RNAi agents for inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9), pharmaceutical compositions thereof, and methods of use

Abstract

The present disclosure relates to RNAi agents, e.g., double-stranded RNAi agents such as small interfering RNA (siRNA) molecules, that can inhibit the expression of the proprotein convertase subtilisin kexin 9 (PCSK9) gene. Pharmaceutical compositions containing the PCSK9 RNAi agents and methods for using the same are also disclosed. The PCSK9 RNAi agents disclosed herein can be conjugated to targeting ligands, including N-acetylgalactosamine-containing ligands, to facilitate delivery to hepatocytes. Delivery of the PCSK9 RNAi agents in vivo inhibits PCSK9 gene expression, thereby reducing PCSK9 protein. The RNAi agents can be used in methods for treating diseases or disorders at least partially mediated by PCSK9 gene expression, including, inter alia, hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, and cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 487,899, filed March 2, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing (compliant with standard ST26), which has been submitted in xml format and is incorporated herein by reference in its entirety. The xml Sequence Listing file is named 30708-WO_SeqListing.xml, was created on February 27, 2024, and is 1581 kb in size.

[0003] The present disclosure relates to RNA interference (RNAi) agents, e.g., double-stranded RNAi agents, for inhibiting proprotein convertase subtilisin kexin 9 (PCSK9) gene expression, pharmaceutical compositions comprising the PCSK9 RNAi agents, and methods of use thereof for the treatment of PCSK9-related diseases and disorders. [Background technology]

[0004] Proprotein convertase subtilisin kexin 9 (PCSK9) plays a key role in plasma cholesterol metabolism and has been identified by both academic and industrial scientists as a target for treating hypercholesterolemia (Peterson et al., J Lipid Res. 2008). Low-density lipoprotein (LDL) is the primary transporter of cholesterol in the bloodstream. LDL cholesterol (LDL-C) is normally removed from the bloodstream by receptor-mediated endocytosis in the liver via the LDL receptor (Horton et al., 2006). Because excessively high LDL-C levels in the blood (hypercholesterolemia) and tissues are associated with the development of atherosclerotic plaques and cardiovascular disease, maintaining adequate levels of LDL receptors is important to prevent LDL-C buildup (Horton et al., 2006; Klein-Szanto & Bassi, 2019). Mutations in the LDL receptor or mutations affecting LDL binding to the receptor have been shown to cause hypercholesterolemia. Furthermore, gain-of-function mutations in PCSK9 have been found to increase LDL levels by promoting the degradation of LDL receptors, while loss-of-function mutations result in hypocholesterolemia, significantly reducing the risk of coronary artery disease (Horton et al., 2006; Klein-Szanto & Bassi, 2019).

[0005] Therefore, PCSK9 has become a therapeutic target for cholesterol-lowering treatment. Statins that increase LDL receptor expression and antibody therapeutics that inhibit PCSK9 are already on the market, but some patients experience tolerability issues that prevent their use as effective treatments. Various small molecule inhibitors of PCSK9 and inhibitors that prevent LDL receptor binding are in clinical development, and small interfering RNA (siRNA) specific for PCSK9 mRNA was recently approved in the UK and the US, but they still have certain drawbacks. Given the role that PCSK9 plays in regulating plasma LDL levels and the severity of the disease when hypercholesterolemia remains uncontrolled, further development of additional inhibitory therapeutics will provide needed options for improving the cardiovascular health of patients with high LDL-C. Summary of the Invention

[0006] Disclosed herein are novel RNAi agents for inhibiting expression of the PCSK9 gene.

[0007] In some embodiments, the PCSK9 RNAi agent is an antisense strand, wherein nucleotides 1-21 of the antisense strand comprise nucleotides 1-21 of the antisense strand sequence of Table 2; and a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand Including, All or substantially all of the nucleotides of the antisense and / or sense strands are modified nucleotides, and the RNAi agent is linked to a targeting ligand that includes an N-acetylgalactosamine.

[0008] In some embodiments, the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides that differs by 0 or 1 nucleotide from 15 contiguous nucleotides of any one of the sense strand sequences in Table 2, Table 4, Table 5C, Table 7B, or Table 8, and the sense strand has a region of at least 85% complementarity with the antisense strand over the 15 contiguous nucleotides.

[0009] In some embodiments, at least one nucleotide of the RNAi agent comprises a modified internucleoside linkage.

[0010] In some embodiments, the modified nucleotide of a PCSK9 RNAi agent disclosed herein is selected from the group consisting of 2'-O-methyl nucleotides, 2'-fluoro nucleotides (also referred to as 2'-deoxy-2'-fluoro nucleotides), 2'-deoxy nucleotides, 2',3'-seconucleotide mimics (also referred to as locked nucleotides or UNAs), locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted 2'-O-methyl nucleotides, inverted 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, 5'-vinyl phosphonate-containing nucleotides, 5'-cyclopropyl phosphonate-containing nucleotides, and 3'-O-methyl nucleotides.

[0011] In other embodiments, all or substantially all of the modified nucleotides of the RNAi agents disclosed herein are 2'-O-methyl nucleotides, 2'-fluoro nucleotides, or a combination thereof.

[0012] In some embodiments, the 5'-end of the antisense strand comprises a 5'-cyclopropylphosphonate-2'-O-methyl nucleotide.

[0013] In some embodiments, the antisense strand consists of, consists essentially of, or comprises the nucleotide sequence of any one of the modified antisense strand sequences in Table 3.

[0014] In some embodiments, the sense strand consists of, consists essentially of, or comprises the nucleotide sequence of any of the modified sense strand sequences in Table 4.

[0015] In some embodiments, the antisense strand comprises the nucleotide sequence of any one of the modified sequences in Table 3, and the sense strand comprises the nucleotide sequence of any one of the modified sequences in Table 4.

[0016] The RNAi agent disclosed herein is linked to the targeting ligand that comprises N-acetylgalactosamine.In further embodiments, the targeting ligand is linked to the sense strand.In some embodiments, the targeting ligand is linked to the 5' end of the sense strand.

[0017] In some embodiments, the sense strand is 15-30 nucleotides in length and the antisense strand is 21-30 nucleotides in length. In other embodiments, the sense strand and antisense strand are each 21-27 nucleotides in length. In other embodiments, the sense strand and antisense strand are each 21-24 nucleotides in length. In yet other embodiments, the sense strand and antisense strand are each 21 nucleotides in length.

[0018] In some embodiments, the RNAi agent has two blunt ends.

[0019] In some embodiments, the sense strand comprises one or two end caps, hi other embodiments, the sense strand comprises one or two inverted abasic residues.

[0020] In some embodiments, the RNAi agent is composed of a sense strand and an antisense strand that form a duplex sequence of a duplex structure shown in Table 5A, Table 5B, Table 5C, or Table 8.

[0021] In some embodiments, the sense strand further comprises an inverted abasic residue at the 3' end of the nucleotide sequence, the 5' end of the nucleotide sequence, or both.

[0022] In some embodiments, the targeting ligand is [ka] Includes.

[0023] In a further embodiment, the targeting ligand is linked to the sense strand of the RNAi agent by a phosphodiester or phosphorothioate bond; [ka] It comprises or consists of:

[0024] In some embodiments, the PCSK9 RNAi agent comprises an antisense strand comprising or consisting of the nucleotide sequence of SEQ ID NO:401, and a sense strand comprising or consisting of the nucleotide sequence of SEQ ID NO:430.

[0025] In some embodiments, a PCSK9 RNAi agent comprises a modified antisense strand that comprises, or consists of, the nucleotide sequence of SEQ ID NO:309, and a modified sense strand that comprises, or consists of, the nucleotide sequence of SEQ ID NO:368.

[0026] Also disclosed herein are compositions comprising the disclosed RNAi agents, further comprising a pharmaceutically acceptable excipient.

[0027] Further provided herein is a method for inhibiting expression of the PCSK9 gene in liver cells in a human subject in vivo, comprising introducing into the subject an effective amount of a disclosed PCSK9 RNAi agent or a disclosed composition.

[0028] Further provided herein are methods for treating a PCSK9-associated disease, disorder, or condition, comprising administering to a human subject in need thereof a therapeutically effective amount of the disclosed compositions.

[0029] In some embodiments, the disease includes hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD), and / or other PCSK9-related diseases.

[0030] In some embodiments, the RNAi agent is administered at a dose of about 0.05 mg to about 5.0 mg per kg of body weight of a human subject. In some embodiments, the PCSK9 RNAi agent disclosed herein is administered in a single fixed-dose injection comprising about 25 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, or about 400 mg of a PCSK9 RNAi agent drug substance listed in Table 8.

[0031] Also provided herein is the use of the disclosed RNAi agents or the disclosed compositions for the treatment of a disease, disorder, or condition mediated at least in part by PCSK9 gene expression.

[0032] Further provided herein is a use of the disclosed RNAi agent or the disclosed composition for the preparation of a pharmaceutical composition for treating a disease, disorder, or condition mediated at least in part by PCSK9 gene expression. [Brief explanation of the drawings]

[0033] [Figures 1A-1D] 1 is a chemical structure representation of PCSK9 RNAi agent AD09327 (SEQ ID NOs: 309 / 368) with a targeting ligand (NAG37) linked to the 5' end of the sense strand, shown as the free acid.

[0034] [Figures 2A-2D] 1 is a chemical structure representation of PCSK9 RNAi agent AD09327 (SEQ ID NOs: 309 / 368) with a targeting ligand (NAG37) linked to the 5' end of the sense strand, shown as the sodium salt. DETAILED DESCRIPTION OF THE INVENTION

[0035] The disclosed RNAi agents, compositions thereof, and methods of use can be more readily understood by reference to the following detailed description, which forms a part of this disclosure. It is to be understood that the present disclosure is not limited to what is specifically described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting.

