Compositions and methods for inhibiting gene expression of lpa

Abstract

RNA interference (RNAi) agents and RNAi agent conjugates for inhibiting LPA (apo(a)) gene expression are described. Also described are pharmaceutical compositions comprising one or more LPA RNAi agents, optionally with one or more additional therapeutic agents. In vivo delivery of the LPA RNAi agents to liver cells provides for inhibition of LPA gene expression and treatment of cardiovascular and cardiovascular-related diseases.

Description

[0001] background

[0002] Lipoprotein(a) [Lp(a)] is a heterogeneous low-density lipoprotein (LDL)-like particle containing a lipid core and apolipoprotein B (apoB-100), as well as a unique component, namely apolipoprotein(a) attached to apoB-100 via disulfide bonds (apo(a)).

[0003] The Apo(a) gene (LPA) is primarily expressed in the liver and is limited to expression in humans and non-human primates. Lp(a) levels in humans are genetically determined and do not significantly change with diet, exercise, or other lifestyle modifications. The length of the LPA varies depending on the number of present Kringle KIV2 domains, and its expression is negatively correlated with the number of present domains. Normal Lp(a) levels range from 0.1 to 25 mg / dL, with approximately 25% of the US population having Lp(a) levels of 30 mg / dL or higher.

[0004] Analysis of Lp(a) levels in multiple studies suggests that high Lp(a) levels are an independent risk factor for cardiovascular disease, stroke, and other related conditions, including atherosclerotic stenosis. Furthermore, genome-wide association studies have also investigated LPA as a genetic risk factor for diseases such as atherosclerotic stenosis.

[0005] When therapeutic lipoprotein serotherapy is used to lower both Lp(a) and LDL levels in patients with hyperlipidemia, a significant reduction in cardiovascular events is observed. Therefore, therapeutic agents and treatments are needed for these and other LPA-related conditions.

[0006] Overview

[0007] This article describes LPA (also known as apo(a)) RNA interference (RNAi) agents (also known as RNAi triggers or triggers) and compositions containing LPA RNAi agents for selectively and effectively inhibiting the expression of the LPA gene. LPA is the name of the gene encoding apolipoprotein(a) (apo(a)) (i.e., a key component of lipoprotein(a) particles (Lp(a))). The LPA RNAi agents described herein can be used to prevent or treat the following diseases or to prepare medicines for the prevention or treatment of the following diseases, including but not limited to: Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapolipoprotein β-lipoproteinemia, cerebrovascular arteriosclerosis, cerebrovascular disease, and venous thrombosis.

[0008] Each LPA RNAi agent contains at least one sense strand and one antisense strand. The sense strand and antisense strand may be partially, substantially, or completely complementary to each other. The sense strand and antisense strand of the RNAi agent described herein may each be 17 to 30 nucleotides in length. In some embodiments, the sense strand and antisense strand are independently 17 to 26 nucleotides in length. The sense strand and antisense strand may be the same length or different lengths. The RNAi agent described herein inhibits the expression of the LPA gene in vitro or in vivo after delivery to cells expressing the LPA gene.

[0009] The sense strand of the LPA RNAi agent comprises a nucleotide sequence having at least 90% identity with the sequence in the LPA mRNA over a core segment of at least 17 consecutive nucleotides. In some embodiments, the sense strand nucleotide sequence having at least 90% identity with the sequence in the LPA mRNA is 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. The antisense strand of the LPA RNAi agent comprises a nucleotide sequence having at least 90% complementarity with the sequences in the LPA mRNA and the corresponding sense strand over a core segment of at least 16 consecutive nucleotides. In some embodiments, the antisense strand nucleotide sequence having at least 90% complementarity with the sequence in the LPA mRNA, or the corresponding sense strand, is 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.

[0010] In some embodiments, one or more LPARNAi agents are delivered to target cells or tissues using any oligonucleotide delivery technology known in the art. Nucleic acid delivery methods include, but are not limited to, encapsulation in liposomes, iontophoresis, or incorporation with other mediators such as hydrogels, cyclodextrins, biodegradable nanocapsules and bioadhesive microspheres, protein carriers, or dynamic polyconjugates. TM (See, for example, WO 2000 / 053722, WO 2008 / 0022309, WO 2011 / 104169 and WO 2012 / 083185, each of which is incorporated herein by reference). In some embodiments, the LPA RNAi agent is conjugated to a targeting group. In some embodiments, the targeting group may include a cell receptor ligand, such as a galactose cluster, including a galactose cluster comprising an N-acetyl-galactosamine trimer.

[0011] In some embodiments, a pharmaceutical composition is described as comprising one or more LPA RNAi agents. In some embodiments, the LPA RNAi agent may optionally be combined with one or more additional (i.e., second, third, etc.) therapeutic agents. The additional therapeutic agent may be another LPA RNAi agent (e.g., an LPA RNAi agent targeting a different sequence within the LPA target). The additional therapeutic agent may also be a small molecule drug, antibody, antibody fragment, and / or vaccine. An LPA RNAi agent, with or without the additional therapeutic agent, may be combined with one or more excipients to form a pharmaceutical composition.

[0012] In some embodiments, compositions for in vivo delivery of LPA RNAi agents to liver cells, particularly hepatocytes, are described, comprising: an LPA RNAi agent conjugated to a target group. In some embodiments, the target group is a desialylate glycoprotein ligand.

[0013] Methods for treating human subjects who have or are at risk of developing a pathological condition mediated at least partially by LPA expression are also described, the methods comprising administering to the subject a therapeutically effective amount of an LPA RNAi agent or a composition containing an LPA RNAi agent. The method of treating the subject with an LPA RNAi agent or a composition containing an LPA RNAi agent may optionally be combined with one or more steps of administering one or more additional (i.e., second) therapeutic agents or treatments. The LPA RNAi agent and the additional therapeutic agent may be administered as a single composition, or they may be administered separately. Examples of additional therapeutic agents include, but are not limited to, HMgCo-A reductase inhibitors (statins), ezetimibe, PCSK-9 inhibitors, CTEP inhibitors, therapies targeting ANGPTL3, therapies targeting APOC3, and niacin.

[0014] The use of the LPA RNAi agent described herein may be used in methods for the therapeutic prevention or treatment of the following diseases, including but not limited to: Burger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapolipoprotein β-lipoproteinemia, cerebrovascular arteriosclerosis, cerebrovascular disease, and venous thrombosis. Such methods involve administering the LPA RNAi agent as described herein to a subject, such as a human or animal subject.

[0015] The pharmaceutical composition can be administered in a variety of ways, depending on whether local or systemic treatment is required and the area to be treated. Administration can be prepared by any method known in the art, such as, but not limited to, local (e.g., via a transdermal patch), pulmonary (e.g., by inhalation or spraying of powder or aerosol, including via nebulizer, intratracheal, or intranasal), percutaneous, transdermal, oral, or parenteral administration. Parenteral administration includes, but is not limited to, intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous (via implanted device), intracranial, intraparenchymal, intrathecal, and intraventricular administration. In some embodiments, the pharmaceutical composition described herein is administered by subcutaneous injection.

[0016] The LPA RNAi agents and / or compositions described herein can be used in the therapeutic treatment of the following diseases, including but not limited to: Burger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapolipoprotein β-lipoproteinemia, cerebrovascular arteriosclerosis, cerebrovascular disease, and venous thrombosis. Such methods involve administering the LPA RNAi agents described herein to a subject, such as a human or animal subject.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials may be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and not restrictive.

[0018] Other features and advantages of the invention will become apparent from the following detailed description and claims. Brief description of the attached diagram

[0020] Figure 1 This diagram illustrates the serum Lp(a) protein levels in Lp(a) transgenic (Tg) mice following a single subcutaneous administration of a specified LPA RNAi agent at 0.5 mg / kg (dashed line) or 2 mg / kg (solid line). Lp(a) levels were normalized to day 1 and the saline control.

[0021] Figure 2 This plot illustrates serum Lp(a) protein levels in Lp(a)Tg mice after three subcutaneous doses of the designated LPARNAi agent (dashed line) or 3 mg / kg (solid line) (administered weekly for three weeks, on days 1, 8, and 15). Lp(a) levels were normalized to day 1 and the saline control.

[0022] Figure 3 A graph depicting Lp(a) particle levels in cynomolgus monkey serum following a single administration of 2 mg / kg AD01196 LPA RNAi agent (administered on day 1 at a 1:1 (w / w) ratio with the delivery polymer). Lp(a) levels were normalized to two pre-dose values ​​(shown on day 0).

[0023] Figure 4 The graphs depict Lp(a) particle levels in cynomolgus monkey serum following administration of 4 mg / kg or 6 mg / kg LPA RNAi agent (administered on days 1 and 71 at a 1:1 (w / w) ratio with the delivery polymer). Lp(a) levels were normalized to pre-dose values ​​(shown on day 0). 4 mg / kg dose = black circle; 6 mg / kg dose = gray square.

[0024] Figure 5 The graph illustrates the Lp(a) particle levels in cynomolgus monkey serum following three weekly subcutaneous doses of 3 mg / kg AD02819 LPA RNAi on days 1, 8, and 15. Lp(a) levels were normalized to the three pre-dose values ​​(shown on day 0).

[0025] Figure 6The graph illustrates the Lp(a) particle levels in cynomolgus monkey serum following a single subcutaneous administration of 3 mg / kg LPA RNAi on day 1. The AD03460 and AD03536 groups received an additional 1 mg / kg dose of LPA RNAi on day 48. Lp(a) levels were normalized to three pre-dose values ​​(shown on day 0).

[0026] Detailed Explanation

[0027] This document describes RNAi agents (referred to herein as LPA RNAi agents) for inhibiting the expression of the LPA gene. Upon delivery to cells expressing the LPA gene, the RNAi agents described herein inhibit or knock down LPA expression in vitro and / or in vivo through the biological process of RNA interference (RNAi). As used herein, unless otherwise specifically indicated, LPA may refer to the LPA gene, LPA mRNA, or LP(a) protein where appropriate.

[0028] LPA RNAi agents comprise a sense strand and an antisense strand. Each sense strand and antisense strand contains a core sequence of 17-23 nucleotides in length. The antisense strand core sequence is 100% (perfectly) complementary or at least 90% (substantially) complementary to the nucleotide sequence present in the LPA mRNA (sometimes referred to as the target sequence). The sense strand core sequence is 100% (perfectly) complementary or at least 90% (substantially) complementary to the sequence in the antisense strand, and therefore the sense strand core sequence is perfectly identical or at least 90% identical to the nucleotide sequence present in the LPA mRNA (target sequence). The sense strand core sequence may have the same length as the corresponding antisense core sequence or may be of a different length. In some embodiments, the antisense strand core sequence is 17, 18, 19, 20, 21, 22, or 23 nucleotides long. In some embodiments, the sense strand core sequence is 17, 18, 19, 20, 21, 22, or 23 nucleotides long.

[0029] The sense and antisense strands of the LPA RNAi agent anneal to form a double helix. The sense and antisense strands of the LPA RNAi agent are partially, substantially, or completely complementary to each other. Within the complementary double helix region, the sense core sequence is at least 90% or 100% complementary to the antisense core sequence. In some embodiments, the sense core sequence contains a sequence of at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides that are at least 90% or 100% complementary to the corresponding 17, 18, 19, 20, or 21 nucleotide sequence of the antisense core sequence (i.e., the sense and antisense core sequences of the LPA RNAi agent have regions of at least 90% or 100% base pairing of at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides).

