDL and / or cholesterol in the subject, and restores arterial pressure and / or SBP in the subject to normal levels, compared to before the subject is administered the composition. In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a first RNAi agent and a second RNAi agent, wherein the method reduces further rising of LDL and / or cholesterol levels in the subject overtime, and reduces further rising of arterial pressure and / or SBP in the subject over time, compared to before the subject is administered the composition. In some embodiments, the reduction is long-lasting (e.g., lasting for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, at least 24 months, or longer) after each administration of the compositions.

[0247] In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a first RNAi agent and a third RNAi agent, wherein the method reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more) the expression level (e.g., mRNA and / or protein level) of PCSK9 and Lp(a) in the subject, compared to before the subject is administered the composition. In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a first RNAi agent and a third RNAi agent, wherein the method reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or more) the levels of LDL and / or cholesterol in the subject, and reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or more) arterial pressure, and reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or more) Lp(a) levels in the subject, compared to before the subject is administered the composition. In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a first RNAi agent and a third RNAi agent, wherein the method restores the levels of LDL and / or cholesterol in the subject, and reduces Lp(a) levels in the subject to normal levels, compared to before the subject is administered the composition. In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a first RNAi agent and a third RNAi agent, wherein the method reduces further rising of LDL and / or cholesterol levels in the subject overtime, and reduces further rising of Lp(a) levels in the subject over time, compared to before the subject is administered the composition. In some embodiments, the reduction is long-lasting (e.g., lasting for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, at least 24 months, or longer) after each administration of the compositions.

[0248] In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a second RNAi agent and a third RNAi agent, wherein the method reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more) the expression level (e.g., mRNA and / or protein level) of AGT and Lp(a) in the subject, compared to before the subject is administered the composition. In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a second RNAi agent and a third RNAi agent, wherein the method reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or more) arterial pressure and / or SBP in the subject, and reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or more) Lp(a) levels in the subject, compared to before the subject is administered the composition. In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a second RNAi agent and a third RNAi agent, wherein the method restores arterial pressure and / or SBP in the subject, and Lp(a) levels in the subject to normal levels, compared to before the subject is administered the composition. In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a second RNAi agent and a third RNAi agent, wherein the method reduces further rising of arterial pressure and / or SBP in the subject overtime, and reduces further rising of Lp(a) levels in the subject over time, compared to before the subject is administered the composition. In some embodiments, the reduction is long-lasting (e.g., lasting for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, at least 24 months, or longer) after each administration of the compositions.

[0249] In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a first RNAi agent, a second RNAi agent, and a third RNAi agent, wherein the method reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more) the expression level (e.g., mRNA and / or protein level) of PCSK9, AGT, and Lp(a) in the subject, compared to before the subject is administered the composition. In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a first RNAi agent, a second RNAi agent, and a third RNAi agent, wherein the method reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or more) the levels of LDL and / or cholesterol in the subject, reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or more) arterial pressure and / or systolic blood pressure (SBP) in the subject, and reduces (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or more) Lp(a) levels in the subject, compared to before the subject is administered the composition. In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a first RNAi agent, a second RNAi agent, and a third RNAi agent, wherein the method restores the levels of LDL and / or cholesterol in the subject, restores arterial pressure and / or SBP in the subject, and / or restores the Lp(a) levels in the subject to normal levels, compared to before the subject is administered the composition. In some embodiments, a method described herein comprises administering (e.g., via subcutaneous injection) to a subject (e.g., a human subject that has high baseline levels of LDL, SBP, and / or Lp(a), has symptoms of a cardiovascular disease, and / or is diagnosed with a cardiovascular disease) a composition comprising a first RNAi agent, a second RNAi agent, and a third RNAi agent, wherein the method reduces further rising of LDL and / or cholesterol levels in the subject overtime, reduces further rising of arterial pressure and / or SBP in the subject over time, and reduces further rising of Lp(a) levels in the subject over time, compared to before the subject is administered the composition. In some embodiments, the reduction is long-lasting (e.g., lasting for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, at least 24 months, or longer) after each administration of the compositions.