[0036] It will be understood that certain features of the disclosure contained herein, which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0037] definition

[0038] As used herein, "RNAi agent" refers to a chemical composition comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can degrade mRNA transcripts of a target messenger RNA (mRNA) in a sequence-specific manner or inhibit the translation of the mRNA (e.g., degrade or inhibit under appropriate conditions). As used herein, an RNAi agent may act via an RNA interference mechanism (i.e., inducing RNA interference via interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) in mammalian cells) or may act by any alternative mechanism or pathway. Although RNAi agents, as the term is used herein, are believed to act primarily via the RNA interference mechanism, the disclosed RNAi agents are not constrained or limited to any particular pathway or mechanism of action. The RNAi agents disclosed herein are composed of a sense strand and an antisense strand and include, but are not limited to, small (or short) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the targeted mRNA (i.e., PCSK9 mRNA). The RNAi agent can contain one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0039] As used herein, the terms "silencing," "reducing," "inhibiting," "downregulating," or "knockdown," when referring to the expression of a given gene, mean that expression of the gene, as measured by the level of RNA transcribed from the gene or the level of a polypeptide, protein, or protein subunit translated from the mRNA, in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is decreased when the cell, group of cells, tissue, organ, or subject is treated with an RNAi agent as described herein, compared to a second cell, group of cells, tissue, organ, or subject that has not been so treated.

[0040] As used herein, the terms "sequence" and "nucleotide sequence" refer to a sequence or order of nucleic acid bases or nucleotides written in consecutive letters using standard nomenclature. A nucleotide sequence can contain unmodified and / or modified nucleotides. An oligonucleotide or nucleic acid molecule can contain unmodified and / or modified nucleotides.

[0041] As used herein, a "base," "nucleotide base," or "nucleobase" refers to a heterocyclic pyrimidine or purine compound that is a component of a nucleotide, including the primary purine bases adenine and guanine, and the primary pyrimidine bases cytosine, thymine, and uracil. Nucleobases may be further modified to include, but are not limited to, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases (see, e.g., Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008). The synthesis of such modified nucleobases (including phosphoramidite compounds containing modified nucleobases) is known in the art.

[0042] As used herein, the term "nucleotide" has the same meaning as commonly understood in the art. Thus, as used herein, the term "nucleotide" refers to a glycoside comprising a sugar moiety, a base moiety, and a covalently attached group (linking group), such as a phosphate or phosphorothioate internucleoside linking group, and encompasses both naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and / or base moieties, also referred to herein as nucleotide analogs. As used herein, a single nucleotide may be referred to as a monomer or unit.

[0043] As used herein, and unless otherwise specified, the term "complementary," when used to describe a first nucleobase or nucleotide sequence (e.g., the sense strand of an RNAi agent or a targeting mRNA) to a second nucleobase or nucleotide sequence (e.g., the antisense strand of an RNAi agent or a single-stranded antisense oligonucleotide), refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base-pair hydrogen bonds under mammalian physiological conditions (or other suitable in vivo or in vitro conditions)) with an oligonucleotide comprising the second nucleotide sequence to form a duplex or double-helix structure under specific standard conditions. Those skilled in the art will be able to select the most appropriate set of conditions for the hybridization test. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, so long as at least the above hybridization requirements are met. Sequence identity or complementarity is independent of modifications. For example, a and Af, as defined herein, are complementary to U (or T) and identical to A for purposes of determining identity or complementarity.

[0044] As used herein, "fully complementary" or "sufficiently complementary" means that in a hybridized pair of nucleic acid base or nucleotide sequence molecules, all (100%) of the bases in a contiguous sequence of a first oligonucleotide hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may include all or a portion of the first or second nucleotide sequence.

[0045] As used herein, "partially complementary" means that in a hybridized pair of nucleic acid base or nucleotide sequence molecules, at least 70% (but not all) of the bases in a contiguous sequence of a first oligonucleotide hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may include all or a portion of the first or second nucleotide sequence.

[0046] As used herein, "substantially complementary" means that in a hybridized pair of nucleic acid or nucleotide sequence molecules, at least 85% (but not all) of the bases in a contiguous sequence of a first oligonucleotide hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may include all or a portion of the first or second nucleotide sequence.

[0047] As used herein, the terms "complementary," "fully complementary," "partially complementary," and "substantially complementary" are used in reference to nucleobase or nucleotide matches between the sense and antisense strands of an RNAi agent or between the antisense strand of an RNAi agent and the sequence of PCSK9 mRNA.

[0048] As used herein, the term "substantially identical" or "substantial identity" as applied to a nucleic acid sequence means that a nucleotide sequence (or a portion of a nucleotide sequence) has at least about 85% or more sequence identity, e.g., at least 90%, at least 95%, or at least 99% identity, compared to a reference sequence. The percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions in both sequences where the same type of nucleobase occurs to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to calculate the percentage of sequence identity. The invention disclosed herein encompasses nucleotide sequences that are substantially identical to the nucleotide sequences disclosed herein.

[0049] As used herein, the terms "individual," "patient," and "subject" are used interchangeably to refer to a member of any animal species, including, but not limited to, birds, humans and other primates, and other mammals, including commercially relevant mammals or animal models, such as mice, rats, monkeys, cows, pigs, horses, sheep, cats, and dogs. Preferably, the subject is a human.

[0050] As used herein, the terms "treat," "treatment," and the like refer to a method or step taken to reduce or alleviate the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, "treat" and "treatment" can include preventing, managing, prophylactic treatment, and / or inhibiting or reducing the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0051] As used herein, "PCSK9-related diseases or disorders" includes any disease or disorder that can be treated by a PCSK9 RNAi agent (i.e., a decrease in expression of the PCSK9 gene, and thereby a decrease in the amount of PCSK9 protein found in a cell or tissue, can provide a therapeutic benefit to a subject), including, but not limited to, hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD), or any other disease or disorder in which a reduction in low-density lipoprotein cholesterol (LDL-C) is desirable or would benefit a subject or patient.

[0052] As used herein, the phrase "introducing into a cell" when referring to an RNAi agent means functionally delivering the RNAi agent to a cell. The phrase "functional delivery" means delivering the RNAi agent to a cell in a manner that allows the RNAi agent to have the expected biological activity (e.g., sequence-specific inhibition of gene expression).

[0053] Unless otherwise noted, symbols used herein [ka] The use of means that any group may be attached in accordance with the scope of the invention described herein.

[0054] As used herein, the term "isomers" refers to compounds that have identical molecular formulae but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers." Stereoisomers that are not mirror images of one another are termed "diastereoisomers," and stereoisomers that are non-superimposable mirror images are termed "enantiomers" or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is termed a "chiral center."

[0055] As used herein, unless a structure is specifically identified as having a particular conformation, for each structure in which asymmetric centers exist and which therefore give rise to enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to represent all such possible isomers, including optically pure and racemic forms. For example, the structures disclosed herein are intended to encompass mixtures of diastereomers as well as single stereoisomers.

[0056] When used in a claim herein, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. When used in a claim herein, the phrase "consisting essentially of" limits the claim to the items or steps specified and to those items or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0057] Those skilled in the art will readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state depending on the environment in which the compound or composition is placed. Therefore, as used herein, the structures disclosed herein contemplate that certain functional groups, such as OH, SH, or NH, may be protonated or deprotonated. As will be readily understood by those skilled in the art, the disclosure herein is intended to encompass the disclosed compounds and compositions regardless of their protonation state based on the environment (e.g., pH). Similarly, it should be understood that compounds described herein having labile protons or basic atoms represent salt forms of the corresponding compounds. The compounds described herein may be in the form of a free acid, a free base, or a salt. It should be understood that pharmaceutically acceptable salts of the compounds described herein are within the scope of the present invention.

[0058] As used herein, the terms "linked" or "conjugated," when referring to a connection between two compounds or molecules, mean that the two compounds or molecules are connected by a covalent bond. Unless otherwise specified, the terms "linked" and "conjugated," as used herein, may refer to a connection between a first compound and a second compound with or without any intervening atoms or groups of atoms.

[0059] As used herein, the term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to." The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although methods and materials similar or equivalent to those described herein can be used in practicing or testing the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are merely illustrative and not limiting.

[0061] Where a value is explicitly recited, it is understood that values ​​that are approximately the same quantity or amount as the recited value are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of the disclosure is disclosed as having multiple alternatives, examples of the disclosure in which each alternative is excluded, alone or in any combination with the other alternatives, are also disclosed herein. More than one element of the disclosure may have such an exclusion, and all combinations of elements with such exclusions are disclosed herein.

[0062] Other objects, features, aspects, and advantages of the present invention will become apparent from the following detailed description, the accompanying drawings, and the claims.

[0063] RNAi agents Described herein are RNAi agents for inhibiting expression of the PCSK9 gene. Each PCSK9 RNAi agent comprises a sense strand and an antisense strand. The sense strand can be 15-49 nucleotides in length. The antisense strand can be 18-49 nucleotides in length. The sense strand and antisense strand can be the same length or different lengths. In some embodiments, the sense strand and antisense strand are each independently 21-27 nucleotides in length. In some embodiments, both the sense strand and the antisense strand are each 21-26 nucleotides in length. In some embodiments, the sense strand and the antisense strand are each 21-24 nucleotides in length. In some embodiments, the sense strand is about 19 nucleotides in length and the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 21 nucleotides in length and the antisense strand is about 23 nucleotides in length. In some embodiments, the sense strand is 23 nucleotides in length and the antisense strand is 21 nucleotides in length. In some embodiments, both the sense strand and the antisense strand are each 21 nucleotides in length. In some embodiments, each antisense strand of an RNAi agent is independently 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, each sense strand of an RNAi agent is independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides in length. The sense and antisense strands are annealed to form a duplex, and in some embodiments the double-stranded RNAi agent has a duplex length of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0064] Examples of nucleotide sequences used to form PCSK9 RNAi agents are provided in Table 2, Table 3, Table 4, Table 5C, Table 7A, Table 7B, and Table 8. Examples of RNAi agent duplexes comprising the sense and antisense strand sequences of Table 2, Table 3, Table 4, Table 5C, Table 7A, and Table 7B are shown in Table 5A, Table 5B, Table 5C, and Table 8.

[0065] In some embodiments, the region of complete, substantial, or partial complementarity between the sense strand and the antisense strand is 15 to 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides in length and is located at or near the 5' end of the antisense strand (e.g., this region can be 0, 1, 2, 3, or 4 nucleotides away from the 5' end of the antisense strand that are not complete, substantial, or partial complementarity).