[0030] As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence (such as a sense strand of an RNAi agent or LPA mRNA) in relation to a second nucleotide sequence (such as an antisense strand of an RNAi agent), refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize (form base pair hydrogen bonds) with an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions and form a double helix or double-stranded structure. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include native or modified nucleotides or nucleotide mimics, provided that the above requirements regarding their hybridization ability are met. "Completely complementary" or "fully complementary" means that all (100%) bases in the sequential sequence of the first polynucleotide will hybridize with the same number of bases in the sequential sequence of the second polynucleotide. The sequential sequence may contain all or part of the first or second nucleotide sequence. As used herein, "partially complementary" means that in the hybridized nucleobase sequence pair, at least 70% of the bases in the first polynucleotide sequence hybridize with the same number of bases in the second polynucleotide sequence. As used herein, "substantially complementary" means that in the hybridized nucleobase sequence pair, at least 85% of the bases in the first polynucleotide sequence hybridize with the same number of bases in the second polynucleotide sequence. The terms "complementary," "fully complementary," and "substantially complementary" as used herein can be used in relation to base matching between the sense and antisense strands of the RNAi agent or between the antisense strand of the RNAi agent and the sequence of the LPA mRNA. Sequence identity or complementarity is independent of modification. For the purpose of determining identity or complementarity, for example, a and Af are complementary to U (or T) and identical to A.

[0031] The LPA RNAi agent described herein has sense and antisense strands that are independently 17 to 30 nucleotides long. In some embodiments, the sense and antisense strands are independently 17 to 26 nucleotides long. In some embodiments, the sense and antisense strands are 19 to 26 nucleotides long. In some embodiments, the RNAi agent has sense and antisense strands that are independently 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides long. The sense and antisense strands may be the same length or they may be different lengths. In some embodiments, the sense and antisense strands are each 26 nucleotides long. In some embodiments, the sense strand is 23 nucleotides long and the antisense strand is 21 nucleotides long. In some embodiments, the sense strand is 22 nucleotides long and the antisense strand is 21 nucleotides long. In some embodiments, the sense strand is 21 nucleotides long and the antisense strand is 21 nucleotides long. In some implementations, the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long.

[0032] The sense strand and / or antisense strand may optionally and independently contain 1, 2, 3, 4, 5, or 6 additional nucleotides (extensions) at the 3′ end, 5′ end, or both 3′ and 5′ ends of the core sequence. If the additional nucleotides of the antisense strand are present, they may or may not be complementary to the corresponding sequence in the LPA mRNA. If the additional nucleotides of the sense strand are present, they may or may not be identical to the corresponding sequence in the LPA mRNA. If the additional nucleotides of the antisense strand are present, they may or may not be complementary to the additional nucleotides of the corresponding sense strand.

[0033] As used herein, the extension comprises 1, 2, 3, 4, 5, or 6 nucleotides at the 5′ and / or 3′ ends of the sense strand core sequence and / or the antisense strand core sequence. The extended nucleotides on the sense strand may or may not be complementary to the corresponding nucleotides (core sequence nucleotides or extended nucleotides) in the antisense strand. Conversely, the extended nucleotides on the antisense strand may or may not be complementary to the corresponding nucleotides (core sequence nucleotides or extended nucleotides) in the sense strand. In some embodiments, both the sense and antisense strands of the RNAi agent contain 3′ and 5′ extensions. In some embodiments, one or more 3′ extended nucleotides of one strand pair with one or more 5′ extended nucleotides of the other strand. In other embodiments, one or more 3′ extended nucleotides of one strand do not pair with one or more 5′ extended nucleotides of the other strand. In some embodiments, the LPA RNAi agent has an antisense strand with a 3′ extension and a sense strand with a 5′ extension.

[0034] In some embodiments, the LPA RNAi agent comprises a 3′ extended antisense strand having a length of 1, 2, 3, 4, 5, or 6 nucleotides. In other embodiments, the LPA RNAi agent comprises a 3′ extended antisense strand having a length of 1, 2, or 3 nucleotides. In some embodiments, one or more antisense strand extending nucleotides comprise uracil or thymidine nucleotides or nucleotides complementary to the corresponding LPAmRNA sequence. In some embodiments, the 3′ antisense strand extension comprises, but is not limited to, the following, but consists of, but is not limited to: Ab, AbAb, AUA, UGCUU, CUG, UG, UGCC, CUGCC, CGU, CUU, UGCCUA, CUGCCU, UGCCU, UGAUU, GCCUAU, T, TT (each listed from 5′ to 3′).

[0035] In some embodiments, the LPA RNAi agent comprises a 5′ antisense strand with a length of 1, 2, 3, 4, or 5 nucleotides. In other embodiments, the LPA RNAi agent comprises a 5′ antisense strand with a length of 1 or 2 nucleotides. In some embodiments, one or more antisense strand extending nucleotides comprise uracil or thymidine nucleotides or nucleotides complementary to the corresponding LPA mRNA sequence. In some embodiments, the 5′ antisense strand extension comprises, but is not limited to, UA, TU, U, T, CUC (each listed as 5′ to 3′). If present, the antisense strand may have a combination of any of the above 3′ extensions with any of the stated 5′ antisense strand extensions.

[0036] In some embodiments, the LPA RNAi agent comprises a 3′ sense strand having a length of 1, 2, 3, 4, or 5 nucleotides. In some embodiments, one or more sense strand extending nucleotides include adenosine, uracil, or thymidine nucleotides, AT dinucleotides, or nucleotides corresponding to nucleotides in the LPA mRNA sequence. In some embodiments, the 3′ sense strand extension comprises, but is not limited to, T, UUAb, UAB, Ab, UT, TT, UUT, TTT, or TTTT (each listed from 5′ to 3′).

[0037] In some embodiments, the LPA RNAi agent comprises a sense strand with a 5′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, one or more sense strand extending nucleotides comprise uracil or adenosine nucleotides or nucleotides corresponding to nucleotides in the LPAmRNA sequence. In some embodiments, the sense strand 5′ extension can be, but is not limited to: CA, AUAGGC, AUAGG, AUAG, AUA, A, AA, AC, GCA, GGCA, GGC, UAUCA, UAUC, Ab, UCA, UAU (each listed from 5′ to 3′). The sense strand may have a 3′ extension and / or a 5′ extension.

[0038] Examples of nucleotide sequences used to form LPA RNAi agents are provided in Tables 1, 2A, 2B, 3A, and 3B. As used herein, the term "sequence" or "nucleotide sequence" refers to a sequence of nucleobases, nucleotides, and / or nucleosides, whether modified or unmodified, described in a series of letters using standard nucleotide nomenclature and a symbol table of modified nucleotides as described herein.

[0039] RNAi agents include, but are not limited to: short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and cleavage enzyme substrates (such as U.S. Patent Nos. 8084599, 8349809, and 8513207).

[0040] The sense and antisense sequences of the unmodified LPA RNAi agent are provided in Table 1. During the formation of the LPA RNAi agent, each nucleotide in each sequence listed in Table 1 may be a modified nucleotide.

[0041] Table 1. Antisense and sense sequences of unmodified LPA RNAi agents.

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048] Ab = non-base nucleotide

[0049] The LPA RNAi agents described herein are formed by annealing the antisense strand with the sense strand. A sense strand containing a sequence listed in Table 1 or Table 2B can be hybridized with any antisense strand containing a sequence listed in Table 1 or Table 2A, provided that the two sequences are at least 90% complementary over 16, 17, 18, 19, 20, or 21 consecutive nucleotide sequences.

[0050] In some embodiments, the antisense strand of the LPA RNAi agent comprises the nucleotide sequence of any sequence in Table 1. In some embodiments, the antisense strand of the LPA RNAi agent comprises the nucleotide sequence of any sequence in Table 1, specifically nucleotides 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24, 2-24, 1-25, 2-25, 1-26, or 2-26. In some embodiments, the sense strand of the LPA RNAi agent comprises the nucleotide sequence of any sequence in Table 1. In some embodiments, the sense strand of the LPA RNAi agent comprises any of the nucleotide sequences 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24, 2-24, 1-25, 2-25, 1-26, or 2-26 from Table 1.

[0051] In some embodiments, the sense and antisense strands of the RNAi agent described herein contain the same number of nucleotides. In some embodiments, the sense and antisense strands of the RNAi agent described herein contain different numbers of nucleotides. In some embodiments, the 5′ end of the sense strand and the 3′ end of the antisense strand of the RNAi agent form blunt ends. In some embodiments, the 3′ end of the sense strand and the 5′ end of the antisense strand of the RNAi agent form blunt ends. In some embodiments, both ends of the RNAi agent form blunt ends. In some embodiments, neither end of the RNAi agent is blunt. As used herein, a blunt end refers to the end of a double-stranded RNAi agent in which the terminal nucleotides of the two annealed strands are complementary (forming complementary base pairs). In some embodiments, the 5′ end of the sense strand and the 3′ end of the antisense strand of the RNAi agent form broken ends. In some embodiments, the 3′ end of the sense strand and the 5′ end of the antisense strand of the RNAi agent form broken ends. In some embodiments, both ends of the RNAi agent form broken ends. In some embodiments, neither end of the RNAi agent is broken. As used herein, a brittle end refers to the end of a double-stranded RNAi agent in which the terminal nucleotides of the two annealed strands form a pair (i.e., no overhang) but are not complementary (i.e., form a non-complementary pair). As used herein, an overhang is a segment of one or more unpaired nucleotides at the end of one strand of a double-stranded RNAi agent. The unpaired nucleotides may be on the sense strand or antisense strand, thus creating a 3′ or 5′ overhang. In some embodiments, the RNAi agent contains: blunt ends and brittle ends, blunt ends and 5′ overhang ends, blunt ends and 3′ overhang ends, brittle ends and 5′ overhang ends, brittle ends and 3′ overhang ends, two 5′ overhang ends, two 3′ overhang ends, 5′ overhang ends and 3′ overhang ends, two brittle ends, or two blunt ends.

[0052] Nucleotide bases (or nucleobases) are heterocyclic pyrimidine or purine compounds that are components of all nucleic acids and include adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). As used herein, the term "nucleotide" may include modified nucleotides or nucleotide mimics, base-free sites (Ab or X), or substituted portions.

[0053] In some implementations, the LPA RNAi agent is prepared as or provided as a salt, a mixed salt, or a free acid.

[0054] Modified nucleotides

[0055] In some embodiments, the LPA RNAi agent contains one or more modified nucleotides. As used herein, "modified nucleotide" is a nucleotide other than a ribonucleotide (2′-hydroxynucleotide). In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the nucleotides are modified. Modified nucleotides include, but are not limited to, deoxynucleotides, nucleotide mimics, baseless nucleotides (represented herein as X, Ab), 2′-modified nucleotides, 3′ to 3-linked (reverse) nucleotides (represented herein as invdN, invN, invn, invX, invAb), nucleotides containing non-natural bases, bridging nucleotides, peptide nucleic acids (PNA), and 2′,3′-broken nucleotide mimics (unlocked nucleobase analogs, represented herein as N). UNA Or NUNA), locked nucleotides (represented as N in this paper) LNA Or NLNA), 3'-O-methoxy (2' nucleoside-linked) nucleotide (represented as 3'-OMen in this paper), 2'-F-arabinonucleotide (represented as NfANA or Nf in this paper). ANA 5′-Me,2′-fluoronucleotides (referred to herein as 5Me-Nf), morpholinonucleotides, phosphonoethylene deoxyribonucleotides (referred to herein as vpdN), phosphonoethylene nucleotides, and phosphonocyclopropyl nucleotides (cPrpN). 2′-Modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2′ position of the five-membered sugar ring) include, but are not limited to, 2′-O-methyl nucleotides (referred to herein as lowercase ′n′ in the nucleotide sequence), 2′-deoxy-2′-fluoronucleotides (referred to herein as Nf, and also as 2′-fluoronucleotides), 2′-deoxynucleotides (referred to herein as dN), 2′-methoxyethyl (2′-O-2-methoxyethyl) nucleotides (referred to herein as NM or 2′-MOE), 2′-amino nucleotides, and 2′-alkyl nucleotides. It is not necessary to uniformly modify all positions in a given compound. Conversely, more than one modification can be incorporated into a single LPA RNAi agent or even into its single nucleotide. The sense and antisense strands of LPA RNAi agents can be synthesized and / or modified using methods known in the art. A modification at one nucleotide is independent of modifications at another nucleotide.