[0250] Some aspects of the present disclosure provide methods of maintaining normal levels of LDL and / or cholesterol, arterial pressure and / or SBP, and / or Lp(a) in a subject (e.g., human subject) by administering to the subject any one of the compositions described herein. In some embodiments, the administration is via subcutaneous injection. In some embodiments, the subject does not exhibit a symptom of a cardiovascular disease and / or has not been diagnosed with a cardiovascular disease. In some embodiments, the subject is at risk of developing a cardiovascular disease.

[0251] In some embodiments, in any one of the methods described herein, the subject is a human subject that is less than or is 40 years old (e.g., 40, less than 40, 35, 30, 25, or 20 years old, or younger). In some embodiments, in any one of the methods described herein, the subject is a human subject that is more than 40 years old (e.g., more than 40, 45, 50, 55, 60, 65, 70, 75, 80 years old, or older). In some embodiments, in a method described herein, a composition described herein is administered (e.g., via subcutaneous injection) to a subject (e.g., human subject) having (e.g., exhibits symptoms of and / or is diagnosed with) a cardiovascular disease, and the composition is administered in an amount effective for treating a cardiovascular disease and / or treating anyone of the symptoms of a cardiovascular disease. In some embodiments, the cardiovascular disease is atherosclerotic cardiovascular disease. In some embodiments, the cardiovascular disease is hypertension. In some embodiments, the subject has suffered a cardiovascular event (e.g., stroke, heart attack).

[0252] In some embodiments, in a method described herein, a composition described herein is administered (e.g., via subcutaneous injection) to a subject (e.g., human subject) at risk of developing a cardiovascular disease, and the composition is administered in an amount effective for deterring the development of the cardiovascular disease, before the subject develops symptoms of the cardiovascular disease. In some embodiments, the cardiovascular disease is atherosclerotic cardiovascular disease and / or hypertension. In some embodiments, the subject does not exhibit symptoms and / or has not been diagnosed with a cardiovascular disease. In some embodiments, the subject has not experienced a cardiovascular event (e.g., stroke, heart attack).

[0253] In some embodiments, a subject treated by a composition described herein that comprises long-acting RNAi agent is administered the composition no more than once every 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months, or even less frequently). In some embodiments, the subject is administered the composition once a year. In some embodiments, the subject is administered the composition no more than once a year. In some embodiments, in a method described herein, the subject is administered the composition no more than twice a year. In some embodiments, composition is administered to the subject for a period of time (e.g., the remainder of the subject’s lifetime).

[0254] Having now described some embodiments in detail, practice of the invention will be more fully understood from the following examples, which are presented herein for illustration only and should not be construed as limiting the invention in any way. EXAMPLES

[0255] The potential cardiovascular effects of siRNAs targeting PCSK9 and AGT, alone or in combination, were studied in spontaneously hypertensive rats (Fig. 1). Animals were divided into five groups (8 animals / group) and dosed with a test material comprising either: (1) 5 mg / kg siRNA targeting PCSK9; (2) 5 mg / kg siRNA targeting AGT; (3) both 5 mg / kg siRNA targeting PCSK9 and 5 mg / kg siRNA targeting AGT; or (4) 25-35 mg / kg Valsartan, an angiotensin II receptor blocker, as a control. The fifth group received phosphate-buffered saline (PBS) as an additional control. Mean arterial pressure was monitored during the entire course of the experiment.

[0256] Following euthanasia, relative (to baseline) expression of PCSK9 and AGT mRNA (Figs. 2A-2B) and protein (Figs. 3A-3B) was determined. The results indicated a reduction in both PCSK9 mRNA (Fig. 2A) and protein (Fig. 3A) levels in animals that received either siRNA targeting PCSK9 or both siRNA targeting PCSK9 and siRNA targeting AGT.

[0257] Similarly, a reduction in AGT mRNA (Fig. 2B) and protein (Fig. 3B) levels in animals that received either siRNA targeting AGT or both siRNA targeting PCSK9 and siRNA targeting AGT was noted. No reduction in the mRNA or protein levels of both PCSK9 and AGT was observed in animals in animals treated with either Valsartan or PBS.