[0066] The sense strand of the PCSK9 RNAi agent described herein comprises at least 15 contiguous nucleotides that are at least 85% identical to a core stretch sequence (also referred to herein as a "core stretch" or "core sequence") of the same number of nucleotides in the PCSK9 mRNA. In some embodiments, the sense strand core stretch sequence is 100% (fully) complementary or at least about 85% (substantially) complementary to the antisense strand core stretch sequence. Thus, the sense strand core stretch sequence is typically fully identical or at least about 85% identical to a nucleotide sequence of the same length present in the PCSK9 mRNA target (e.g., sometimes referred to as a target sequence). In some embodiments, the sense strand core stretch is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, the sense strand core stretch is 17 nucleotides in length. In some embodiments, the sense strand core stretch is 19 nucleotides in length. In some embodiments, the sense strand core stretch is 21 nucleotides in length.

[0067] The antisense strand of the PCSK9 RNAi agent described herein comprises at least 15 consecutive nucleotides that are at least 85% complementary to a core stretch of the same number of nucleotides in PCSK9 mRNA and a core stretch of the same number of nucleotides in the corresponding sense strand. In some embodiments, the antisense strand core stretch is 100% (fully) complementary or at least about 85% (substantially) complementary to a nucleotide sequence of the same length present in the PCSK9 mRNA target (e.g., target sequence). In some embodiments, the antisense strand core stretch is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, the antisense strand core stretch is 21 nucleotides in length. In some embodiments, the antisense strand core stretch is 19 nucleotides in length. The sense strand core stretch sequence may be the same length as the corresponding antisense core sequence, or may be a different length.

[0068] The sense and antisense strands of a PCSK9 RNAi agent anneal to form a duplex. The sense and antisense strands of a PCSK9 RNAi agent can be partially, substantially, or completely complementary to each other. Within the complementary duplex region, the sense strand core stretch sequence is at least 85% complementary, or 100% complementary, to the antisense core stretch sequence. In some embodiments, the sense strand core stretch sequence comprises a sequence of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides that is at least 85% or 100% complementary to a corresponding 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide sequence of the antisense strand core stretch sequence (i.e., the sense and antisense core stretch sequences of the PCSK9 RNAi agent have a region of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or nucleotides that are at least 85% base-paired or 100% base-paired).

[0069] In some embodiments, the antisense strand of a PCSK9 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 2, Table 3, Table 5C, Table 7A, or Table 8. In some embodiments, the sense strand of a PCSK9 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 2, Table 4, Table 5C, Table 7B, or Table 8.

[0070] In some embodiments, the sense strand and / or the antisense strand can optionally and independently comprise 1, 2, 3, 4, 5, or 6 additional nucleotides (extensions) at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the core stretch sequence. The additional nucleotides of the antisense strand, if present, may or may not be complementary to the corresponding sequence in PCSK9 mRNA. The additional nucleotides of the sense strand, if present, may or may not be identical to the corresponding sequence in PCSK9 mRNA. The additional nucleotides of the antisense strand, if present, may or may not be complementary to the corresponding additional nucleotides (if present) of the sense strand.

[0071] As used herein, an extension comprises 1, 2, 3, 4, 5, or 6 nucleotides at the 5'-end and / or 3'-end of the sense strand core stretch sequence and / or the antisense strand core stretch sequence. The extension nucleotides of the sense strand may or may not be complementary to any of the nucleotides of the core stretch sequence or extension nucleotides in the corresponding antisense strand. Conversely, the extension nucleotides of the antisense strand may or may not be complementary to any of the nucleotides of the core stretch sequence or extension nucleotides in the corresponding sense strand. In some embodiments, both the sense and antisense strands of an RNAi agent comprise a 3'-extension and a 5'-extension. In some embodiments, one or more of the 3'-extension nucleotides of one strand are base-paired with one or more 5'-extension nucleotides of the other strand. In other embodiments, one or more of the 3'-extension nucleotides of one strand are not base-paired with one or more 5'-extension nucleotides of the other strand. In some embodiments, the PCSK9 RNAi agent has an antisense strand with a 3' extension and a sense strand with a 5' extension. In some embodiments, the extended nucleotides are unpaired and form an overhang. As used herein and in the art, the term "overhang" refers to an extension of a stretch of one or more unpaired nucleotides located at the end of either the sense strand or the antisense strand that does not form part of the hybridized or duplex portion of the RNAi agent disclosed herein.

[0072] In some embodiments, the PCSK9 RNAi agent comprises an antisense strand having a 3' extension that is 1, 2, 3, 4, 5, or 6 nucleotides in length. In other embodiments, the PCSK9 RNAi agent comprises an antisense strand having a 3' extension that is 1, 2, or 3 nucleotides in length. In some embodiments, one or more of the antisense strand extension nucleotides comprises a nucleotide that is complementary to the corresponding PCSK9 mRNA sequence. In some embodiments, one or more of the antisense strand extension nucleotides comprises a nucleotide that is not complementary to the corresponding PCSK9 mRNA sequence.

[0073] In some embodiments, the PCSK9 RNAi agent comprises a sense strand having a 3' extension of 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprises an adenosine, uracil, or thymidine nucleotide, an AT dinucleotide, or a nucleotide that corresponds to or is identical to a nucleotide in the PCSK9 mRNA sequence. In some embodiments, the 3' sense strand extension comprises or consists of, but is not limited to, one of the following sequences: T, UT, TT, UU, UUT, TTT, or TTTT (each listed in the 5' to 3' direction).

[0074] The sense strand can have a 3' extension and / or a 5' extension. In some embodiments, the PCSK9 RNAi agent comprises a sense strand having a 5' extension that is 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprises a nucleotide that corresponds to or is identical to a nucleotide in the PCSK9 mRNA sequence.

[0075] Examples of sequences used to form PCSK9 RNAi agents are provided in Table 2, Table 3, Table 4, Table 5C, Table 7A, Table 7B, and Table 8. In some embodiments, the antisense strand of the PCSK9 RNAi agent comprises any of the sequences in Table 2, Table 3, Table 5C, Table 7A, or Table 8. In certain embodiments, the antisense strand of the PCSK9 RNAi agent comprises or consists of any one of the modified sequences in Table 3. In some embodiments, the antisense strand of the PCSK9 RNAi agent comprises the nucleotide sequence (5' end to 3' end) 1-17, 2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21 of any of the sequences in Table 2, Table 3, Table 5C, Table 7A, or Table 8. In some embodiments, the PCSK9 RNAi agent sense strand comprises any of the sequences in Table 2, Table 4, Table 5C, Table 7B, or Table 8. In some embodiments, the sense strand of the PCSK9 RNAi agent comprises a nucleotide sequence (5' end to 3' end) 1-18, 1-19, 1-20, 1-21, 2-19, 2-20, 2-21, 3-20, 3-21, or 4-21 of any of the sequences in Table 2, Table 4, Table 5C, Table 7B, or Table 8. In particular embodiments, the sense strand of the PCSK9 RNAi agent comprises or consists of a modified sequence of any one of the modified sequences in Table 4.

[0076] In some embodiments, the sense strand and antisense strand of an RNAi agent described herein comprise the same number of nucleotides. In some embodiments, the sense strand and antisense strand of an RNAi agent described herein comprise a different number of nucleotides. In some embodiments, the 5' end of the sense strand and the 3' end of the antisense strand of an RNAi agent form a blunt end. In some embodiments, the 3' end of the sense strand and the 5' end of the antisense strand of an RNAi agent form a blunt end. In some embodiments, both ends of an RNAi agent form a blunt end. In some embodiments, neither end of an RNAi agent is blunt. As used herein, "blunt end" refers to the ends of a double-stranded RNAi agent in which the terminal nucleotides of the two annealed strands are complementary (form complementary base pairs).

[0077] In some embodiments, the 5' end of the sense strand and the 3' end of the antisense strand of an RNAi agent form frayed ends. In some embodiments, the 3' end of the sense strand and the 5' end of the antisense strand of an RNAi agent form frayed ends. In some embodiments, both ends of an RNAi agent form frayed ends. In some embodiments, neither end of an RNAi agent is a frayed end. As used herein, "frayed end" refers to the end of a double-stranded RNAi agent in which the terminal nucleotides of the two annealed strands are paired (i.e., do not form an overhang) but are not complementary (i.e., form a non-complementary pair). In some embodiments, one or more unpaired nucleotides at the end of one strand of a double-stranded RNAi agent form an overhang. The unpaired nucleotides may be present in the sense strand or the antisense strand and form a 3' or 5' overhang. In some embodiments, the RNAi agent comprises a blunt end and a frayed end, a blunt end and a 5' overhanging end, a blunt end and a 3' overhanging end, a frayed end and a 5' overhanging end, a frayed end and a 3' overhanging end, two 5' overhanging ends, two 3' overhanging ends, a 5' overhanging end and a 3' overhanging end, two frayed ends, or two blunt ends. Typically, the overhangs, if present, are located at the 3' end of the sense strand, the antisense strand, or both the sense and antisense strands.

[0078] The PCSK9 RNAi agents disclosed herein may also be composed of one or more modified nucleotides. In some embodiments, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand of the PCSK9 RNAi agent are modified nucleotides. The PCSK9 RNAi agents disclosed herein may further be composed of one or more modified internucleoside linkages, for example, one or more phosphorothioate linkages. In some embodiments, the PCSK9 RNAi agent comprises one or more modified nucleotides and one or more modified internucleoside linkages. In some embodiments, a 2'-modified nucleotide is combined with a modified internucleoside linkage.

[0079] In some embodiments, the PCSK9 RNAi agent is prepared or provided as a salt, mixed salt, or free acid. In some embodiments, the PCSK9 RNAi agent is prepared as a pharmaceutically acceptable salt. In some embodiments, the PCSK9 RNAi agent is prepared as a pharmaceutically acceptable sodium salt. Such forms known in the art are within the scope of the invention disclosed herein.

[0080] Modified Nucleotides Modified nucleotides, when used in various oligonucleotide constructs, can maintain the activity of the compounds within cells while increasing the serum stability of these compounds and can also minimize the potential for activating interferon activity in humans upon administration of the oligonucleotide construct.