[0056] Modified nucleotides also include nucleotides having modified nucleobases. Modified nucleobases include, but are not limited to, synthetic and natural nucleobases, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halogenated uridine... Pyrimidines and cytosines, 5-propynyluracil and cytosines, 6-azouracil, cytosine and thymines, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogenated, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazoguanine and 7-deazoadenine, and 3-deazoguanine and 3-deazoadenine.

[0057] Modified nucleoside interlinking

[0058] In some embodiments, one or more nucleotides of the LPA RNAi agent are linked by non-standard linkages or the backbone (i.e., modified nucleoside linkages or modified backbones). In some embodiments, the modified nucleoside linkages are non-phosphate-containing covalent nucleoside linkages. Modified internucleotide linkages or backbones include, but are not limited to, 5′-thiophosphate groups with normal 3′-5′ linkages (represented herein as lowercase 's′ before the nucleotide, such as in sN, sn, sNf, or sdN), chiral thiophosphates, thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl-phosphate triesters, methyl phosphonates, and other alkyl esters including 3′-alkylenephosphonates and chiral phosphonates, phosphinates, aminophosphates including 3′-aminoaminophosphates and aminoalkylaminophosphates, thiocarbonylaminophosphates, thiocarbonylalkyl-phosphonates, thiocarbonylalkyl phosphate triesters, morpholino linkages, and borane phosphates; 2′-5′ linkage analogs of these substances; and those with reverse polarity, wherein adjacent pairs of nucleoside units are 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′ linkages. In other embodiments, the modified internucleotide links or backbones are phosphorus-free. Phosphorus-free modified internucleotide links include, but are not limited to, intra-linkage of short-chain alkyl or cycloalkyl sugars, intra-linkage of mixed heteroatoms and alkyl or cycloalkyl sugars, and intra-linkage of one or more short-chain heteroatoms or heterocyclic sugars. In some embodiments, the modified internucleotide backbones include, but are not limited to, siloxane backbones, sulfide, sulfone, and sulfone backbones; formacetyl and thioformyl backbones, methyleneformyl and thioformyl backbones, olefin-containing backbones, aminosulfonate backbones, methyleneimino and methylenehydrazine backbones, sulfonate and sulfonamide backbones, amide backbones; and others having mixed N, O, S, and CH2 component moieties.

[0059] In some implementations, the sense strand of the LPA RNAi agent may contain 1, 2, 3, or 4 phosphate thioester bonds, and the antisense strand of the LPA RNAi agent may contain 1, 2, 3, or 4 phosphate thioester bonds, or both the sense strand and the antisense strand may independently contain 1, 2, 3, or 4 phosphate thioester bonds.

[0060] In some embodiments, the sense strand of the LPA RNAi agent contains two phosphate-thioester nucleoside links. In some embodiments, the two phosphate-thioester nucleoside links are between nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, the two phosphate-thioester nucleoside links are between nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, or 6-8 from the 5' end of the sense strand. In some embodiments, the antisense strand of the LPA RNAi agent contains four phosphate-thioester nucleoside links. In some embodiments, the four phosphate-thioester nucleoside links are between nucleotides at positions 1-3 from the 5' end of the sense strand, and between nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end of the sense strand. In some implementations, the LPA RNAi agent contains two thiophosphate nucleoside links in the sense strand and four thiophosphate nucleoside links in the antisense strand.

[0061] In some embodiments, the LPA RNAi agent contains one or more modified nucleotides and one or more modified nucleoside links. In some embodiments, the 2′-modified nucleotide is combined with the modified nucleoside link.

[0062] LPA RNAi agents with modified nucleotides

[0063] Examples of antisense strands containing modified nucleotides are provided in Table 2A. Examples of sense strands containing modified nucleotides are provided in Table 2B. In Tables 2A and 2B, the following symbols are used to indicate modified nucleotides:

[0064]

[0065]

[0066] In addition, the following targeting and linking groups are listed in Tables 2A and 2B: (Alk-PEG5-C6), (C11-PEG3-NAG3), (C12), (C6-PEG4-NAG3), (C6-SS-Alk-Me), (Chol-TEG), (Dy540), (NAG13), (NAG18), (NAG24), (NAG25), (NAG26), (NAG27), (NAG28), (NAG29), (NAG30), (NAG31), (NAG32), (NAG33), (NAG34), (NAG35), (NAG36), (NAG37), (NAG4), (PAZ), (Sp18), (Steryl), (Alk-SMPT-C6). Each sense and / or antisense strand may have any of the above-specified targeting or linking groups, as well as other targeting or linking groups conjugated to the 5′ and / or 3′ ends of the sequence. The chemical structures of these groups are provided in Table 4.

[0067] Table 2A. Antisense strands of LPA RNAi agents with modified nucleotides.

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] Table 2B. Sense strands of LPA RNAi agents with modified nucleotides.

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093] A sense strand containing the sequences listed in Table 2B can be hybridized to any antisense strand containing the sequences listed in Table 2A, provided that the two sequences have at least 90% complementarity over a consecutive 16, 17, 18, 19, 20, or 21 nucleotide sequence. Representative LPA RNAi agents are indicated by the duplex ID No. shown in Tables 3A and 3B.

[0094] In some embodiments, the LPA RNAi agent comprises any of the duplexes D presented herein. In some embodiments, the LPA RNAi agent comprises any of the duplexes ID presented herein. In some embodiments, the LPA RNAi agent comprises the sense and antisense nucleotide sequences of any of the duplexes ID presented herein. In some embodiments, the LPA RNAi agent comprises the sense and antisense nucleotide sequences of any of the duplexes ID presented herein, and a targeting group and / or a linker group, wherein the targeting group and / or linker group are covalently linked (i.e., conjugated) to the sense or antisense strand. In some embodiments, the LPA RNAi agent comprises modified nucleotide sequences of the sense and antisense strands of any of the duplexes ID presented herein. In some embodiments, the LPA RNAi agent comprises modified nucleotide sequences of the sense and antisense strands of any of the duplexes ID presented herein, and a targeting group and / or a linker group, wherein the targeting group and / or linker group are covalently linked to the sense or antisense strand.

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1242 (TCGUAUAACAAUAAGGGGC). In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1244 (UCGUAUAACAAUAAGGGG). In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1246 (UCGUAUAACAAUAAGGG). In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1248 (TGAGAAUGAGCCUCGAUAA). In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1250 (UGAGAAUGAGCCUCGAUA). In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1252 (UGAGAAUGAGCCUCGAU). In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1254 (UGUAUAACAAUAAGGGG). In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1280 (CGUAUAACAAUAAGGGGC). In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1281 (GAGAAUGAGCCUCGAUAA). In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1282 (UCGUAUAACAAUAAGGGGC). In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1283 (UGAGAAUGAGCCUCGAUAA). In some embodiments, one or more nucleotides are modified.

[0119] In some embodiments, the LPA RNAi agent comprises a sense strand containing the nucleotide sequence of SEQ ID NO: 1243 (GCCCCUUAUUGUUAUACGA). In some embodiments, the LPA RNAi agent comprises a sense strand containing the nucleotide sequence of SEQ ID NO: 1245 (CCCCUUAUUGUUAUACGA). In some embodiments, the LPA RNAi agent comprises a sense strand containing the nucleotide sequence of SEQ ID NO: 1247 (CCCUUAUUGUUAUACGA). In some embodiments, the LPA RNAi agent comprises a sense strand containing the nucleotide sequence of SEQ ID NO: 1249 (UUAUCGAGGCUCAUUCUCA). In some embodiments, the LPA RNAi agent comprises a sense strand containing the nucleotide sequence of SEQ ID NO: 1251 (UAUCGAGGCUCAUUCUCA). In some embodiments, the LPA RNAi agent comprises a sense strand containing the nucleotide sequence of SEQ ID NO: 1253 (AUCGAGGCUCAUUCUCA). In some embodiments, the LPA RNAi agent comprises a sense strand containing the nucleotide sequence of SEQ ID NO: 1255 (CCCCUUAUUGUUAUACA). In some embodiments, the LPA RNAi agent comprises a sense strand containing the nucleotide sequence of SEQ ID NO: 1284 (GCCCCUUAUUGUUAUACG). In some embodiments, the LPA RNAi agent comprises a sense strand containing the nucleotide sequence of SEQ ID NO: 1285 (UUAUCGAGGCUCAUUCUC). In some embodiments, one or more nucleotides are modified.

[0120] In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1242 and a sense strand containing the nucleotide sequence of SEQ ID NO: 1243. In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1244 and a sense strand containing the nucleotide sequence of SEQ ID NO: 1245. In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1246 and a sense strand containing the nucleotide sequence of SEQ ID NO: 1247. In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1248 and a sense strand containing the nucleotide sequence of SEQ ID NO: 1249. In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1250 and a sense strand containing the nucleotide sequence of SEQ ID NO: 1251. In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1252 and a sense strand containing the nucleotide sequence of SEQ ID NO: 1253. In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1254 and a sense strand containing the nucleotide sequence of SEQ ID NO: 1255. In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1280 and a sense strand containing the nucleotide sequence of SEQ ID NO: 1284. In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1281 and a sense strand containing the nucleotide sequence of SEQ ID NO: 1285. In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1282 and a sense strand containing the nucleotide sequence of SEQ ID NO: 1259. In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO: 1283 and a sense strand containing the nucleotide sequence of SEQ ID NO: 1249. In some embodiments, one or more nucleotides are modified.

[0121] In some embodiments, the LPA RNAi agent comprises an antisense strand containing the nucleotide sequence of SEQ ID NO. 156, 164, or 188. In some embodiments, the LPA RNAi agent comprises a sense strand containing the nucleotide sequence of SEQ ID NO. 310, 357, 384, or 376. In some embodiments, the LPA RNAi agent comprises both an antisense strand and a sense strand containing the nucleotide sequence of SEQ ID No: 156 / 310, SEQ ID No: 164 / 357, SEQ ID No: 188 / 384, SEQ ID No: 164 / 376, or SEQ ID No: 164 / 384. In some embodiments, one or more nucleotides are modified.

[0122] In some embodiments, the LPA RNAi agent comprises an antisense strand containing a nucleotide sequence of SEQ ID NO: 637, 709, 790, 787, or 788. In some embodiments, the LPA RNAi agent comprises a sense strand containing a nucleotide sequence of SEQ ID NO: 1132, 1135, 1189, 1191, or 1186. In some embodiments, the LPA RNAi agent comprises both an antisense strand and a sense strand containing nucleotide sequences of SEQ ID No: 637 / 1132, SEQ ID No: 709 / 1135, SEQ ID No: 790 / 1189, SEQ ID No: 787 / 1191, or SEQ ID No: 788 / 1186.

[0123] In some embodiments, the LPA RNAi agent comprises SEQ ID NO: 637, 709, 790, 787, or 788. In some embodiments, the LPA RNAi agent comprises SEQ ID NO: 1132, 1135, 1189, 1191, or 1186. In some embodiments, the LPA RNAi agent comprises SEQ ID No: 637 / 1132, SEQ ID No: 709 / 1135, SEQ ID No: 790 / 1189, SEQ ID No: 787 / 1191, or SEQ ID No: 788 / 1186.

[0124] In some embodiments, the LPA RNAi agent comprises SEQ ID NO: 637, 709, 790, 787, or 788. In some embodiments, the LPA RNAi agent comprises SEQ ID NO: 1132, 1135, 1189, 1191, or 1186. In some embodiments, the LPA RNAi agent comprises SEQ ID No: 637 / 1132, SEQ ID No: 709 / 1135, SEQ ID No: 790 / 1189, SEQ ID No: 787 / 1191, or SEQ ID No: 788 / 1186.