[0258] Analysis of cholesterol, relative to baseline, showed decreased levels in animals that were treated with either siRNA targeting PCSK9 or both siRNA targeting PCSK9 and siRNA targeting AGT (Fig. 4). No change from baseline was noted in animals that received either siRNA targeting AGT, Valsartan, or PBS.

[0259] No change in mean arterial pressure was noted in animals that received either siRNA targeting PCSK9 or phosphate-buffered saline, while animals that received either siRNA targeting AGT, siRNA targeting PCSK9 and siRNA targeting AGT, Valsartan, or PBS all displayed reduced mean arterial pressure as a function of time (Fig. 5).

Claims

CLAIMS1. A composition comprising:(i) a first RNAi agent that targets proprotein convertase subtilisin / kexin type 9 (PCSK9) and a second RNAi agent that targets angiotensinogen (AGT);(ii) a first RNAi agent that targets PCSK9 and a third RNAi agent that targets apolipoprotein A (LPA);(iii) a first RNAi agent that targets PCSK9, a second RNAi agent that targets AGT, and the third RNAi agent that targets LPA; or(iv) a second RNAi agent that targets AGT and a third RNAi agent that targets LPA.

2. The composition of claim 1, wherein the first RNAi agent, the second RNAi agent, and / or the third RNAi agent is an siRNA.

3. The composition of claim 2, wherein the siRNA is double stranded.

4. The composition of any one of claims 1-3, wherein the first RNAi agent comprises an antisense strand comprising a region of complementarity to a PCSK9 RNA, and a sense strand that is complementary to the antisense strand.

5. The composition of any one of claims 1-4, wherein the second RNAi agent comprises an antisense strand comprising a region of complementarity to an AGT RNA, and a sense strand that is complementary to the antisense strand.

6. The composition of any one of claims 1-5, wherein the third RNAi agent comprises an antisense strand comprising a region of complementarity to an LPA RNA, and a sense strand that is complementary to the antisense strand.

7. The composition of any one of claims 4-6, wherein the region of complementarity is at least 8 nucleosides in length.

8. The composition of any one of claims 4-7, wherein the antisense strand is 15-35 nucleosides in length and / or the sense strand is 15-35 nucleosides in length.

9. The composition of any one of claims 4-8, wherein the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises an overhang of 0, 1, 2, 3, 4 or 5 nucleosides at the 3’ and / or 5’ end of the sense strand and / or antisense strand.

10. The composition of any one of claims 1-8, wherein the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one modified nucleoside comprising a chemical modification in the base of the nucleoside.

11. The composition of any one of claims 1-8, wherein the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one modified nucleoside comprising a chemical modification in the sugar moiety of the nucleoside.

12. The composition of claim 10 or 11, wherein at least one modified nucleoside comprises a 2’-modified nucleoside.

13. The composition of claim 12, wherein the 2’ modified nucleoside is a 2’-O-methyl modified nucleoside or 2’-F modified nucleoside.

14. The composition of any one of claims 1-13, wherein the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises at least one modified internucleoside linkage.

15. The composition of claim 14, wherein at least one modified internucleoside linkage comprises a phosphorothioate internucleoside linkage.

16. The composition of any one of claims 1-15, wherein the first RNAi agent, the second RNAi agent, and / or the third RNAi agent comprises a biostable phosphate mimic, optionally wherein the biostable phosphate mimic is a vinylpho sphonate moiety.

17. The composition of claim 16, wherein the biostable phosphate mimic is at the 5’ end of the antisense strand.

18. The composition of any one of claims 1-17, wherein the first RNAi agent, the second RNAi agent, and / or the third RNAi agent is conjugated to a targeting moiety.

19. The composition of any one of claims 1-18, wherein the composition comprises the first RNAi agent and the second RNAi agent, wherein the first RNAi agent and the second RNAi agent are not linked.

20. The composition of any one of claims 1-18, wherein the composition comprises the first RNAi agent and the second RNAi agent, wherein the first RNAi agent and the second RNAi agent are covalently linked.