[0081] In some embodiments, the PCSK9 RNAi agent comprises one or more modified nucleotides. As used herein, a "modified nucleotide" is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, inverted nucleotides, modified nucleobase-containing nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2',3'-seconucleotide mimics (non-locked nucleobase analogs or non-locked nucleotides), locked nucleotides, 3'-O-methoxy (2' internucleoside linkage) nucleotides, 2'-F-arabinonucleotides, 5'-Me, 2'-fluoronucleotides, morpholino nucleotides, vinylphosphonate deoxyribonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides. 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 (also referred to herein or in the art as 2'-methoxy nucleotides), 2'-fluoro nucleotides (also referred to herein or in the art as 2'-deoxy-2'-fluoro nucleotides), 2'-deoxy nucleotides, 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides (also referred to herein or in the art as 2'-MOE nucleotides), 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, two or more modifications can be incorporated into a single PCSK9 RNAi agent or into that single nucleotide. The sense and antisense strands of a PCSK9 RNAi agent can be synthesized and / or modified by methods known in the art.A modification at one nucleotide is independent of a modification at another nucleotide.

[0082] Modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiothiazol-1-yl ... Included are synthetic and natural nucleobases such as ocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0083] In some embodiments, the 5'-end and / or 3'-end of the antisense strand can contain an abasic residue (Ab), which may also be referred to as an "abasic site" or "abasic nucleotide." An abasic residue (Ab) is a nucleotide or nucleoside lacking a nucleobase at the 1'-position of the sugar moiety. In some embodiments, the abasic residue can be positioned within the nucleotide sequence. In some embodiments, Ab or AbAb can be added to the 3'-end of the antisense strand. In some embodiments, the 5'-end of the sense strand can contain one or more additional abasic residues (e.g., (Ab) or (AbAb)). In some embodiments, UUAb, UAb, or Ab is added to the 3'-end of the sense strand. In some embodiments, the abasic (deoxyribose) residue can be replaced with a ribitol (abasic ribose) residue.

[0084] In some embodiments, all or substantially all of the nucleotides of an RNAi agent are modified nucleotides. As used herein, an RNAi agent in which substantially all of the nucleotides present are modified nucleotides is an RNAi agent that has four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides that are ribonucleotides (i.e., unmodified) in both the sense strand and the antisense strand. As used herein, a sense strand in which substantially all of the nucleotides present are modified nucleotides is a sense strand that has two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand that are unmodified ribonucleotides. As used herein, an antisense strand in which substantially all of the nucleotides present are modified nucleotides is an antisense strand that has two or fewer (i.e., 0, 1, or 2) nucleotides in the antisense strand that are unmodified ribonucleotides. In some embodiments, one or more nucleotides of an RNAi agent are unmodified ribonucleotides. The chemical structures of certain modified nucleotides are set forth in Table 6 herein.

[0085] Modified internucleoside linkages In embodiments, one or more nucleotides of a PCSK9 RNAi agent are joined by a non-canonical bond or backbone (ie, a modified internucleoside bond or a modified backbone). Modified internucleoside linkages or backbones include, but are not limited to, phosphorothioate groups (represented herein by a lowercase "s"), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkylphosphonates (e.g., methylphosphonates or 3'-alkylenephosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate, aminoalkylphosphoramidate, or thionophosphoramidate), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates with reversed polarity in which adjacent pairs of nucleoside units are linked 3'-5' then 5'-3' or 2'-5' then 5'-2'. In some embodiments, the modified internucleoside linkage or backbone lacks a phosphorus atom. Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short-chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short-chain heteroatom or heterocyclic intersugar linkages. In some embodiments, modified internucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH moieties.

[0086] In some embodiments, the sense strand of a PCSK9 RNAi agent can comprise 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, the antisense strand of a PCSK9 RNAi agent can comprise 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand can independently comprise 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, the sense strand of a PCSK9 RNAi agent can comprise 1, 2, 3, or 4 phosphorothioate linkages, the antisense strand of a PCSK9 RNAi agent can comprise 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand can independently comprise 1, 2, 3, or 4 phosphorothioate linkages.

[0087] In some embodiments, the sense strand of the PCSK9 RNAi agent contains at least two phosphorothioate internucleoside linkages. In some embodiments, the phosphorothioate internucleoside linkages are located between nucleotides 1 to 3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate internucleoside linkage is located at the 5' end of the sense strand nucleotide sequence, and another phosphorothioate linkage is located at the 3' end of the sense strand nucleotide sequence. In some embodiments, two phosphorothioate internucleoside linkages are located at the 5' end of the sense strand, and another phosphorothioate linkage is located at the 3' end of the sense strand. In some embodiments, the sense strand does not contain phosphorothioate internucleoside linkages between nucleotides, but does contain one, two, or three phosphorothioate linkages between the terminal nucleotides at both the 5' and 3' ends, and optionally between any inverted abasic residue end caps present. In some embodiments, a targeting ligand is attached to the sense strand via a phosphorothioate linkage.

[0088] In some embodiments, the antisense strand of the PCSK9 RNAi agent comprises four phosphorothioate internucleoside linkages. In some embodiments, the four phosphorothioate internucleoside linkages are present between nucleotides 1-3 from the 5' end of the antisense strand and between nucleotides 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end of the antisense strand. In some embodiments, three phosphorothioate internucleoside linkages are located between positions 1-4 from the 5' end of the antisense strand, and a fourth phosphorothioate internucleoside linkage is located between positions 20-21 from the 5' end of the antisense strand. In some embodiments, the PCSK9 RNAi agent comprises at least three or four phosphorothioate internucleoside linkages in the antisense strand.

[0089] Capping residues or moieties In some embodiments, the sense strand may include one or more capping residues or moieties, sometimes referred to in the art as "caps," "terminal caps," or "capping residues." As used herein, a "capping residue" is a non-nucleotide compound or other moiety that can be incorporated at one or more ends of the nucleotide sequence of an RNAi agent disclosed herein. Capping residues can, in some cases, confer certain beneficial properties to the RNAi agent, such as protection against exonuclease degradation. In some embodiments, an inverted abasic residue (invAb) (also referred to in the art as an "inverted abasic site") is added as a capping residue (see, e.g., F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16; U.S. Pat. No. 5,998,203). Capping residues are generally known in the art and include, for example, inverted abasic residues and terminal PCSK9H7(propyl), C6H 13 (hexyl), or C 12 H 25(dodecyl) group. In some embodiments, the capping residue is present at the 5'-end, the 3'-end, or both the 5'-end and the 3'-end of the sense strand. In some embodiments, the 5'-end and / or the 3'-end of the sense strand may contain one or more inverted abasic deoxyribose moieties as capping residues.

[0090] In some embodiments, one or more inverted abasic residues (invAb) are added to the 3'-end of the sense strand. In some embodiments, one or more inverted abasic residues (invAb) are added to the 5'-end of the sense strand. In some embodiments, one or more inverted abasic residues or inverted abasic sites are inserted between the targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. In some embodiments, the activity or other desired property of the RNAi agent is enhanced by including one or more inverted abasic residues or inverted abasic sites at or near the end of the sense strand of the RNAi agent.

[0091] In some embodiments, one or more inverted abasic residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more inverted abasic residues can be inserted between the targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. The inverted abasic residues can be linked by a phosphate linkage, a phosphorothioate linkage (e.g., referred to herein as (invAb)s), or other internucleoside linkage. In some embodiments, the activity or other desired properties of the RNAi agent can be enhanced by including one or more inverted abasic residues at or near the end of the sense strand of the RNAi agent. In some embodiments, the inverted abasic (deoxyribose) residue can be replaced with an inverted ribitol (abasic ribose) residue. In some embodiments, the 3' end of the antisense strand core stretch sequence or the 3' end of the antisense strand sequence can include an inverted abasic residue. The chemical structure of an inverted abasic deoxyribose residue is shown in Table 6 below.