[0125] In some implementations, the LPA RNAi agent comprises an antisense strand and a sense strand having a nucleotide sequence of either an antisense / sense duplex as shown in Table 3A or 3B.

[0126] In some embodiments, the LPA RNAi agent comprises an antisense strand and a sense strand having a nucleotide sequence having any of the antisense / sense duplexes in Table 3A or 3B, and also comprises a desialylate glycoprotein receptor ligand targeting group.

[0127] In some embodiments, the LPA RNAi agent comprises an antisense strand and a sense strand having a nucleotide sequence having any of the antisense / sense duplexes of Table 3A or 3B, and further comprises a targeting group selected from the group consisting of: , (C11-PEG3-NAG3), (C6-PEG4-NAG3), (NAG 3), (NAG4), , (NAG3-Palm), (NAG13), (NAG18), (NAG24), (NAG25), (NAG25)s, (NAG26), (NAG27), (NAG28), (NAG29), (N AG30), (NAG30)s, (NAG31), (NAG13), (NAG31s), (NAG32), (NAG33), (NAG34), (NAG35), (NAG36) and (NAG37).

[0128] In some implementations, the LPA RNAi agent comprises an antisense strand and a sense strand of a modified nucleotide sequence having any of the antisense / sense duplexes in Table 3A or 3B.

[0129] In some embodiments, the LPA RNAi agent comprises an antisense strand and a sense strand of a modified nucleotide sequence having any of the antisense / sense duplexes in Table 3A or 3B, and also comprises a desialylate glycoprotein receptor ligand targeting group.

[0130] In some embodiments, the LPA RNAi agent comprises an antisense strand and a sense strand of a modified nucleotide sequence having any of the antisense / sense duplexes in Table 3A or 3B, and further comprises a targeting group selected from the group consisting of: (C11-PEG3-NAG3), (C11-PEG3-NAG3), (C6-PEG4-NAG3), (NAG3), (NAG4), (NAG3-AA2), (NAG3-Palm), (NAG13), (NAG18), (NAG24), (NAG25), (NAG25)s, (NAG26), (NAG27), (NAG28), (NAG29), (NAG30), (NAG30)s, (NAG31), (NAG13), (NAG31s), (NAG32), (NAG33), (NAG34), (NAG35), (NAG36), and (NAG37).

[0131] In some implementations, the LPA RNAi agent comprises any double strand from Table 3A or 3B.

[0132] In some implementations, the LPA RNAi agent consists of any double strand from Table 3A or 3B.

[0133] In some implementations, the LPA RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences AD03460, AD03536, AD03851, AD03853, AD3847, or AD04110.

[0134] In some embodiments, the LPA RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences AD03460, AD03536, AD03851, AD03853, AD3847 or AD04110, and also comprises a desialylate glycoprotein receptor ligand targeting group.

[0135] In some embodiments, the LPA RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences AD03460, AD03536, AD03851, AD03853, AD3847, or AD04110, and further comprises a targeting group selected from the group consisting of: (C11-PEG3-NAG3), (C11-PEG3-NAG3), (C6-PEG4-NAG3), (NAG... 3), (NAG4), (NAG3-AA2), (NAG3-Palm), (NAG13), (NAG18), (NAG24), (NAG25), (NAG25)s, (NAG26), (NAG27), (NAG28), (NAG29), (NAG30), (NAG30)s, (NAG31), (NAG13), (NAG31s), (NAG32), (NAG33), (NAG34), (NAG35), (NAG36), and (NAG37).

[0136] In some implementations, the LPA RNAi agent comprises an antisense strand and a sense strand having modified nucleotide sequences AD03460, AD03536, AD03851, AD03853, AD3847, or AD04110.

[0137] In some embodiments, the LPA RNAi agent comprises an antisense strand and a sense strand having modified nucleotide sequences AD03460, AD03536, AD03851, AD03853, AD3847 or AD04110, and also comprises a desialylate glycoprotein receptor ligand targeting group.

[0138] In some embodiments, the LPA RNAi agent comprises an antisense strand and a sense strand having modified nucleotide sequences AD03460, AD03536, AD03851, AD03853, AD3847, or AD04110, and further comprises a targeting group selected from the group consisting of: (C11-PEG3-NAG3), (C11-PEG3-NAG3), (C6-PEG4-NAG3), (NAG3), (NAG4), (NAG3-AA2), (NAG3... -Palm), (NAG13), (NAG18), (NAG24), (NAG25), (NAG25)s, (NAG26), (NAG27), (NAG28), (NAG29), (NAG3 0), (NAG30)s, (NAG31), (NAG13), (NAG31s), (NAG32), (NAG33), (NAG34), (NAG35), (NAG36) and (NAG37).

[0139] In some implementations, the LPA RNAi agent comprises AD03460, AD03536, AD03851, AD03853, AD3847, or AD04110.

[0140] In some implementations, the LPA RNAi agent consists of AD03460, AD03536, AD03851, AD03853, AD3847, or AD04110.

[0141] Non-nucleotide groups

[0142] In some embodiments, the LPA RNAi agent contains or is conjugated to one or more nonnucleotide groups, including but not limited to targeting groups, linker groups, delivery polymers, or delivery mediators. The nonnucleotide groups can enhance the targeting, delivery, or attachment of the RNAi agent. Examples of targeting groups and linker groups are provided in Table 4. The nonnucleotide groups can be covalently linked to the 3′ and / or 5′ ends of the sense strand and / or antisense strand. In some embodiments, the LPA RNAi agent contains nonnucleotide groups linked to the 3′ and / or 5′ ends of the sense strand. In some embodiments, the nonnucleotide groups are linked to the 5′ end of the sense strand of the LPA RNAi agent. The nonnucleotide groups can be directly or indirectly attached to the RNAi agent via a linker / connector group. In some embodiments, the nonnucleotide groups are attached to the RNAi agent via an unstable, cleavable, or reversible bond or linker.

[0143] In some embodiments, the nonnucleotide group enhances the pharmacokinetic or biodistribution properties of the RNAi agent or conjugate to which it is attached, thereby improving the cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, the nonnucleotide group enhances the endocytosis of the RNAi agent.

[0144] Targeting group

[0145] The targeting group can be monovalent, divalent, trivalent, tetravalent, or have a higher valence. Representative targeting groups include, but are not limited to, compounds with affinity for cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity for somatic cell surface molecules. In some embodiments, a linker such as a PEG linker or one, two, or three base-free and / or ribitol groups is used to attach the targeting group to the RNAi agent. In some embodiments, the targeting group includes a galactose cluster.

[0146] The LPA RNAi agents described herein can be synthesized with a reactive group (e.g., an amino group) at the 5′ end. This reactive group can be used to subsequently attach the target moiety using methods typical in the art.

[0147] In some embodiments, the targeting group includes a desialylate glycoprotein receptor ligand. In some embodiments, the desialylate glycoprotein receptor ligand comprises one or more galactose derivatives or galactose clusters, or is composed of one or more galactose derivatives or galactose clusters. As used herein, the term galactose derivative includes both galactose and galactose derivatives having an affinity for the desialylate glycoprotein receptor (equal to or greater than that of galactose). Galactose derivatives include, but are not limited to, galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, and N-iso-butyrylgalactosamine (see, for example: Iobst, ST, and Drickamer, KJBC 1996, 271, 6686). Galactose derivatives and galactose clusters that can be used to target oligonucleotides and other molecules to the liver in vivo are known in the art (see, e.g., Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Galactose derivatives have been used in vivo to target molecules to hepatocytes by binding to the asialic acid glycoprotein receptor (ASGPr) expressed on the surface of hepatocytes. Binding of ASGPr ligands to ASGPr promotes cell-specific targeting to hepatocytes and internalization of molecules into hepatocytes. Galactose clusters can be attached to the 3′ or 5′ end of RNAi polynucleotides using methods known in the art.

[0148] As used herein, a galactose cluster comprises a molecule having two or four terminal galactose derivatives. The terminal galactose derivatives are attached to the molecule via their C-1 carbon. In some embodiments, the galactose cluster is a galactose derivative trimer, a triacylgalactose derivative, or a trivalent galactose derivative. In some embodiments, the galactose cluster comprises N-acetylgalactosamine (GalNAc). In some embodiments, the galactose cluster comprises trivalent N-acetyl-galactosamine. In some embodiments, the galactose cluster is a galactose derivative tetramer, a tetraacylgalactose derivative, or a tetravalent galactose derivative. In some embodiments, the galactose cluster comprises tetravalent N-acetyl-galactosamine.

[0149] As used herein, a galactose trimer contains three galactose derivatives, each attached to a central branch point. As used herein, a galactose derivative tetramer contains four galactose derivatives, each attached to a central branch point. The galactose derivatives can be attached to the central branch point via the C-1 carbon of the sugar. In some embodiments, the galactose derivatives are attached to the branch point via a linker or spacer. In some embodiments, the linker or spacer is a flexible hydrophilic spacer, such as a PEG group (see, for example, U.S. Patent No. 5,885,968; Biessen et al., J. Med. Chem. 1995, Vol. 39, pp. 1538-1546). In some embodiments, the PEG spacer is a PEG3 spacer. The branch point can be any small molecule that allows attachment of the three galactose derivatives and further allows attachment of the branch point to an RNAi agent. Examples of branch point groups are 2-lysine or 2-glutamic acid. Attachment of the branch point to the RNAi agent can occur via a linker or spacer. In some embodiments, the connector or spacer comprises a flexible hydrophilic spacer, such as, but not limited to, a PEG spacer. In some embodiments, the PEG spacer is a PEG3 spacer (three ethylene units). In other embodiments, the PEG spacer has 1 to 20 ethylene units (PEG1 to PEG2). 20 In some embodiments, the galactose derivative comprises N-acetylgalactosamine (GalNAc or NAG). In some embodiments, the galactose cluster consists of a galactose derivative tetramer, which may be, for example, an N-acetyl-galactosamine tetramer.

[0150] In some embodiments, pharmaceutical compositions for in vivo delivery of LPA RNAi agents to liver cells are described. Such pharmaceutical compositions may comprise, for example, an LPA RNAi agent conjugated to a galactose cluster. In some embodiments, the galactose cluster consists of a galactose derivative trimer (which may be, for example, an N-acetyl-galactosamine trimer) or a galactose derivative tetramer (which may be, for example, an N-acetyl-galactosamine tetramer).

[0151] Targeting groups include, but are not limited to, (Chol-TEG), (TEG-Chol), (C11-PEG3-NAG3), (C m -PEG n -NAG3), (C x -PEG z-NAG3), (NAG 3), (NAG4), (NAG3-AA2), (MAG3-Palm), (NAG13), (NAG18), (NAG24), (NAG25), (NAG25)s, (NAG26), (NAG27), (NAG28), (NAG29), (NAG30), (NAG30)s, (NAG31), (NAG13), (NAG31s), (NAG32), (NAG33), (NAG34), (NAG35), (NAG36), and (NAG37).

[0152] Linking group

[0153] In some embodiments, a linker group is conjugated to the RNAi agent. The linker group promotes covalent bonding of the agent to a target group or a delivery polymer or delivery medium. The linker group may be conjugated to the 3′ or 5′ end of the sense or antisense strand of the RNAi agent. In some embodiments, the linker group is conjugated to the sense strand of the RNAi agent. In some embodiments, the linker group is conjugated to the 5′ or 3′ end of the sense strand of the RNAi agent. In some embodiments, the linker group is conjugated to the 5′ end of the sense strand of the RNAi agent. Examples of linker groups include, but are not limited to: Alk-SMPT-C6, Alk-SS-C6, DBCO-TEG, Me-Alk-SS-C6, and C6-SS-Alk-Me, reactive groups such as primary amines and alkynes, alkyl groups, non-basic ribose, ribitols, and / or PEG groups.