21. The composition of claim 20, wherein the first RNAi agent and the second RNAi agent are covalently linked via a linker, optionally wherein the linker comprises one or more nucleosides, further optionally wherein the linker is a cleavable linker.

22. The composition of any one of claims 1-18, wherein the composition comprises the first RNAi agent and the third RNAi agent, wherein the first RNAi agent and the third RNAi agent are not linked.

23. The composition of any one of claims 1-18, wherein the composition comprises the first RNAi agent and the third RNAi agent, wherein the first RNAi agent and the third RNAi agent are covalently linked.

24. The composition of claim 23, wherein the first RNAi agent and the third RNAi agent are covalently linked via a linker, optionally wherein the linker comprises one or more nucleosides, further optionally wherein the linker is a cleavable linker.

25. The composition of any one of claims 1-18, wherein the composition comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the first RNAi agent, the second RNAi agent, and the third RNAi agent are not linked to each other.

26. The composition of any one of claims 1-18, wherein the composition comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the first RNAi agent is covalently linked to the second RNAi agent, and the third RNAi agent is not linked to the first RNAi agent or to the second RNAi agent.

27. The composition of claim 26, wherein the first RNAi agent and the second RNAi agent are covalently linked via a linker, optionally wherein the linker comprises one or more nucleosides, further optionally wherein the linker is a cleavable linker.

28. The composition of any one of claims 1-18, wherein the composition comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the first RNAi agent is covalently linked to the third RNAi agent, and the second RNAi agent is not linked to the first RNAi agent or to the third RNAi agent.

29. The composition of claim 28, wherein the first RNAi agent and the third RNAi agent are covalently linked via a linker, optionally wherein the linker comprises one or more nucleosides, further optionally wherein the linker is a cleavable linker.

30. The composition of any one of claims 1-18, wherein the composition comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the second RNAi agent is covalently linked to the third RNAi agent, and the first RNAi agent is not linked to the second RNAi agent or to the third RNAi agent.

31. The composition of claim 30, wherein the second RNAi agent and the third RNAi agent are covalently linked via a linker, optionally wherein the linker comprises one or more nucleosides, further optionally wherein the linker is a cleavable linker.

32. The composition of any one of claims 1-18, wherein the composition comprises the first RNAi agent, the second RNAi agent, and the third RNAi agent, wherein the first RNAi agent, the second RNAi agent, and the third RNAi agent are covalently linked to each other.

33. The composition of claim 32, wherein the first RNAi agent, the second RNAi agent, and the third RNAi agent are covalently linked to each other via linkers, optionally wherein each linker comprises one or more nucleosides, further optionally wherein each linker is a cleavable linker.

34. The composition of any one of claims 1-18, wherein the composition comprises the second RNAi agent and the third RNAi agent, wherein the second RNAi agent and the third RNAi agent are not linked.

35. The composition of any one of claims 1-18, wherein the composition comprises the second RNAi agent and the third RNAi agent, wherein the second RNAi agent and the third RNAi agent are covalently linked.

36. The composition of claim 35, wherein the second RNAi agent and the third RNAi agent are covalently linked via a linker, optionally wherein the linker comprises one or more nucleosides, further optionally wherein the linker is a cleavable linker.

37. The composition of any one of claims 1-36, wherein the composition reduces levels of low-density lipoprotein (LDL), systolic blood pressure (SBP), and / or Lipoprotein(a) (Lp(a)) in a subject with high baseline levels of LDL, SBP, and / or Lp(a), and / or maintains normal levels of LDL, SPB, and / or Lp(a) in the subject.

38. The composition of any one of claims 1-37, wherein the composition provides long-lasting reduction of LDL, SBP, and / or Lp(a) in a subject with high baseline levels of LDL, SBP, and / or Lp(a), and / or provides long-lasting maintenance of normal levels of LDL, SBP, and / or Lp(a) in the subject.

39. The composition of claim 37 or claim 38, wherein the subject is human.

40. The composition of claim 39, wherein the human subject is less than 40 years old.

41. The composition of claim 39, wherein the human subject is more than 40 years old.

42. A method of reducing levels of low-density lipoprotein (LDL), systolic blood pressure (SBP), and / or Lipoprotein(a) (Lp(a)) in a subject, the method comprising administering to the subject the composition of any one of claims 1-38, wherein the subject has high baseline levels of LDL, SBP, and / or Lp(a).