[0092] PCSK9 RNAi agents The PCSK9 RNAi agents disclosed herein are designed to target specific locations on the PCSK9 gene (eg, SEQ ID NO: 1). NM_174936.4, Homo sapiens, proprotein convertase subtilisin / kexin type 9 (PCSK9), mRNA transcript (SEQ ID NO: 1): 1 agcgacgtcg aggcgctcat ggttgcaggc gggcgccgcc gttcagttca gggtctgagc 61 ctggaggat gagccaggca gtgagactgg ctcgggcggg ccgggacgcg tcgttgcagc 121 agcggctccc agctcccagc caggattccg cgcgcccctt cacgcgccct gctcctgaac 181 ttcagctcct gcacagtcct ccccaccgca aggctcaagg cgccgccggc gtggaccgcg 241 cacggcctct aggtctcctc gccaggacag caacctctcc cctggccctc atgggcaccg 301 tcagctccag gcggtcctgg tggccgctgc cactgctgct gctgctgctg ctgctcctgg 361 gtcccgcggg cgcccgtgcg caggaggacg aggacggcga ctacgaggag ctggtgctag 421 ccttgcgttc cgaggaggac ggcctggccg aagcacccga gcacggaacc acagccacct 481 tccaccgctg cgccaaggat ccgtggaggt tgcctggcac ctacgtggtg gtgctgaagg 541 aggagaccca cctctcgcag tcagagcgca ctgcccgccg cctgcaggcc caggctgccc 601 gccggggata cctcaccaag atcctgcatg tcttccatgg ccttcttcct ggcttcctgg 661 tgaagatgag tggcgacctg ctggagctgg ccttgaagtt gccccatgtc gactacatcg 721 aggaggactc ctctgtcttt gcccagagca tcccgtgga cctggagcgg attackccctc 781 cacggtaccg ggcggatgaa taccagcccc ccgacggagg cagcctggtg gaggtgtatc 841 tcctagacac cagcatacag agtgaccacc gggaaatcga gggcagggtc atggtcaccg 901 acttcgagaa tgtgcccgag gaggacggga cccgcttcca cagacaggcc agcaagtgtg 961 acagtcatgg cacccacctg gcaggggtgg tcagcggccg ggatgccggc gtggccaagg 1021 gtgccagcat gcgcagcctg cgcgtgctca actgccaagg gaagggcacg gttagcggca 1081 ccctcatagg cctggagttt attcggaaaa gccagctggt ccgcctgtg gggccactgg 1141 tggtgctgct gccctggcg ggtgggtaca gccgcgtcct caacgccgcc tgccagcgcc 1201 tggcgagggc tggggtcgtg ctggtcaccg ctgccggcaa cttccgggac gatgcctgcc 1261 tctactcccc agcctcagct cccgaggtca tcacagttgg ggccaccaat gcccaagacc 1321 agccggtgac cctggggact ttggggacca actttggccg ctgtgtggac ctctttgccc 1381 caggggagga catcattggt gcctccagcg actgcagcac ctgctttgtg tcacagagtg 1441 ggacatcaca ggctgctgcc cacgtggctg gcattgcagc catgatgctg tctgccgagc 1501 cggagctcac cctggccgag ttgaggcaga gactgatcca cttctctgcc aaagatgtca 1561 tcaatgaggc ctggttccct gaggaccagc gggtactgac ccccaacctg gtggccgccc 1621 tgccccccag cacccatggg gcaggttggc agctgttttg caggactgta tggtcagcac 1681 actcggggcc tacacggatg gccacagccg tcgcccgctg cgccccagat gaggagctgc 1741 tgagctgctc cagtttctcc aggagtggga agcggcgggg cgagcgcatg gaggcccaag 1801 ggggcaagct ggtctgccgg gcccacaacg cttttggggg tgagggtgtc tacgccattg 1861 ccaggtgctg cctgctaccc caggccaact gcagcgtcca cacagctcca ccagctgagg 1921 ccagcatggg gacccgtgtc cactgccacc aacagggcca cgtcctcaca ggctgcagct 1981 cccactggga ggtggaggac cttggcaccc acaagccgcc tgtgctgagg ccacgaggtc 2041 agcccaacca gtgcgtgggc cacagggagg ccagcatcca cgcttcctgc tgccatgccc 2101 caggtctgga atgcaaagtc aaggagcatg gaatcccggc ccctcaggag caggtgaccc 2161 tggcctgcga ggagggctgg accctgactg gctccagtgc cctccctggg acctcccag 2221 tcctggggc ctacgccgta gacaacgt gtgtagtcag gagccgggac gtcagcacta 2281 caggcagcac cagcgaaggg gccgtgacag ccgttgccat ctgctgccgg agccggcacc 2341 tggcgcaggc ctcccaggag ctccagtgac agccccatcc caggatgggt gtctggggag 2401 ggtcaagggc tggggctgag ctttaaaatg gttccgactt gtccctctct cagccctcca 2461 tggcctggca cgaggggatg gggatgcttc cgcctttccg gggctgctgg cctggccctt 2521 gagtggggca gcctccttgc ctggaactca ctcactctgg gtgcctcctc cccaggtgga 2581 ggtgccagga agctccctcc ctcactgtgg ggcatttcac cattcaaaca ggtcgagctg 2641 tgctcgggtg ctgccagctg ctcccaatgt gccgatgtcc gtgggcagaa tgacttttat 2701 tgagctcttg ttccgtgcca ggcattcaat cctcaggtct ccaccaagga ggcaggattc 2761 ttcccatgga taggggaggg ggcggtaggg gctgcaggga caaacatcgt tggggggtga 2821 gtgtgaaagg tgctgatggc cctcatctcc agctaactgt ggagaagccc ctgggggctc 2881 cctgattaat ggaggcttag ctttctggat ggcatctagc cagaggctgg agacaggtgc 2941 gcccctggtg gtcacaggct gtgccttggt ttcctgagcc acctttactc tgctctatgc 3001 caggctgtgc tagcaacacc caaaggtggc ctgcggggag ccatcaccta ggactgactc 3061 ggcagtgtgc agtggtgcat gcactgtctc agccaacccg ctccactacc cggcagggta 3121 cacattcgca cccctacttc acagaggaag aaacctggaa ccagaggggg cgtgcctgcc 3181 aagctcacac agcaggaact gagccagaaa cgcagattgg gctggctctg aagccaagcc 3241 tcttcttact tcacccggct gggctcctca tttttacggg taacagtgag gctgggaagg 3301 ggaacacaga ccaggaagct cggtgagtga tggcagaacg atgcctgcag gcatggaact 3361 ttttccgtta tcacccaggc ctgattcact ggcctggcgg agatgcttct aaggcatggt 3421 cgggggagag ggccaacaac tgtccctcct tgagcaccag ccccacccaa gcaagcagac 3481 atttatcttt tgggtctgtc ctctctgttg ccttttaca gccaactttt ctagacctgt 3541 tttgcttttg taacttgaag atatttattc tgggttttgt agcattttta ttaatatggt 3601 gactttttaa aataaaaaca aacaaacgtt gtcctaa

[0093] As defined herein, the antisense strand sequence is designed to target the PCSK9 gene at a given position on the gene when the 5'-end nucleobase of the antisense strand is aligned with a position 21 nucleotides downstream (towards the 3' end) from the position on the gene when base-pairing with the gene. For example, as exemplified in Table 1 and Table 2 herein, in an antisense strand sequence designed to target the PCSK9 gene at position 3536, the 5'-end nucleobase of the antisense strand must be aligned with position 3556 of the PCSK9 gene when base-pairing with the gene.

[0094] As provided herein, in a PCSK9 RNAi agent, the nucleobase at position 1 (5'→3') of the antisense strand need not be complementary to the gene, provided there is at least 85% complementarity between the antisense strand and the gene (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity) over a core stretch sequence of at least 15 contiguous nucleotides. For example, in the case of a PCSK9 RNAi agent disclosed herein that is designed to target position 3536 of the PCSK9 gene, the 5'-terminal nucleobase of the antisense strand of the PCSK9 RNAi agent must be aligned with position 3556 of the gene, but the 5'-terminal nucleobase of the antisense strand may be complementary to position 3556 of the PCSK9 gene, provided there is at least 85% complementarity between the antisense strand and the gene (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity) over a core stretch sequence of at least 15 contiguous nucleotides. As particularly demonstrated by the examples disclosed herein, and as is well known in the art, the specific site of binding of a gene by the antisense strand of a PCSK9 RNAi agent (e.g., whether the PCSK9 RNAi agent is designed to target the PCSK9 gene at position 3536 or some other position) is important to the level of inhibition achieved by the PCSK9 RNAi agent and the toxicity profile achieved by the molecule (see, e.g., Kamola et al., The siRNA Non-seed Region and Its Target Sequences are Auxiliary Determinants of Off-Target Effects, PLOS Computational Biology, 11(12), Figure 1 (2015)).

[0095] In some embodiments, a PCSK9 RNAi agent disclosed herein targets the PCSK9 gene at or near the location of the PCSK9 gene sequence shown in Table 1. In some embodiments, the antisense strand of a PCSK9 RNAi agent disclosed herein comprises a core stretch sequence that is fully, substantially, or at least partially complementary to a target PCSK9 21-mer sequence disclosed in Table 1. [Table 1]

[0096] In some embodiments, a PCSK9 RNAi agent comprises an antisense strand, and position 21 (5'→3') of the antisense strand can base pair with position 1 of a 21mer target sequence disclosed in Table 1. In some embodiments, a PCSK9 RNAi agent comprises an antisense strand, and position 1 (5'→3') of the antisense strand can base pair with position 21 of a 21mer target sequence disclosed in Table 1.

[0097] In some embodiments, a PCSK9 RNAi agent comprises an antisense strand, wherein position 2 (5'→3') of the antisense strand can base pair with position 20 of a 21mer target sequence disclosed in Table 1. In some embodiments, a PCSK9 RNAi agent comprises an antisense strand, wherein positions 2-18 (5'→3') of the antisense strand can base pair with each respective complementary base located at positions 18-2 of a 21mer target sequence disclosed in Table 1.

[0098] In the case of the RNAi agents disclosed herein, the nucleotide at position 1 of the antisense strand (5'->3') can be perfectly complementary to the PCSK9 gene or can be non-complementary to the PCSK9 gene. In some embodiments, the nucleotide at position 1 of the antisense strand (5'->3') is U, A, or dT. In some embodiments, the nucleotide at position 1 of the antisense strand (5'->3') forms an A:U or U:A base pair with the sense strand.

[0099] In some embodiments, the antisense strand of the PCSK9 RNAi agent comprises the nucleotide sequence (5' end to 3' end) 2-18, 2-19, 2-20, or 2-21 of any of the antisense strand sequences in Table 2, Table 3, Table 5C, Table 7A, or Table 8. In some embodiments, the sense strand of the PCSK9 RNAi comprises the nucleotide sequence (5' end to 3' end) 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 of any of the sense strand sequences in Table 2, Table 4, Table 5C, Table 7B, or Table 8.

[0100] In some embodiments, the antisense strand of the PCSK9 RNAi agent comprises the nucleotide sequence (5' end to 3' end) 2-18, 2-19, 2-20, or 2-21 of any of the antisense strand sequences in Table 2, Table 3, Table 5C, Table 7A, or Table 8. In some embodiments, the sense strand of the PCSK9 RNAi comprises the nucleotide sequence (5' end to 3' end) 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 of any of the sense strand sequences in Table 2, Table 4, Table 5C, Table 7B, or Table 8.

[0101] In some embodiments, the PCSK9 RNAi agent is composed of (i) an antisense strand comprising nucleotide sequence (5' end to 3' end) 2 to 18 or 2 to 19 of any of the antisense strand sequences in Table 2 or Table 3, and (ii) a sense strand comprising nucleotide sequence (5' end to 3' end) 3 to 21, 2 to 21, 1 to 21, 3 to 20, 2 to 20, 1 to 20, 3 to 19, 2 to 19, 1 to 19, 3 to 18, 2 to 18, or 1 to 18 of any of the sense strand sequences in Table 2 or Table 4.

[0102] In some embodiments, the PCSK9 RNAi agent is composed of (i) an antisense strand comprising nucleotide sequence (5' end to 3' end) 2 to 18 or 2 to 19 of any of the antisense strand sequences in Table 2 or Table 3, and (ii) a sense strand comprising nucleotide sequence (5' end to 3' end) 3 to 21, 2 to 21, 1 to 21, 3 to 20, 2 to 20, 1 to 20, 3 to 19, 2 to 19, 1 to 19, 3 to 18, 2 to 18, or 1 to 18 of any of the sense strand sequences in Table 2 or Table 4.

[0103] In some embodiments, the PCSK9 RNAi agent comprises a core 21mer nucleotide sequence shown in Table 2 below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0104] The sense and antisense strands of a PCSK9 RNAi agent that comprises or consists of a sequence in Table 2 can be modified or unmodified nucleotides. In some embodiments, a PCSK9 RNAi agent having sense and antisense strand sequences that comprise or consist of a sequence in Table 2 is all or substantially all modified nucleotides.