[0154] A linker or connecting group is a connection between two atoms that links one target chemical group (such as an RNAi agent) or segment to another target chemical group (such as a targeting group or delivery polymer) or segment via one or two covalent bonds. Unstable linkages contain unstable bonds. Linkages may optionally include spacers that increase the distance between the two linking atoms. Spacers may also add flexibility and / or length to the linkage. Spacers may include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, areneyl groups, and arynyl groups; each of these may contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and sugars. Spacer groups are well known in the art, and the foregoing list is not intended to limit the scope of this specification.

[0155] Linking groups include, but are not limited to, alkyl, PEG, (C6-PEG) n -Alk), (C n -SMPT-Alk) and (C n -SS-Alk-Me).

[0156] Any LPA RNAi agent listed in Tables 2A and 2B containing a 3′ or 5′ targeting group or linker group may alternatively lack a 3′ or 5′ targeting group or linker group, or may contain different 3′ or 5′ targeting groups or linker groups, including but not limited to those described in Table 4. Any LPA RNAi agent nucleotide sequence listed in Tables 1, 2A, and 2B, whether modified or unmodified, may contain a 3′ or 5′ targeting group or linker group, including but not limited to those described in Table 4. Any LPA RNAi agent duplex listed in Tables 3A and 3B, whether modified or unmodified, may further contain a targeting group or linker group, including but not limited to those described in Table 4, and the targeting group or linker group may be attached to the 3′ or 5′ end of the sense or antisense strand of the LPA RNAi agent duplex.

[0157] Table 4 shows the structures of various modified nucleotides, targeting groups, and linking groups.

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170] In each of the above structures, NAG comprises N-acetyl-galactosamine or another ASGPR ligand. Each (NAGx) can be attached to an LPA RNAi agent via a phosphate ester group (such as in (NAG25), (NAG30), and (NAG31)) or a thiophosphate ester group (such as in (NAG25)s, (NAG30)s, and (NAG31)s) or another linker group. Alternatively, other linkers known in the art can be used.

[0171]

[0172] Delivery medium

[0173] In some embodiments, a delivery medium can be used to deliver RNAi agents to cells or tissues. A delivery medium is a compound that improves the delivery of RNAi agents to cells or tissues. Delivery mediators may include, but are not limited to, polymers such as amphiphilic polymers, membrane-active polymers, peptides, meliofuscin peptides, meliofuscin-like peptides (MLPs), lipids, reversibly modified polymers or peptides, or reversibly modified membrane-active polyamines.

[0174] In some embodiments, the RNAi agent may be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs, or other delivery systems available in the art. The RNAi agent may also be chemically conjugated to a target group, lipids (including but not limited to cholesterol and cholesterol-based derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example, WO 2000 / 053722, WO 2008 / 0022309, WO 2011 / 104169 and WO 2012 / 083185, WO 2013 / 032829, WO 2013 / 158141, each incorporated herein by reference) or other delivery systems available in the art.

[0175] Pharmaceutical Composition

[0176] This document describes a method for in vivo delivery of an LPA RNAi agent to mammalian hepatocytes. In some embodiments, a delivery medium may be used. A delivery medium is a compound that improves the delivery of an RNAi agent to cells. Delivery mediators may be, but are not limited to: polymers (such as amphiphilic polymers, membrane-active polymers), peptides (such as bee venom or bee venom-like peptides, reversibly modified polymers or peptides), or lipids. In some embodiments, the LPA RNAi agent is ligated to a targeting ligand comprising a desialylate glycoprotein ligand. In some embodiments, the LPA RNAi agent is ligated to a targeting ligand comprising or composed of galactose clusters.

[0177] LPA RNAi agents can be used to inhibit the expression of LPA in cells, cell populations, or tissues, such as those of a subject. In some embodiments, LPA RNAi agents are used to formulate compositions, i.e., pharmaceutical compositions or drugs, for administration to a subject. As used herein, a pharmaceutical composition or drug comprises a pharmacologically effective amount of at least one of the said LPA RNAi agents and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than an active pharmaceutical ingredient (API, therapeutic product, such as an LPA RNAi agent) that has been appropriately safety evaluated and is intentionally included in a drug delivery system. The excipient does not exert or is not intended to exert a therapeutic effect at a predetermined dose. Excipients may be used to a) facilitate the handling of the drug delivery system during manufacturing, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API, c) facilitate product identification, and / or d) enhance any other properties of the overall safety and effectiveness of the API delivery during storage or use. Pharmaceutically acceptable excipients may or may not be inert substances.

[0178] Excipients include, but are not limited to: absorption promoters, anti-adhesion agents, defoamers, antioxidants, adhesives, buffers, carriers, coating agents, pigments, delivery promoters, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavorings, flow aids, humectants, lubricants, oils, polymers, preservatives, brine, salt, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, tensioning agents, mediators, hydrophobic agents, and wetting agents.

[0179] Pharmaceutical compositions may contain other additional components commonly found in pharmaceutical compositions. These additional components include, but are not limited to, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents (such as antihistamines, diphenhydramine, etc.). It is also envisioned that cells, tissues, or isolated organs expressing or containing RNAi agents as defined herein may be used as “pharmaceutical compositions.” As used herein, “pharmacologically effective amount,” “therapeuticly effective amount,” or simply “effective amount,” refers to the amount of RNAi agent that produces the expected pharmacological, therapeutic, or prophylactic results.

[0180] In some embodiments, the LPA RNAi agent is combined with one or more additional therapeutic agents or treatments, including but not limited to: a second LPA RNAi agent or other RNAi agents, small molecule drugs, antibodies, antibody fragments, and / or vaccines. Examples of additional therapeutic agents include, but are not limited to, HMgCo-A reductase inhibitors (statins), ezetimibe, PCSK-9 inhibitors, CTEP inhibitors, therapies targeting ANGPTL3, therapies targeting APOC3, and niacin.

[0181] The RNAi agent and pharmaceutical compositions containing the LPA RNAi agent disclosed herein can be packaged or included in kits, containers, packages, or dispensers. The LPA RNAi agent and pharmaceutical compositions containing the LPA RNAi agent can be packaged in pre-filled syringes or vials.

[0182] In some embodiments, a pharmaceutical composition comprising at least one of the LPA RNAi agents described above is considered. These pharmaceutical compositions can be used to inhibit the expression of the LPA gene in cells, tissues, or organisms. In some embodiments, the pharmaceutical composition is used to treat a subject suffering from a disease, condition, or symptom that would benefit from reduced or inhibited LPA expression. In some embodiments, the pharmaceutical composition is used to treat a subject at risk of developing a disease, condition, or symptom that would benefit from reduced or inhibited LPA expression. Diseases, conditions, or symptoms that would benefit from reduced or inhibited LPA expression include, but are not limited to: Burglar's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapolipoprotein β-lipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease, and venous thrombosis. In some embodiments, the subject is a mammal, including but not limited to human patients.

[0183] Consider cells, tissues, and non-human organisms comprising at least one LPA RNAi agent described herein. The cells, tissues, or non-human organisms are prepared by delivering the RNAi agent to the cells, tissues, or non-human organisms in any manner available in the art. In some embodiments, the cells are mammalian cells, including but not limited to human cells. The cells, tissues, or non-human organisms may be used for research or as research tools (such as drug testing or diagnostics).

[0184] Treatment

[0185] In some embodiments, the LPA RNAi agents described herein may be used to treat subjects who have a disease, disorder, or condition that would benefit from reduced or suppressed LPA expression, or who are at risk of having a disease, disorder, or condition that would benefit from reduced or suppressed LPA expression. Treatment of subjects who would benefit from reduced and / or suppressed LPA gene expression includes therapeutic and / or prophylactic treatment. Examples of diseases, disorders, or conditions include, but are not limited to: Burger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapolipoprotein β-lipoproteinemia, cerebrovascular arteriosclerosis, cerebrovascular disease, and venous thrombosis. In some embodiments, the method includes administering a composition, such as a pharmaceutical composition comprising the LPA RNAi agent described herein, to the mammal to be treated.

[0186] In some embodiments, a therapeutically effective amount of one or more of the LPA RNAi agents is administered to a subject to inhibit LPA expression in the subject (e.g., to effectively inhibit the amount of LPA expressed in the subject). In some embodiments, one or more of the LPA RNAi agents described herein are used to treat a subject suffering from a disease or condition that would benefit from reduced or inhibited LPA expression. In some embodiments, the LPA RNAi agent is used to treat or prevent at least one symptom in a subject suffering from a disease or condition that would benefit from reduced or inhibited LPA expression. A therapeutically effective amount of any one or more of the RNAi agents is administered to the subject to treat the symptom. In some embodiments, a preventatively effective amount of any one or more of the RNAi agents is administered to the subject to prevent at least one symptom.

[0187] In some embodiments, LPA RNAi agents are used to treat or manage clinical manifestations, wherein a therapeutically or preventively effective amount of one or more LPA RNAi agents or a composition containing an LPA RNAi agent described herein is administered to a subject requiring such treatment, prevention, or management. In some embodiments, the method includes administering a composition containing an LPA RNAi agent described herein to a mammal to be treated.

[0188] In some embodiments, the method further includes the step of administering a second therapeutic agent or treatment. In some embodiments, the second therapeutic agent is another LPA RNAi agent (e.g., an LPA RNAi agent targeting a different sequence within the LPA target). In other embodiments, the second therapeutic agent may be selected from the group consisting of small molecule drugs, antibodies, antibody fragments, and vaccines.

[0189] The route of administration is the way in which an RNAi agent comes into contact with the body. Generally, methods of administration for drugs and nucleic acids used to treat subjects are well known in the art and can be applied to the administration of the compositions described herein. The compounds described herein can be administered via any suitable route in a formulation appropriately tailored to the specific route. Thus, the compounds described herein can be administered by injection, such as intravenous, intramuscular, intradermal, subcutaneous, or intraperitoneal injection.

[0190] In some embodiments, the LPA RNAi agents or compositions described herein can be delivered to cells, cell populations, tissues, or subjects using oligonucleotide delivery technologies known in the art. Generally, any suitable method recognized in the art for delivering nucleic acid molecules (in vitro or in vivo) can be applied to the LPA RNAi agents described herein. For example, delivery can be via local administration (e.g., direct injection, implantation, or topical administration), systemic administration, or subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrasheathal), intramuscular, transdermal, airway (aerosol), nasal, rectal, or local (including buccal and sublingual) administration. In some embodiments, the composition is administered via subcutaneous or intravenous infusion or injection.

[0191] In some embodiments, the RNAi agent can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs, or other delivery systems available in the art. The RNAi agent can also be chemically conjugated to a targeting group, lipids (including but not limited to cholesterol and cholesterol-based derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example, WO 2000 / 053722, WO 2008 / 0022309, WO 2011 / 104169 and WO 2012 / 083185, each incorporated herein by reference) or other delivery systems available in the art. The LPA RNAi agent can be conjugated to a delivery polymer. In some embodiments, the delivery polymer is a reversibly masked / modified amphiphilic membrane-active polyamine.

[0192] Suppression of expression

[0193] As used herein, the terms “silence,” “reduction,” “inhibition,” “downregulation,” or “knockdown of gene expression” when referring to the LPA gene mean that when cells, cell populations, or tissues are treated with the LPA RNAi agent, the expression of the gene (measured, for example, by the level of RNA transcribed from the gene or the level of polypeptides, proteins, or protein subunits translated from mRNA) is reduced compared to the same cells, cell populations, or tissues before the application of the LPA RNAi agent.