43. A method of maintaining normal levels of low-density lipoprotein (LDL), systolic blood pressure (SBP), and / or Lipoprotein(a) (Lp(a)) in a subject, the method comprising administering to the subject the composition of any one of claims 1-38.

44. A method of treating cardiovascular disease in a subject, the method the comprising administering to the subject the composition of any one of claims 1-38.

45. The method of any one of claims 42-44, wherein the subject is less than 40 years old.

46. The method of any one of claims 42-44, wherein the subject is more than 40 years old.

47. The method of any one of claims 42-46, wherein the subject has a cardiovascular disease or is at risk of developing a cardiovascular disease.

48. The method of any one of claims 43-47, wherein the administration begins before the subject develops symptoms of the cardiovascular disease or before the subject is diagnosed with a cardiovascular disease.

49. The method of any one of claims 42-48, wherein the subject is administered the composition no more than once or twice a year.

50. The method of any one of claims 42-48, wherein the subject is administered the composition once a year.

51. The method of any one of claims 42-50, wherein the composition is administered to the subject for a period of time.

52. The method of claim 51, wherein the period of time is the remainder of the subject’s lifetime.

53. The method of any one of claims 42-52, wherein the method reduces levels of LDL, SBP, and / or Lp(a) in a subject with high baseline levels of LDL, SBP, and / or Lp(a), and / or maintains normal levels of LDL, SBP, and / or Lp(a) in a subject with normal baseline levels of LDL, SBP, and / or Lp(a).

54. The method of any one of claims 42-53, wherein the method provides long-lasting reduction of levels of LDL, SBP, and / or Lp(a) in a subject with high baseline levels of LDL, SBP, and / or Lp(a), and / or provides long-lasting maintenance of normal levels of LDL, SBP, and / or Lp(a) in a subject with normal baseline levels of LDL, SBP, and / or Lp(a).

55. The method of any one of claims 44-54, wherein the cardiovascular disease is atherosclerotic cardiovascular disease and / or hypertension.

56. The method of any one of claims 42-55, further comprising measuring the levels of LDL, SBP, and / or Lp(a) in the subject before and / or after administration of the composition.

57. A composition comprising two or more RNAi agents, wherein each of at least two of the RNAi agents targets a different gene selected from: proprotein convertase subtilisin / kexin type 9 (PCSK9), angiotensinogen (AGT), apolipoprotein A (LPA).

58. The composition of claim 57, wherein each of the two or more RNAi agents is an siRNA, optionally a double- stranded siRNA.

59. The composition of claim 57 or 58, wherein each of the two or more RNAi agents has a length of 15-35 nucleosides.

60. The composition of any one of claims 57-59, wherein each of the two or more RNAi agents is chemically modified.

61. The composition of claim 60, wherein each of the two or more RNAi agents comprises a 2’-modified nucleoside, optionally a 2’-O-methyl modified nucleoside or 2’-F modified nucleoside.

62. The composition of any one of claims 57-61, wherein each of the two or more RNAi agents comprises at least one modified intemucleoside linkage, optionally a phosphorothioate intemucleoside linkage.

63. The composition of any one of claims 57-62, wherein each of the two or more RNAi agents comprises a biostable phosphate mimic, optionally a vinylpho sphonate moiety.

64. The composition of any one of claims 57-63, wherein each of the two or more RNAi agents is conjugated to a targeting moiety.

65. The composition of any one of claims 57-64, wherein two or more RNAi agents are covalently linked to each other, optionally via a linker.

66. A method comprising administering to a subject the composition of any one of claims 57-65, wherein the subject has normal or high baseline levels of LDL, SBP, and / or Lp(a).

67. A method of treating cardiovascular disease in a subject, the method comprising administering to the subject the composition of any one of claims 57-65.

68. The method of claim 66 or 67, wherein the subject is older than 40 years old and / or has a cardiovascular disease or is at risk of developing a cardiovascular disease.

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