[0105] In some embodiments, the antisense strand of a PCSK9 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 2. In some embodiments, the sense strand of a PCSK9 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 2.

[0106] As used herein, each N listed in the sequences disclosed in Table 2 may be independently selected from any and all nucleobases (including those found in both modified and unmodified nucleotides). In some embodiments, the N nucleotide listed in the sequences disclosed in Table 2 has a nucleobase that is complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, the N nucleotide listed in the sequences disclosed in Table 2 has a nucleobase that is not complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, the N nucleotide listed in the sequences disclosed in Table 2 has the same nucleobase as the N nucleotide at the corresponding position on the other strand. In some embodiments, the N nucleotide listed in the sequences disclosed in Table 2 has a different nucleobase than the N nucleotide at the corresponding position on the other strand.

[0107] Particular modified antisense strands of PCSK9 RNAi agents and their underlying unmodified nucleobase sequences are provided in Table 3. Particular modified sense strands of PCSK9 RNAi agents and their underlying unmodified nucleobase sequences are provided in Table 4. When forming a PCSK9 RNAi agent, each nucleotide in the underlying base sequence listed in Tables 3 and 4 and Table 2 above can be a modified nucleotide.

[0108] The PCSK9 RNAi agents described herein are formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 2 or Table 4 can hybridize with any antisense strand containing a sequence listed in Table 2 or Table 3, provided that the two sequences have a region of at least 85% complementarity over a contiguous 15, 16, 17, 18, 19, 20, or 21 nucleotide sequence.

[0109] In some embodiments, the antisense strand of a PCSK9 RNAi agent comprises the nucleotide sequence of any of the sequences in Table 2 or Table 3.

[0110] In some embodiments, a PCSK9 RNAi agent comprises or consists of a duplex having the sense and antisense strand nucleobase sequences of any of the sequences in Table 2, Table 3, or Table 4. In some embodiments, a PCSK9 RNAi agent comprises or consists of a duplex sequence that is prepared or provided as a sodium salt, mixed salt, or free acid.

[0111] Examples of antisense strands containing modified nucleotides are provided in Tables 3 and 5C. Examples of sense strands containing modified nucleotides are provided in Tables 4 and 5C.

[0112] As used in Tables 3, 4, 5C, 7A, 7B, and 8, the following notation is used to indicate modified nucleotides and linking groups: A = adenosine-3'-phosphate; C = cytidine-3′-phosphate; G = guanosine-3′-phosphate; U = uridine-3'-phosphate; I = inosine-3′-phosphate; a = 2'-O-methyladenosine-3'-phosphate; as = 2'-O-methyladenosine-3'-phosphorothioate; c = 2'-O-methylcytidine-3'-phosphate; cs = 2'-O-methylcytidine-3'-phosphorothioate; g = 2'-O-methylguanosine-3'-phosphate; gs = 2'-O-methylguanosine-3'-phosphorothioate; t = 2'-O-methyl-5-methyluridine-3'-phosphate; ts = 2'-O-methyl-5-methyluridine-3'-phosphorothioate; u = 2'-O-methyluridine-3'-phosphate; us = 2'-O-methyluridine-3'-phosphorothioate; i = 2'-O-methylinosine-3'-phosphate; is = 2'-O-methylinosine-3'-phosphorothioate; Af = 2'-fluoroadenosine-3'-phosphate; Afs = 2'-fluoroadenosine-3'-phosphorothioate; Cf = 2'-fluorocytidine-3'-phosphate; Cfs = 2'-fluorocytidine-3'-phosphorothioate; Gf = 2'-fluoroguanosine-3'-phosphate; Gfs = 2'-fluoroguanosine-3'-phosphorothioate; Tf = 2'-fluoro-5'-methyluridine-3'-phosphate; Tfs = 2′-fluoro-5′-methyluridine-3′-phosphorothioate; Uf = 2'-fluorouridine-3'-phosphate; Ufs = 2'-fluorouridine-3'-phosphorothioate; A UNA = 2',3'-seco-adenosine-3'-phosphate (see Table 6); A UNA s = 2',3'-seco-adenosine-3'-phosphorothioate (see Table 6); C UNA = 2',3'-seco-cytidine-3'-phosphate (see Table 6); C UNA s = 2',3'-seco-cytidine-3'-phosphorothioate (see Table 6); G UNA = 2',3'-seco-guanosine-3'-phosphate (see Table 6); G UNA s = 2',3'-seco-guanosine-3'-phosphorothioate (see Table 6); U UNA = 2',3'-seco-uridine-3'-phosphate (see Table 6); U UNA s = 2',3'-seco-uridine-3'-phosphorothioate (see Table 6); a_2N = 2'-O-methyl-2-aminoadenosine-3'-phosphate (see Table 6) a_2Ns = 2'-O-methyl-2-aminoadenosine-3'-phosphorothioate (see Table 6); (invAb) = inverted abasic deoxyribonucleotide (see Table 6); (invAb)s = inverted abasic deoxyribonucleotide-5'-phosphorothioate (see Table 6); cPrpa = 5'-cyclopropylphosphonate-2'-O-methyladenosine-3'-phosphate (see Table 6); cPrpas = 5'-cyclopropylphosphonate-2'-O-methyladenosine-3'-phosphorothioate (see Table 6); cPrpu = 5'-cyclopropylphosphonate-2'-O-methyluridine-3'-phosphate (see Table 6); cPrpus = 5'-cyclopropylphosphonate-2'-O-methyluridine-3'-phosphorothioate (see Table 6);

[0113] As those skilled in the art will readily understand, unless otherwise indicated by the sequence (e.g., by a phosphorothioate linkage "s"), nucleotide monomers, when present in an oligonucleotide, are linked to one another by a 5'-3'-phosphodiester bond. As those skilled in the art will clearly understand, the inclusion of a phosphorothioate linkage shown in the modified nucleotide sequences disclosed herein replaces a phosphodiester linkage typically present in an oligonucleotide. Furthermore, those skilled in the art will readily understand that the terminal nucleotide at the 3' end of a given oligonucleotide sequence typically has a hydroxyl (-OH) group at the 3' position of each given monomer ex vivo in place of a phosphate moiety. Furthermore, in the embodiments disclosed herein, when each strand is viewed from 5' to 3', an inverted abasic residue is inserted such that the 3' position of the deoxyribose is linked to the 3' end of the preceding monomer in each strand (see, e.g., Table 6). Furthermore, as those skilled in the art will readily understand and appreciate, while the chemical structures of phosphorothioates depicted herein typically show an anion on the sulfur atom, the invention disclosed herein encompasses all phosphorothioate tautomers and resonance structures (e.g., when the sulfur atom bears a double bond and the anion is on the oxygen atom). Unless otherwise expressly stated herein, such understanding of those skilled in the art will be used when describing the PCSK9 RNAi agents and compositions of PCSK9 RNAi agents disclosed herein.

[0114] Specific examples of targeting ligands, targeting groups, and linking groups for use in the PCSK9 RNAi agents disclosed herein are provided below in Table 6. More specifically, targeting groups and linking groups (which together can form a targeting ligand) include (NAG37) and (NAG37), the chemical structures of which are provided below in Table 6. Each sense strand and / or antisense strand can have any targeting ligand, targeting group, or linking group listed herein, as well as other groups conjugated to the 5' and / or 3' ends of the sequence. [Table 3-1] [Table 3-2] [Table 3-3] [Table 4-1] [Table 4-2] [Table 4-3]

[0115] The PCSK9 RNAi agent described herein is formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 2, Table 4, Table 5C, Table 7B, or Table 8 can hybridize with any antisense strand containing a sequence listed in Table 2, Table 3, Table 5C, Table 7A, or Table 8, provided that the two sequences have a region of at least 85% complementarity over a continuous 15, 16, 17, 18, 19, 20, or 21 nucleotide sequence.

[0116] In some embodiments, the antisense strand of a PCSK9 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 3 or Table 5C. In some embodiments, the sense strand of a PCSK9 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 4 or Table 5C.

[0117] In some embodiments, the antisense strand of the PCSK9 RNAi agent comprises the nucleotide sequence of any of the sequences in Table 2, Table 3, or Table 5C. In some embodiments, the antisense strand of the PCSK9 RNAi agent comprises the nucleotide sequence (5' end to 3' end) 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21 of any of the sequences in Table 2, Table 3, or Table 5C. In certain embodiments, the antisense strand of the PCSK9 RNAi agent comprises or consists of the modified sequence of any one of the modified sequences in Table 3 or Table 5C.

[0118] In some embodiments, the PCSK9 RNAi agent sense strand comprises the nucleotide sequence of any of the sequences in Table 2, Table 4, or Table 5C. In some embodiments, the sense strand of the PCSK9 RNAi agent comprises the nucleotide sequence (5' end to 3' end) 1-17, 2-17, 3-17, 4-17, 1-18, 2-18, 3-18, 4-18, 1-19, 2-19, 3-19, 4-19, 1-20, 2-20, 3-20, 4-20, 1-21, 2-21, 3-21, or 4-21 of any of the sequences in Table 2, Table 4, or Table 5C. In certain embodiments, the sense strand of the PCSK9 RNAi agent comprises or consists of a modified sequence of any one of the modified sequences in Table 4 or Table 5C.

[0119] In the case of the PCSK9 RNAi agents disclosed herein, the nucleotide at position 1 (5'->3') of the antisense strand can be perfectly complementary to the PCSK9 gene or can be non-complementary to the PCSK9 gene. In some embodiments, the nucleotide at position 1 (5'->3') of the antisense strand is U, A, or dT (or modified forms thereof). In some embodiments, the nucleotide at position 1 (5'->3') of the antisense strand forms an A:U or U:A base pair with the sense strand.

[0120] A sense strand comprising a sequence listed in Table 2, Table 4, Table 5C, Table 7B, or Table 8 can hybridize to any antisense strand comprising a sequence listed in Table 2, Table 3, Table 5C, Table 7A, or Table 8, provided the two sequences have a region of at least 85% complementarity over a contiguous 15, 16, 17, 18, 19, 20, or 21 nucleotide sequence. In some embodiments, a PCSK9 RNAi agent has a sense strand consisting of any of the modified sequences in Table 4 or Table 5C and an antisense strand consisting of any of the modified sequences in Table 3 or Table 5C. Certain representative sequence pairings are exemplified by the duplex ID numbers shown in Tables 5A, 5B, 5C, and 8.