[0194] In some embodiments, the gene expression level and / or mRNA level of LPA in subjects administered the LPA RNAi agent is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% compared to subjects before administration of the LPA RNAi agent or subjects who did not receive the LPA RNAi agent. The gene expression level and / or mRNA level in the subjects may be reduced in the subjects' cells, cell populations, and / or tissues. In some embodiments, the protein level of LPA in subjects administered the LPA RNAi agent is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% compared to subjects before administration of the LPA RNAi agent or those who did not receive the LPA RNAi agent. The protein level in the subject may be reduced in the subject's cells, cell populations, tissues, blood, and / or other fluids. The reduction in gene expression, mRNA, or protein levels can be assessed by any method known in the art. The reduction or decrease in LPA mRNA and / or protein levels is collectively referred to herein as a reduction or decrease in LPA, or inhibition or reduction of LPA expression.

[0195] When referring to RNAi agents, "introduction into cells" means the functional delivery of the RNAi agent into cells. Functional delivery means that the RNAi agent is delivered into cells and has the intended biological activity (e.g., sequence-specific inhibition of gene expression).

[0196] Cells and tissues and non-human organisms

[0197] Consider cells, tissues, and non-human organisms containing at least one LPA RNAi agent described herein. Prepare the cells, tissues, or non-human organisms by delivering the RNAi agent to them.

[0198] The above-provided implementation schemes and terms are now illustrated by the following non-limiting examples. Example

[0199] Example 1. Synthesis of RNAi agents.

[0200] A) Synthesis. LPA RNAi agents are synthesized using a solid-phase phosphoramide technique for oligonucleotide synthesis. Depending on scale, MerMade96E (Bioautomation) or MerMadel2 (Bioautomation) is used. A solid support (CPG) made of controlled-porosity glass is used. or The nucleotides were synthesized from Prime Synthesis, Aston, PA, USA. All DNA, 2′-modified RNA, and UNA phosphoramide were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the following 2′-O-methylphosphoramide was used: (5′-O-dimethoxytriphenylmethyl-N... 6 -(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphamide, 5'-O-dimethoxy-triphenylmethyl-N 4 -(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphamide, (5′-O-dimethoxytriphenylmethyl-N 2 -(isobutyryl)-2'-O-methyl-guanosine-3′-O-(2-cyano-ethyl-N,N-diisopropylamino)phosphamide and 5'-O-dimethoxy-triphenylmethyl-2'-O-methyl-uridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide. 2′-deoxy-2′-fluoro-phospho-imide carries the same protecting group as 2'-O-methylRNAimide. The following UNA phosphorus amides were used: 5′-(4,4′-dimethoxytriphenylmethyl)-N-benzoyl-2′,3′-dextrin-adenosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoryl-imide, 5′-(4,4′-dimethoxytriphenylmethyl)-N-acetyl-2′,3′-dextrin-cyto ...5′-phos Phosphoramides, including 5′-(4,4′-dimethoxytriphenylmethyl)-N-isobutyryl-2′,3′-dextrin-guanosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, and 5′-(4,4′-dimethoxytriphenylmethyl)-2′,3′-dextrin-uridine, were dissolved in anhydrous acetonitrile (50 mM) and molecular sieves were added. To introduce TEG-cholesterol at the 5′ end of the oligomer, 1-dimethoxytriphenylmethyloxy-3-O-(N-cholesterol-3-aminopropyl)-triethylene glycol-glycero-2-O-(2-cyanoethyl)-(N,N,-diisopropyl)-phosphoramide from Glen Research (Sterling, VA, USA) was used. The 5′ modification was introduced without any changes to the synthetic cycle. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) was used as the activator solution. Coupling times were 10 min (RNA), 180 s (cholesterol), 90 s (2′OMe and UNA), and 60 s (2′F and DNA). To introduce thiophosphate linkages, a 100 mM solution of 3-phenyl-1,2,4-diathiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used. See Tables 1, 2A, and 2B for specific sequences.

[0201] B. Cleavage and deprotection of support-bound oligomers. After solid-phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt% methylamine in water and 28% ammonium hydroxide (Aldrich) at 30°C for 2 hours. The solution was evaporated and the solid residue was reconstituted in water (see below).

[0202] C. Purification. Crude cholesterol-containing oligomers were purified by reversed-phase HPLC using a Waters XBridge BEH300 C4 5u pre-column and a Shimadzu LC-8 system. Buffer A was 100 mM TEAA, pH 7.5, containing 5% acetonitrile, and buffer B was 100 mM TEAA containing 95% acetonitrile. UV traces were recorded at 260 nm. Appropriate fractions were then run on size exclusion HPLC using a GE Healthcare XK 16 / 40 column packed with Sephadex G-25 media, with a run buffer of 100 mM ammonium bicarbonate, pH 6.7, and 20% acetonitrile. Other crude oligomers were purified by anion-exchange HPLC using a TKSgel SuperQ-5pW 13u column and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0, containing 20% ​​acetonitrile, and buffer B was the same as buffer A with 1.5 M sodium chloride added. Record the UV trace at 260 nm. Collect the appropriate fractions and then run them on size exclusion HPLC as described for cholesterol-containing oligomers.

[0203] D. Annealing. The complementary strands were mixed to form an RNAi agent by combining equimolar solutions (sense and antisense) in 0.2×PBS (phosphate-buffered saline, 1×, Corning, Cellgro). The solution was placed in a 70°C hot mixer, heated to 95°C, held at 95°C for 5 minutes, and then slowly cooled to room temperature. Some of the RNAi agent was lyophilized and stored at -15 to -25°C. The duplex concentration was determined by measuring the absorbance of the solution in 0.2×PBS on a UV-Vis spectrometer. The duplex concentration was then determined by multiplying the absorbance at 260 nm by the conversion factor and dilution factor. Unless otherwise specified, all conversion factors were 0.037 mg / (mL·cm). For some experiments, the conversion factor was calculated from the experimentally determined extinction coefficient.

[0204] Example 2. Preliminary in vitro analysis of LPA RNAi agents. Candidate sequences identified by computer analysis as having cross-reactivity with humans and non-human primates were screened. 108 potential LPA RNAi agents identified by computer were synthesized and their in vitro efficacy was screened in three groups. For screening purposes, the human LPA cDNA sequence (accession number NM_005577.1) was subcloned from a commercially available mammalian expression vector (Origene, Rockville, MD) into a commercially available reporter-based screening plasmid psiCHECK2 (Promega, Madison, WI) to generate Renida luciferase / LPA fusion mRNA. For the efficacy of LPA RNAi agents in a human background, Hep3B cells (human hepatocellular carcinoma line) were plated in a 96-well format at approximately 10,000 cells / well. Each of the 108 LPA RNAi agents was co-transfected at two or three concentrations (1 nM and 0.1 nM, or 0.02, 0.2, and 2 nM) with 50–100 ng of LPA-psiCHECK2 plasmid DNA per well and 0.2 μL of LipoFectamine 2000 per well. Gene knockdown was determined by measuring Renilla luciferase levels normalized to constitutively expressed firefly luciferase levels also present on the psiCHECK2 plasmid using a dual luciferase reporter assay (Promega, Madison, WI) (Tables 5A and 5B).

[0205] Table 5A. In vitro analysis of LPA RNAi agents and inhibition of LPA expression.

[0206]

[0207]

[0208] Table 5B. In vitro analysis of LPA RNAi agents and inhibition of LPA expression.

[0209]

[0210]

[0211] Table 5. In vitro analysis of 10 mM LPA RNAi agent, inhibition of LPA expression.

[0212]

[0213] Table 5. In vitro analysis of 1 mM LPA RNAi agent, inhibition of LPA expression.

[0214]

[0215]

[0216]

[0217]

[0218]

[0219] Example 3. LPA RNAi agent EC 50 Assay. Ten EC were generated using the same cells and transfection conditions. 50 The curves show LPA RNAi concentrations ranging from 150 fM to 3 nM. EC was measured using GraphPad Prism software. 50 (Table 6)

[0220] Table 6. EC50 of the specified LPA RNAi agent as determined in vitro 50 Value (nM).

[0221]

[0222] Example 4. In vitro analysis of structure-activity relationship (SAR) designed LPA RNAi agents. SAR groups of LPA RNAi agents (364 sequences based on AD01532 and 351 sequences based on AD01533) were synthesized and their efficacy was screened in vitro. For screening purposes, a 2756 bp human LPA cDNA sequence from KIV-3 to KIV-9 (accession number NM_005577.1) was synthesized and cloned (GeneWiz, South Plainfield, NJ) into the commercially available reporter-based screening plasmid psiCHECK2 (Promega, Madison, WI), generating Renida luciferase / LPA fusion mRNA. For the efficacy of LPA RNAi agents in a human background, HuH7 cells (human hepatocellular carcinoma line) were plated at approximately 7500 cells / well in a 96-well format. Each LPA RNAi agent was co-transfected at two concentrations (1 nM and 0.1 nM, or 10 nM and 1 nM) with 25 ng LPA-psiCHECK2 plasmid DNA per well and 0.2 μL LipoFectamine 2000 per well. Gene knockdown was determined by measuring Renilla luciferase levels normalized to constitutively expressed firefly luciferase levels also present on the psiCHECK2 plasmid using a dual luciferase reporter assay (Promega, Madison, WI) (Tables 7A and 7B).

[0223] Table 7A. In vitro efficacy screening results of LPA RNAi agents as determined by dual luciferase reporter assay.

[0224]

[0225]

[0226]

[0227] Table 7B. In vitro efficacy screening results of LPA RNAi agents, as determined by dual luciferase reporter assay.

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234] Example 5. In vivo analysis of the efficacy of RNAi agents in transient transgenic and transgenic mice.

[0235] A) Administration and Sample Collection. To evaluate the in vivo efficacy of the LPA RNAi agent, transient transgenic mice were used. Wild-type mice were injected with a plasmid containing the SEAP gene under the control of the mouse albumin promoter via hydrodynamic tail vein injection at least 30 days prior to administration of the cholesterol-conjugated LPA RNAi agent. The LPA gene target sequence was cloned into the 3′UTR of the plasmid. These mice were designated SEAP-LPA HTV mice. The cholesterol-conjugated LPA RNAi agent was administered to the mice on day 1 using an MLP delivery polymer (WO 2012 / 083185, incorporated herein by reference; bee venom was synthesized and modified with CDM-NAG to produce the MLP delivery polymer as described). Each mouse received 200–250 μL of a solution containing a given dose of the LPA RNAi agent + MLP delivery polymer (in most cases, 1:1 w / w RNAi agent: MLP delivery polymer) via intravenous (IV) injection into the tail vein. In some experiments, the LPA RNAi agent was directly conjugated to the delivery polymer. The polymer-conjugated LPA RNAi agent was similarly injected into the tail vein. The specified LPA RNAi agent (Table 8B) was conjugated to a polyacrylate polymer ARF1164-106A-5 (MW 41962 g / mol, a polymer synthesized as described in WO 2013 / 158141) having 54.4% ethoxyethylaminoacrylate (EEAA) amine monomer and 45.6% propyl acrylate propyl monomer, and masked with 3× ACit-NAG and 6× ACit-PEG (masked polymers as described in WO 2012 / 092373 and PCT / US16 / 34512). Control serum (pretreated) samples were obtained from mice pre-injected on day -4 or day -1. Post-injection serum samples were obtained from mice on days 4, 8, 15, 22, 29, 36, and 43. For some mice, samples were collected on day 3 or 5 instead of day 4.

[0236] In another experiment, transient transgenic mice expressing full-length LPA were used to evaluate LPA RNAi agents in vivo. At least 30 days prior to administration of the cholesterol-targeting LPA RNAi agent, immunocompromised (Nod. scid) mice were injected via hydrodynamic tail vein injection with microcircuits containing LPA cDNA controlled by the mouse albumin promoter. These mice were designated LPA mc HTV mice. On day 1, mice were administered either the cholesterol-targeting or NAG (also known as GalNAc)-conjugated LPA RNAi agent. For the cholesterol-targeting RNAi agent, each mouse received an intravenous (IV) injection of 200–250 μL of a solution containing a dose of the RNAi agent plus MLP delivery polymer (1:1 w / w RNAi agent: MLP delivery polymer) into the tail vein. For the NAG-conjugated LPA RNAi agent, mice received a subcutaneous (SC) injection of 300 μL of a solution containing a dose of the RNAi agent in buffered saline into the loose skin on the back between the shoulders. For some samples, the LPA RNAi agent was administered with or without the MLP delivery peptide. The RNAi agent delivered with the MLP delivery peptide was administered via intravenous (IV) injection of 200–250 μL of a solution containing a given dose of RNAi agent + MLP delivery polymer (in most cases, 1:2 w / w RNAi agent: MLP delivery polymer) into the tail vein. Control serum (pretreated) samples were obtained from mice on day -4 or day -1 after pre-injection. Post-injection serum samples were obtained from mice on days 4, 8, 15, 22, 29, 36, and 43. For some mice, samples were collected on day 3 or day 5 instead of day 4.

[0237] In another experiment, LPA RNAi agents were administered to apo(a) and Lp(a) transgenic mice (Frazer KA et al. 1995, Nature Genetics 9: 424-431). These mice expressed human apo(a) from a YAC (encoding the apo(a) protein) containing the complete LPA gene with additional sequences at both 5′ and 3′. Lp(a) mice were bred by crossing mice containing apo(a) YAC with mice expressing human apoB-100 (Callow MJ et al. 1994, PNAS 91: 2130-2134). Cholesterol-targeted RNAi agents and NAG-conjugated RNAi agents were administered as described above. Control serum (pretreated) samples were obtained from mice pre-injected on day -1. Post-injection serum samples were obtained from mice on days 4, 8, 15, 22, 29, 36, 43, 50, 57, and 64.

[0238] B) LPA expression knockdown analysis. For SEAP-LPA HTV mice, LPA expression knockdown was analyzed using the Phospha-Light chemiluminescent reporter gene assay system. TM (Life Technologies) measures mouse serum to monitor SEAP protein levels. For normalization, the SEAP level in each animal at a given time point is divided by the pretreatment level expressed in that animal to determine the "normalized to pretreatment" expression ratio. Expression at a specific time point is then averaged among individuals within the group.

[0239] For LPA mc HTV mice and transgenic mice, serum levels of human apo(a) protein were monitored using an ELISA assay of apo(a)(Abcam). For normalization, the apo(a) level in each animal at a time point was divided by the pretreatment level expressed in that animal (in this case, day 1) to determine the "normalized to day 1" expression ratio. Expression at specific time points was then normalized to the saline control group by dividing the "normalized to day 1" ratio of each animal by the average "normalized to day 1" ratio of all mice in the saline control group. This resulted in expression at each time point being normalized to the expression in the control group.

[0240] Following the manufacturer's recommendations, Lp(a) levels were measured on a Cobas Integra 400 (Roche Diagnostics). For normalization, the apo(a) level of each animal at a time point was divided by the pretreatment level expressed in that animal (in this case, day 1) to determine the "normalized to day 1" expression ratio. Expression at specific time points was then normalized to the saline control group by dividing the "normalized to day 1" ratio of each animal by the average "normalized to day 1" ratio of all mice in the saline control group. This resulted in expression at each time point being normalized to the expression in the control group.

[0241] Table 8A. Relative LPA levels in mice after intravenous administration of a cholesterol-conjugated LPA RNAi agent + MLP-delivered polymer.

[0242]

[0243]

[0244] Table 8B. Relative LPA levels in mice after intravenous administration of a polymer-conjugated LPA RNAi agent.

[0245]

[0246] Table 8C. Relative LPA levels in mice after subcutaneous administration of NAG-conjugated LPA RNAi agent.

[0247]

[0248]

[0249]

[0250]

[0251] Example 6. In vivo screening of LPA RNAi agents and the timeline of SEAP knockdown. Cholesterol-conjugated LPA RNAi agents were administered to transient transgenic mice as described above. Each mouse received a single intravenous (IV) dose of 8 mg / kg LPA RNAi agent and 8 mg / kg MLP delivery polymer. Serum SEAP protein levels were monitored for up to 36 days. Knockdown levels and duration of response are shown in Table 9. A reduction in serum SEAP protein levels of greater than 85% was achieved after administration of all tested LPA RNAi agents; all tested LPA RNAi agents, except for the two tested, showed a knockdown of greater than 99.4%. AD01196 and AD01199 showed >95% knockdown on day 36.

[0252] Example 7. In vivo screening of LPA RNAi agents and the time course of LPA knockdown at lower doses of LPA RNAi agents. Cholesterol-conjugated LPA RNAi agents were administered to transient transgenic mice as described above. Each mouse received a single intravenous (IV) dose of 2 mg / kg LPA RNAi agent and 2 mg / kg MLP delivery polymer. Serum SEAP protein levels were monitored for up to 43 days (Table 10).

[0253] Example 8. In vivo testing of LPA RNAi agent in apo(a) transgenic (Tg) mice. AD01196 was administered to mice as described above. Each mouse received a single intravenous (IV) dose of 2 mg / kg LPA RNAi agent with 2 mg / kg MLP delivery polymer or saline. Serum human apo(a) levels were monitored for up to 64 days (Table 11). On day 15, animals in the saline group received a single IV dose of 2 mg / kg control mouse factor VII (F7) RNAi agent with 2 mg / kg MLP delivery polymer. On day 22, F7 levels were measured in all animals. Seven days after administration, F7 activity was knocked down by 99%, with no effect on serum apo(a) levels. AD01196 showed a knockdown of >3 log10 of apo(a) levels at the nadir and >80% knockdown was observed after 3 weeks (Table 11).

[0254] Table 9. Serum SEAP protein levels in SEAP-LPA HTV mice after administration of 8 mg / kg chol-RNAi agent and 8 mg / kg MLP-delivered peptide. SEAP levels were normalized to day -1 and saline control.

[0255]

[0256]

[0257] Table 11. Serum apo(a) protein levels in apo(a)Tg mice after administration of 2 mg / kg cholesterol-conjugated LPA RNAi agent and 2 mg / kg MLP polymer delivery. Apo(a) levels were normalized to day -1 and saline control.

[0258]

[0259] Example 9. Knockdown of apolipoprotein (a) (apo(a)) in non-human primates following LPA RNAi delivery of an MLP delivery polymer. The MLP delivery polymer and LPA RNAi agent were prepared and combined in a pharmaceutically acceptable buffer as described above. On day 1, two cynomolgus macaque primates (both males, 5.0 kg and 8.15 kg, respectively) were injected with 2 mg / kg AD01196 + 2 mg / kg MLP delivery polymer into the saphenous vein. For each injection, the LPA RNAi agent + MLP delivery polymer (2 ml / kg) was injected into the saphenous vein using a 22-25 gauge intravenous catheter. Blood samples were collected at designated time points (shown in Table 12) and apo(a) levels, lipid levels, and toxicity markers were analyzed. Blood was collected from the femoral vein, and the primates were fasted overnight before all blood collections were performed. Blood tests for blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, total cholesterol (TC), and triglycerides (TG) were performed using an automated chemistry analyzer at Meriter Laboratories. Blood tests for lipoprotein(a) (Lp(a)) and low-density lipoprotein (LDL) were measured using an automated analyzer. Serum apo(a) levels were measured by ELISA. Significant knockdown of apo(a) was observed on day 22, with a mean maximum knockdown of 94.5%. A mean maximum knockdown of Lp(a) was observed on day 15, with a mean maximum knockdown of 91.5%. Figure 3 No dose-related toxicity was observed in the treated animals.

[0260] Table 12. Serum apo(a) protein, lipoprotein(a) (mg / dL), low-density lipoprotein (LDL), total cholesterol and triglyceride levels in cynomolgus macaque primates after administration of 2 mg / kg AD01196 and 2 mg / kg MLP polymer delivery. Apo(a) levels were normalized to pre-dose levels.

[0261]

[0262] Table 13. Levels of urea nitrogen, creatinine, alanine aminotransferase, and aspartate aminotransferase in cynomolgus macaque primates after administration of 2 mg / kg AD01196 and 2 mg / kg MLP for polymer delivery.

[0263]

[0264]

[0265] Example 10. In vivo screening of NAG-conjugated LPA RNAi agents and knockdown duration. NAG-conjugated LPA RNAi agents were administered to transient transgenic mice as described above. Each mouse received a single intravenous (IV) dose of 2 mg / kg LPA RNAi agent with 1 mg / kg MLP delivery polymer, or a single subcutaneous (SC) dose of 10 mg / kg NAG-conjugated LPA RNAi agent. Serum SEAP protein levels were monitored for up to 22 days. Knockdown levels and response durations are shown in Tables 14-15. AD01529, AD01532, and AD01533 showed ≥85% knockdown of SEAP levels after IV administration with the MLP delivery polymer, and ≥60% maximum knockdown of SEAP levels after SC administration with the NAG-conjugated LPA RNAi agent alone.

[0266] Table 14. Serum SEAP protein levels in SEAP-LPA HTV mice after administration of 10 mg / kg NAG-conjugated LPA RNAi agent. SEAP levels were normalized to day -1 and saline control.

[0267]

[0268] Table 15. Serum SEAP protein levels in SEAP-LPAHTV mice after IV administration of 1 mg / kg NAG-conjugated LPA RNAi agent + 2 mg / kg MLP polymer delivery. SEAP levels were normalized to day -1 and saline control.

[0269]

[0270] Example 11. In vivo screening of modified NAG-conjugated LPA RNAi agents and knockdown timelines. The specified NAG-conjugated LPA RNAi agents were administered to SEAP-LPA HTV mice as described above. Each mouse received a single subcutaneous (SC) dose of 10 mg / kg of the NAG-conjugated LPA RNAi agent. Serum SEAP protein levels were monitored for up to 22 days. Knockdown levels and response durations are shown in Tables 16-17. AD01765 and AD01768 showed 89% knockdown activity.

[0271] Table 16. Serum SEAP protein levels in SEAP-LPA HTV mice after administration of 10 mg / kg NAG-conjugated LPA RNAi agent. SEAP levels were normalized to day -1 and saline control.

[0272]

[0273] Table 17. Serum SEAP protein levels in SEAP-LPA HTV mice after administration of 10 mg / kg NAG-conjugated LPA RNAi agent. SEAP levels were normalized to day -1 and saline control.

[0274]

[0275] Example 12. In vivo testing of a NAG-conjugated LPA RNAi agent in apo(a)Tg mice. A NAG-conjugated LPA RNAi agent was administered to apo(a)Tg mice as described above. Each mouse received a single subcutaneous (SC) dose of 10 mg / kg LPA RNAi agent or saline. Serum levels of human apo(a) were monitored for up to 22 days (Table 18). AD01765 showed the greatest knockdown of apo(a) levels on day 8, with a 96% knockdown, and >74% knockdown was observed 3 weeks post-administration.

[0276] Table 18. Serum apo(a) protein levels in apo(a)Tg mice after administration of 10 mg / kg NAG-conjugated LPA RNAi agent. Apo(a) levels were normalized to day -1 and saline control.

[0277]

[0278] Example 13. In vivo testing of NAG-conjugated LPA RNAi agents in LPA mc HTV mice. The specified NAG-conjugated LPA RNAi agents were administered to LPA mc HTV mice as described above. Each mouse received a single subcutaneous (SC) dose of 10 mg / kg LPA RNAi agent or saline. Serum human apo(a) levels were analyzed on day 4 (Table 19). AD02001, AD01765, and AD01768 showed >90% knockdown on day 4.

[0279] Table 19. Serum apo(a) protein levels in LPA mc HTV mice after administration of 10 mg / kg NAG-conjugated LPA RNAi agent. Apo(a) levels were normalized to day -1 and saline control.