[0121] In some embodiments, the PCSK9 RNAi agent comprises, consists of, or consists essentially of a duplex represented by any one of the duplex ID numbers described herein. In some embodiments, the PCSK9 RNAi agent comprises the sense and antisense strand nucleotide sequences of any of the duplexes represented by any of the duplex ID numbers described herein. In some embodiments, the PCSK9 RNAi agent comprises the sense and antisense strand nucleotide sequences of any of the duplexes represented by any of the duplex ID numbers described herein and a targeting group and / or linking group, wherein the targeting group and / or linking group is covalently attached (i.e., conjugated) to the sense strand or the antisense strand. In some embodiments, the PCSK9 RNAi agent comprises modified nucleotide sequences of the sense and antisense strands of any of the duplex ID numbers described herein. In some embodiments, the PCSK9 RNAi agent comprises modified nucleotide sequences of the sense and antisense strands of any of the duplex ID numbers described herein and a targeting group and / or linking group, wherein the targeting group and / or linking group is covalently attached to the sense strand or the antisense strand.

[0122] In some embodiments, a PCSK9 RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequence of any of the antisense strand / sense strand duplexes in Table 2 or Tables 5A, 5B, and 5C, and further comprises a targeting group or targeting ligand. In some embodiments, a PCSK9 RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequence of any of the antisense strand / sense strand duplexes in Table 2 or Tables 5A, 5B, and 5C, and further comprises an asialoglycoprotein receptor ligand targeting group.

[0123] A targeting group, with or without a linker, can be attached to the 5' or 3' end of the sense strand and / or antisense strand disclosed in Table 2, Table 3, Table 4, or Table 5C. A linker, with or without a targeting group, can be attached to the 5' or 3' end of the sense strand and / or antisense strand disclosed in Table 2, Table 3, Table 4, and Table 5C.

[0124] In some embodiments, the PCSK9 RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequence of any of the antisense strand / sense strand duplexes in Table 2 or Tables 5A, 5B, and 5C, and further comprises a targeting ligand selected from the group consisting of (NAG37) and (NAG37), respectively, as defined in Table 6.

[0125] In some embodiments, a PCSK9 RNAi agent comprises an antisense strand and a sense strand having a modified nucleotide sequence of any of the antisense strand and / or sense strand nucleotide sequences in Table 3 or Table 4.

[0126] In some embodiments, a PCSK9 RNAi agent comprises an antisense strand and a sense strand having a modified nucleotide sequence of any of the antisense strand nucleotide sequences and / or sense strand nucleotide sequences of any of the duplexes in Table 5A, Table 5B, and Table 5C, and further comprises an asialoglycoprotein receptor ligand targeting group.

[0127] In some embodiments, the PCSK9 RNAi agent comprises, consists of, or consists essentially of any of the duplexes of Table 5A, Table 5B, and Table 5C. [Table 5A-1] [Table 5A-2] [Table 5B-1] [Table 5B-2] [Table 5C-1] [Table 5C-2] [Table 5C-3] [Table 5C-4]

[0128] In some embodiments, the PCSK9 RNAi agents are formulated or provided as a salt, mixed salt, or free acid. The RNAi agents described herein inhibit or knock down expression of one or more PCSK9 genes in vivo and / or in vitro when delivered to cells expressing the PCSK9 gene.

[0129] Targeting Ligands or Groups, Linking Groups, and Delivery Vehicles In some embodiments, the PCSK9 RNAi agent is conjugated to one or more non-nucleotide groups, including, but not limited to, a targeting group, a linking group, a targeting ligand, a delivery polymer, or a delivery vehicle. The non-nucleotide group can enhance targeting, delivery, or binding of the RNAi agent. Examples of targeting groups and linking groups are provided in Table 6. The non-nucleotide group can be covalently attached to the 3' and / or 5' end of the sense strand and / or the antisense strand. In some embodiments, the PCSK9 RNAi agent comprises a non-nucleotide group linked to the 3' and / or 5' end of the sense strand. In some embodiments, the non-nucleotide group is linked to the 5' end of the PCSK9 RNAi agent sense strand. The non-nucleotide group may be linked directly or indirectly to the RNAi agent via a linker / linking group. In some embodiments, the non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker.

[0130] In some embodiments, the non-nucleotide group enhances the pharmacokinetic or biodistribution properties of the RNAi agent or conjugate to which it is attached, improving cell- or tissue-specific distribution and cell-specific uptake of the RNAi agent or conjugate, hi some embodiments, the non-nucleotide group enhances endocytosis of the RNAi agent.

[0131] Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of the conjugate or RNAi agent to which they are attached, improving cell-specific (and in some cases organ-specific) distribution and cell-specific (or organ-specific) uptake of the conjugate or RNAi agent. Targeting groups can be monovalent, divalent, trivalent, tetravalent, or higher with respect to the target to which they are directed. Exemplary 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 mimetics with affinity for cell surface molecules.

[0132] In some embodiments, the targeting group is linked to the RNAi agent using a linker, such as a PEG linker, or one, two, or three abasic and / or ribitol (abasic ribose) residues, which in some cases can function as linkers. In some embodiments, the targeting ligand comprises a galactose derivative cluster.

[0133] The PCSK9 RNAi agents described herein can be synthesized with reactive groups, such as amino groups (also referred to herein as amines), at the 5' and / or 3' ends, which can later be used to attach targeting moieties using methods typical in the art.

[0134] In some embodiments, the targeting group comprises an asialoglycoprotein receptor ligand. As used herein, an asialoglycoprotein receptor ligand is a ligand comprising a moiety having affinity for the asialoglycoprotein receptor. As noted herein, the asialoglycoprotein receptor is highly expressed on hepatocytes. In some embodiments, the asialoglycoprotein receptor ligand comprises or consists of one or more galactose derivatives. As used herein, the term galactose derivative includes both galactose and galactose derivatives having affinity for the asialoglycoprotein 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, Nn-butanoylgalactosamine, and N-isobutanoylgalactosamine (see, e.g., S.T. Iobst and K. Drickamer, JBC, 1996, 271, 6686). Galactose derivatives and clusters of galactose derivatives useful for targeting 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).

[0135] Galactose derivatives have been used to target molecules to hepatocytes in vivo by binding to the asialoglycoprotein receptor expressed on the surface of hepatocytes. Binding of an asialoglycoprotein receptor ligand to the asialoglycoprotein receptor promotes cell-specific targeting to hepatocytes and endocytosis of molecules into hepatocytes. The asialoglycoprotein receptor ligand can be a monomer (e.g., having a single galactose derivative, also referred to as monovalent or monodentate) or a polymer (e.g., having multiple galactose derivatives). Galactose derivatives or galactose derivative clusters can be attached to the 3'-end or 5'-end of the sense strand or antisense strand of an RNAi agent using methods known in the art.

[0136] The preparation of targeting ligands such as galactose derivative clusters is described, for example, in Arrowhead Pharmaceuticals, Inc., International Patent Application Publication No. WO2018 / 044350, and Arrowhead Pharmaceuticals, Inc., International Patent Application Publication No. WO2017 / 156012, the contents of both of which are incorporated herein by reference in their entireties.

[0137] As used herein, a galactose derivative cluster includes a molecule having two to four terminal galactose derivatives. The terminal galactose derivative is attached to the molecule through the C-1 carbon. In some embodiments, the galactose derivative cluster is a galactose derivative trimer (also referred to as a triantennary galactose derivative or a trivalent galactose derivative). In some embodiments, the galactose derivative cluster includes an N-acetyl-galactosamine moiety. In some embodiments, the galactose derivative cluster includes three N-acetylgalactosamine moieties. In some embodiments, the galactose derivative cluster is a galactose derivative tetramer (also referred to as a tetraantennary galactose derivative or a tetravalent galactose derivative). In some embodiments, the galactose derivative cluster includes four N-acetylgalactosamine moieties.

[0138] As used herein, a galactose derivative trimer comprises three galactose derivatives, each linked to a central branch point. As used herein, a galactose derivative tetramer comprises four galactose derivatives, each linked 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 linked 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, e.g., U.S. Pat. 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 for the attachment of three galactose derivatives and further allows for the attachment of the branch point to an RNAi agent. Examples of branch point groups are dilysine or diglutamic acid. The branch point can be attached to the RNAi agent via a linker or spacer. In some embodiments, the linker or spacer comprises a flexible hydrophilic spacer, such as, but not limited to, a PEG spacer. In some embodiments, the linker comprises a rigid linker, such as a cyclic group. In some embodiments, the galactose derivative comprises or consists of N-acetyl-galactosamine. In some embodiments, the galactose derivative cluster is composed of a galactose derivative tetramer, which can be, for example, an N-acetyl-galactosamine tetramer.

[0139] The present disclosure also provides a pharmaceutical composition for delivering a PCSK9 RNAi agent to liver cells in vivo.The pharmaceutical composition can include, for example, a PCSK9 RNAi agent conjugated to a galactose derivative cluster.In some embodiments, the galactose derivative cluster is composed of a galactose derivative trimer, which can be, for example, an N-acetyl-galactosamine trimer, or a galactose derivative tetramer, which can be, for example, an N-acetyl-galactosamine tetramer.

[0140] The targeting ligand or targeting group can be linked to the 3' or 5' end of the sense or antisense strand of a PCSK9 RNAi agent disclosed herein.

[0141] Targeting ligands include, but are not limited to, (NAG37) and (NAG37)s as defined in Table 6. Other targeting groups and targeting ligands, including galactose cluster targeting ligands, are known in the art.

[0142] In some embodiments, a linking group is conjugated to the RNAi agent. The linking group facilitates covalent attachment of the RNAi agent to a targeting group, delivery polymer, or delivery vehicle. The linking group can be attached to the 3'-end and / or 5'-end of the sense or antisense strand of the RNAi agent. In some embodiments, the linking group is attached to the sense strand of the RNAi agent. In some embodiments, the linking group is attached to the 5'-end or 3'-end of the RNAi agent sense strand. In some embodiments, the linking group is attached to the 5'-end of the RNAi agent sense strand. Examples of linking groups include, but are not limited to, reactive groups such as primary amines and alkynes, alkyl groups, abasic nucleotides, ribitol (abasic ribose), and / or PEG groups.