[0280]

[0281] Example 14. Knockdown of apolipoprotein (a) (apo(a)) in non-human primates following LPA RNAi delivery of an MLP delivery polymer. The MLP delivery polymer and LPA RNAi agent were prepared and combined in a pharmaceutically acceptable buffer. On days 1 and 71, two cynomolgus macaque (crab-eating macaque) primates were injected with either 4 mg / kg AD01196 + 4 mg / kg MLP delivery polymer or 6 mg / kg AD01196 + 6 mg / kg MLP delivery polymer. For each injection, the LPA RNAi agent + MLP delivery polymer was injected into the saphenous vein using a 22-25 gauge intravenous catheter. As previously described, at designated time points ( Figure 4 Blood samples were collected and apo(a) levels, lipid levels, and toxicity markers were analyzed. Significant knockdown of apo(a) was observed after the first dose, with a mean maximum knockdown of 96% observed on day 15 at the 4 mg / kg dose and on day 15 at the 6 mg / kg dose. The mean maximum knockdown of Lp(a) after the first dose was 98.5% on day 29 at the 4 mg / kg dose and 98.5% on day 22 at the 6 mg / kg dose. No dose-related toxicities were observed in the treated animals.

[0282] Example 15. In vivo testing of NAG-conjugated LPA RNAi agent-dose response in Lp(a)Tg mice. NAG-conjugated LPA RNAi agents were administered to Lp(a)Tg mice as described above. Each mouse received a single subcutaneous (SC) dose of the LPA RNAi agent (0.5 mg / kg or 2 mg / kg dose level) or saline. Serum Lp(a) levels were monitored until day 43. Figure 1 , Figure 2 All LPA RNAi agents showed a dose-response relationship, with higher doses showing a better knockdown with the lowest point between day 15 and day 22.

[0283] Example 16. Knockdown of apolipoprotein (a) (apo(a)) in non-human primates following delivery of an apo(a)-specific LPA RNAi agent. The LPA RNAi agent was prepared in a pharmaceutically acceptable buffer for subcutaneous (SC) injection as described herein. Two cynomolgus macaque (crab-eating macaque) primates were subcutaneously injected with 3 mg / kg AD02713, AD02819, AD02820, or AD02821 on days 1, 7, and 15. Additionally, monkeys treated with AD02819 were again administered 3 mg / kg AD02819 on days 57 and 85. Blood samples were drawn as previously described, and apo(a) levels, lipid levels, and toxicity markers were analyzed. Significant knockdowns of Lp(a) were observed, with an average maximum knockdown of 89% for AD02713 observed on day 43, 87% for AD02819 observed on day 36, 79% for AD02820 observed on day 36, and 95% for AD02821 observed on day 29. Figure 5 No dose-related toxicity was observed in the treated animals over time.

[0284] Example 17. Knockdown of apolipoprotein (a) (apo(a)) in non-human primates after LPA RNAi agent delivery. LPA RNAi agents were prepared in pharmaceutically acceptable buffers for subcutaneous (SC) injection as described herein. On day 1, two cynomolgus macaque (crab-eating macaque) primates were subcutaneously injected with 3 mg / kg of AD03272, AD03462, AD03549, AD03547, AD03668, AD03460, or AD03536. Additionally, monkeys treated with AD03460 and AD03536 were re-administered with 1 mg / kg of each LPA RNAi agent on day 48. Blood samples were drawn as previously described, and apo(a) levels, lipid levels, and toxicity markers were analyzed. Significant knockdowns of Lp(a) were observed, with the average maximum knockdown of AD03272 observed at day 15 (62%), AD03462 at day 15 (28%), AD03549 at day 29 (47%), AD03547 at day 15 (44%), AD03668 at day 22 (53%), AD03460 at day 29 (79%), and AD03536 at day 22 (71%). Figure 6 No dose-related toxicity was observed in the treated animals over time.

[0285] Example 18. Knockdown of apolipoprotein (a) (apo(a)) in primates after delivery of an apo(a)-specific LPA RNAi agent. An LPA RNAi agent was prepared and combined with a pharmaceutically acceptable buffer for subcutaneous (SQ) injection as described above. On day 1, cynomolgus macaques (crab-eating macaques) were subcutaneously injected with saline or 2 mg / kg AD03460, AD03536, AD03851, AD03853, or AD04110. Blood samples were collected on days 8 and 15 as previously described, and apo(a) levels, lipid levels, and toxicity markers were analyzed. Lp(a) levels were normalized to the average of three pre-dose values.

[0286] Table 20. Lipoprotein (a) levels in cynomolgus primates after administration of saline or 2 mg / kg AD03460, AD03536, AD03851, AD03853 or AD04110.

[0287]

Claims

1. An LPA RNA interference (RNAi) agent comprising a sense strand and an antisense strand, each of the sense strand and the antisense strand being 19-26 nucleotides in length, wherein the antisense strand comprises the sequence of SEQ ID NO:1280, and wherein the sense strand comprises a sequence complementary to the antisense strand sequence.

2. The LPA RNAi agent of claim 1, wherein the sense strand and the antisense strand are each 26 nucleotides in length.

3. The LPA RNAi agent of claim 1, wherein the sense strand and the antisense strand are each 21 nucleotides in length.

4. The LPA RNAi agent of claim 1, wherein the LPA RNAi agent comprises at least one protruding end.

5. The LPA RNAi agent of claim 4, wherein the LPA RNAi agent comprises a protruding end at the 3′ end of the antisense strand.

6. The LPA RNAi agent of claim 4, wherein the LPA RNAi agent comprises a protrusion at the 3′ end of the antisense strand and a protrusion at the 3′ end of the sense strand.

7. The LPA RNAi agent of claim 1, wherein the LPA RNAi agent comprises at least one blunt end.

8. The LPA RNAi agent of claim 7, wherein the LPA RNAi agent comprises a blunt end and a broken end.

9. The LPA RNAi agent of claim 7, wherein the LPA RNAi agent comprises two blunt ends.

10. The LPA RNAi agent according to any one of claims 1-9, wherein the sense strand, antisense strand, or both the sense strand and antisense strand comprise one or more modified nucleotides.

11. The LPA RNAi agent of claim 10, wherein the one or more modified nucleotides are independently selected from: 2′ modified nucleotides, locked nucleotides, baseless nucleotides, reverse deoxynucleotides, morpholinonucleotides, 2′,3′ cut nucleotide mimics, or nucleotides containing non-natural bases.

12. The LPA RNAi agent of claim 11, wherein the 2′ modified nucleotide is: 2′-O-methyl nucleotide, 2′-deoxy-2′-fluoronucleotide, 2′-deoxynucleotide, 2′-methoxyethyl nucleotide, 2′-amino nucleotide or 2′ alkyl nucleotide.

13. The LPA RNAi agent according to any one of claims 1-9, wherein the LPA RNAi agent comprises one or more phosphate thioester nucleoside linkages.

14. The LPA RNAi agent of claim 13, wherein both the sense strand and the antisense strand independently comprise 1, 2, 3 or 4 phosphate thioester nucleoside links.

15. The LPA RNAi agent according to any one of claims 1-9, wherein the LPA RNAi agent further comprises a targeting group.

16. The LPA RNAi agent of claim 15, wherein the targeting group comprises a desialylate glycoprotein receptor ligand.

17. The LPA RNAi agent of claim 16, wherein the desialyl glycoprotein receptor ligand comprises galactose, galactosamine, N-acetyl-galactosamine, or a galactose derivative.

18. The LPA RNAi agent of claim 15, wherein the targeting group is: (Chol-TEG), (TEG-Chol), (C11-PEG3-NAG3), (C6-PEG4-NAG3), (NAG3), (NAG4), (NAG3-AA2), (NAG3-Palm), (NAG13), (NAG18), (NAG24), (NAG25), (NAG25)s, (NAG26), (NAG27), (NAG28), (NAG29), (NAG30), (NAG30)s, (NAG31), (NAG31s), (NAG32), (NAG33), (NAG34), (NAG35), (NAG36) or (NAG37).

19. The LPA RNAi agent of claim 18, wherein the targeting group comprises: NAG is N-acetylgalactosamine, and X is selected from O and S.

20. The LPA RNAi agent of claim 15, wherein the targeting group is conjugated to the 3′ end of the sense strand.

21. The LPA RNAi agent of claim 15, wherein the targeting group is conjugated to the 5′ end of the sense strand.

22. The LPA RNAi agent according to any one of claims 1-9, wherein the antisense strand comprises the sequence of SEQ ID NO:1280 and the sense strand comprises the sequence of SEQ ID NO:1284.

23. The LPA RNAi agent according to any one of claims 1-9, wherein a) The antisense strand contains the sequence of SEQ ID NO:156 and the sense strand contains the sequence of SEQ ID NO:

310. b) The antisense strand contains the sequence of SEQ ID NO:164 and the sense strand contains the sequence of SEQ ID NO:

357. c) The antisense strand contains the sequence of SEQ ID NO:188 and the sense strand contains the sequence of SEQ ID NO:

384. d) The antisense strand contains the sequence of SEQ ID NO:164 and the sense strand contains the sequence of SEQ ID NO:376, or e) The antisense chain contains the sequence of SEQ ID NO:164 and the sense chain contains the sequence of SEQ ID NO:

384.

24. The LPA RNAi agent according to any one of claims 1-9, wherein a) The antisense strand comprises the modified nucleotide sequence of SEQ ID NO:790 and the sense strand comprises the modified nucleotide sequence of SEQ ID NO:1189; b) The antisense strand comprises the modified nucleotide sequence of SEQ ID NO:637 and the sense strand comprises the modified nucleotide sequence of SEQ ID NO:1132; c) The antisense strand comprises the modified nucleotide sequence of SEQ ID NO:709 and the sense strand comprises the modified nucleotide sequence of SEQ ID NO:1135; d) The antisense strand comprises the modified nucleotide sequence of SEQ ID NO:787 and the sense strand comprises the modified nucleotide sequence of SEQ ID NO:1191, or e) The antisense strand comprises the modified nucleotide sequence of SEQ ID NO:788 and the sense strand comprises the modified nucleotide sequence of SEQ ID NO:1189.

25. The LPA RNAi agent of claim 24, wherein the antisense strand consists of a modified nucleotide sequence of SEQ ID NO:790, and the sense strand consists of a modified nucleotide sequence of SEQ ID NO:1189.

26. The LPA RNAi agent of claim 25, wherein the LPA RNAi agent further comprises a (NAG25) or (NAG25)s targeting group conjugated to the 5′ end of the sense strand.

27. The LPA RNAi agent of claim 24, wherein the LPA RNAi agent further comprises a (NAG25) or (NAG25)s targeting group conjugated to the 5′ end of the sense strand.

28. A pharmaceutical composition comprising an LPA RNAi agent as described in any one of claims 1-27 and a pharmaceutically acceptable excipient.

29. Use of the LPA RNAi agent as described in any one of claims 1-27 in the preparation of a medicament for treating cardiovascular disease in a subject in need.

30. Use of the LPA RNAi agent as described in any one of claims 1-27 in the preparation of a medicament for reducing serum Lp(a) levels in subjects who have cardiovascular disease or are at risk of having cardiovascular disease.

31. Use of the LPA RNAi agent as described in any one of claims 1-27 in the preparation of a medicament for treating peripheral artery disease in a subject in need.

32. Use of the LPA RNAi agent as described in any one of claims 1-27 in the preparation of a medicament for treating coronary artery disease in a subject in need.

33. Use of the LPA RNAi agent as described in any one of claims 1-27 in the preparation of a medicament for treating acute coronary syndrome in a subject in need.

34. Use of the LPA RNAi agent as described in any one of claims 1-27 in the preparation of a medicament for treating aortic stenosis in a subject in need.

35. Use of the LPA RNAi agent as described in any one of claims 1-27 in the preparation of a medicament for treating heterozygous or homozygous familial hypercholesterolemia in subjects of need.

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