[0143] In some embodiments, the targeting group is linked internally to a nucleotide of the sense and / or antisense strand of the RNAi agent. In some embodiments, the targeting group is linked to the RNAi agent via a linker.

[0144] A linker or linking group is a connection between two atoms that connects one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as a targeting group or delivery polymer) or segment of interest through one or more covalent bonds. Labile linkages include labile bonds. A linkage can optionally include a spacer that increases the distance between the two linked atoms. A spacer can further add flexibility and / or length to the linkage. Spacers include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups, each of which can include one or more heteroatoms, heterocycles, amino acids, nucleotides, and sugars. Spacer groups are well known in the art, and the above list is not intended to limit the scope of this description.

[0145] In some embodiments, when two or more RNAi agents are included in a single composition, each RNAi agent may be linked to the same targeting group or to two different targeting groups (i.e., targeting groups with different chemical structures). In some embodiments, the targeting group is linked to a PCSK9 RNAi agent disclosed herein without the use of an additional linker. In some embodiments, the targeting group itself is designed to readily include a linker or other moiety to facilitate conjugation. In some embodiments, when two or more PCSK9 RNAi agents are included in a single molecule, each RNAi agent may utilize the same linker or different linkers (i.e., linkers with different chemical structures).

[0146] Any of the PCSK9 RNAi agent nucleotide sequences listed in Table 2, Table 3, Table 4, Table 5C, Table 7A, Table 7B, or Table 8, whether modified or unmodified, can include a 3' and / or 5' targeting group or linking group. Alternatively, any of the PCSK9 RNAi agent sequences listed in Table 3 or 4 or otherwise described herein that include a 3' or 5' targeting group or linking group can include no 3' or 5' targeting group or linking group, or can include a different 3' or 5' targeting group or linking group, including, but not limited to, those shown in Table 6. Any of the PCSK9 RNAi agent duplexes listed in Table 5A, Table 5B, Table 5C, and Table 8, whether modified or unmodified, can further include a targeting group or linking group, including, but not limited to, a targeting group or linking group listed in Table 6, where the targeting group or linking group can be attached to the 3' or 5' end of either the sense or antisense strand of the PCSK9 RNAi agent duplex.

[0147] Examples of targeting groups and linking groups, which when combined can form targeting ligands, are provided in Table 6. Tables 4, 5C, and 8 provide specific embodiments of PCSK9 RNAi agent sense strands having targeting groups or linking groups attached to the 5' or 3' terminus. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0148] In each of the above structures in Table 6, NAG comprises N-acetylgalactosamine. In some embodiments, the NAG shown in Table 6 above can comprise another galactose derivative that has affinity for the asialoglycoprotein receptor present on hepatocytes, as would be understood by one of skill in the art to bind in light of the above structures and the description provided herein. Other linking groups known in the art may also be used.

[0149] In some embodiments, a delivery vehicle can be used to deliver an RNAi agent to a cell or tissue. A delivery vehicle is a compound that improves delivery of an RNAi agent to a cell or tissue. The delivery vehicle can include, but is not limited to, a polymer, such as an amphiphilic polymer, a membrane-active polymer, a peptide, a melittin peptide, a melittin-like peptide (MLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane-active polyamine. In some embodiments, the RNAi agent can be combined with a lipid, a nanoparticle, a polymer, a liposome, a micelle, a DPC, or other delivery systems available in the art. RNAi agents can also be chemically conjugated to targeting groups, lipids (including, but not limited to, cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, e.g., WO2000 / 053722, WO2008 / 0022309, WO2011 / 104169, and WO2012 / 083185, WO2013 / 032829, WO2013 / 158141, each of which is incorporated herein by reference), hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, proteinaceous vectors, or other delivery systems suitable for nucleic acid or oligonucleotide delivery known and available in the art.

[0150] Pharmaceutical Composition The PCSK9 RNAi agents disclosed herein can be prepared as pharmaceutical compositions or formulations (also referred to herein as "medicines"). In some embodiments, pharmaceutical compositions comprise at least one PCSK9 RNAi agent. These pharmaceutical compositions are particularly useful for inhibiting the expression of target mRNA in a target cell, cell population, tissue, or organism (such as a human).

[0151] The pharmaceutical composition can be used to treat subjects or patients with diseases, disorders, or conditions that would benefit from reducing the level of PCSK9 mRNA and / or the PCSK9 protein translated from the PCSK9 mRNA. The pharmaceutical composition can be used to treat subjects at risk of developing diseases, disorders, symptoms, or conditions that would benefit from reducing the level of target mRNA or inhibiting the expression of a target gene. In one embodiment, the method comprises administering to the subject to be treated a PCSK9 RNAi agent linked to a targeting ligand described herein. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) are added to the pharmaceutical composition comprising the PCSK9 RNAi agent, thereby forming a pharmaceutical formulation or medicament suitable for in vivo delivery to a subject, including a human.

[0152] Pharmaceutical compositions and methods including the PCSK9 RNAi agents disclosed herein reduce the level of target mRNA in a cell, a group of cells, a tissue, an organ, or a subject by administering a therapeutically effective amount of a PCSK9 RNAi agent described herein to a subject, thereby inhibiting PCSK9 mRNA expression or translation in the subject. In some embodiments, the subject has previously been identified as having pathogenic upregulation of the target gene in liver cells. In some embodiments, the subject has previously been identified or diagnosed as having hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD), and / or other PCSK9-related diseases. In some embodiments, the subject suffers from symptoms associated with hypercholesterolemia. In some embodiments, the subject would benefit from reduced PCSK9 gene expression in the subject's liver.

[0153] In some embodiments, the described pharmaceutical compositions comprising a PCSK9 RNAi agent are used to treat or manage clinical conditions related to or including hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD), and / or other PCSK9-related diseases. Other diseases or conditions for which a PCSK9 RNAi agent may be useful include coronary artery disease, polygenic dyslipidemia, or heart disease. In some embodiments, a therapeutically effective amount (including a prophylactically effective amount) of one or more pharmaceutical compositions is administered to a subject in need of such treatment. In some embodiments, administration of any of the disclosed PCSK9 RNAi agents can be used to reduce the number, severity, and / or frequency of disease symptoms in a subject.

[0154] The pharmaceutical compositions described, comprising PCSK9 RNAi agents, can be used to treat at least one symptom in a subject with a disease or disorder that would benefit from a reduction or inhibition of PCSK9 mRNA expression and / or a reduction in PCSK9 protein level. Measurement of PCSK9 levels can be performed according to established methods known in the art. Examples disclosed herein are provided.

[0155] In some embodiments, a subject is administered a therapeutically effective amount of one or more pharmaceutical compositions comprising a PCSK9 RNAi agent to thereby treat a symptom, hi other embodiments, a prophylactically effective amount of one or more PCSK9 RNAi agents is administered to a subject to thereby prevent or inhibit at least one symptom.

[0156] The administration route is the route by which the PCSK9 RNAi agent comes into contact with the body. Generally, the methods for administering drugs and oligonucleotides and nucleic acids to treat mammals are well known in the art and can be applied to the administration of the compositions described herein. The PCSK9 RNAi agent disclosed herein can be administered via any suitable route in a preparation appropriately tailored to a specific route. Thus, the pharmaceutical compositions described herein can be administered, for example, intravenously, intramuscularly, intradermally, subcutaneously, intraarticularly, or intraperitoneally by injection. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection.

[0157] Pharmaceutical compositions comprising the PCSK9 RNAi agents described herein can be delivered to cells, cell groups, tissues, or subjects using oligonucleotide delivery techniques known in the art. Generally, any suitable art-recognized method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the compositions described herein. For example, delivery can be by local administration (e.g., direct injection, implantation, or topical administration), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by subcutaneous infusion or injection or intravenous infusion or injection.

[0158] In some embodiments, the pharmaceutical compositions described herein comprise one or more pharmaceutically acceptable excipients. The pharmaceutical compositions described herein are formulated for administration to a subject.

[0159] As used herein, a pharmaceutical composition or medicament comprises a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than an active pharmaceutical ingredient (API, therapeutic product, e.g., PCSK9 RNAi agent) that is intentionally included in a drug delivery system. The excipient does not exert, or is not intended to exert, a therapeutic effect at the intended dosage. An excipient can function to a) aid in the processing of the drug delivery system during manufacturing, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API, c) assist in product identification, and / or d) enhance any other attribute of the overall safety or efficacy of the API delivery during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0160] Excipients include, but are not limited to, absorption enhancers, anti-adherents, anti-foaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, water repellents, and wetting agents.

[0161] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). Suitable carriers must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be achieved by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0162] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent containing one or a combination of the ingredients listed above as needed, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients listed above.For the preparation of sterile powder for preparing sterile injectable solution, methods for preparation include vacuum drying and freeze-drying, which can obtain the powder of active ingredient and any additional ingredients that are desired from the solution that has been previously sterile-filtered.

[0163] In some embodiments, pharmaceutical formulations comprising the PCSK9 RNAi agent disclosed herein suitable for subcutaneous administration can be prepared in aqueous sodium phosphate buffer (e.g., PCSK9 RNAi agent formulated in 0.5 mM monosodium phosphate, 0.5 mM disodium phosphate in water). In some embodiments, pharmaceutical formulations comprising the PCSK9 RNAi agent disclosed herein suitable for subcutaneous administration can be prepared in water for injection (sterile water). PCSK9 RNAi agent disclosed herein suitable for subcutaneous administration can be prepared in isotonic saline (0.9%).

[0164] Formulations suitable for intra-articular administration may be in the form of a sterile aqueous preparation of the drug, which may be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems may also be used for delivery of the drug for both intra-articular and intraocular administration.

[0165] The formulation suitable for oral administration of the PCSK9 RNAi agent disclosed herein can also be prepared.In some embodiments, the PCSK9 RNAi agent disclosed herein is orally administered.In some embodiments, the PCSK9 RNAi agent disclosed herein is formulated into capsule for oral administration.

[0166] The active compound can be prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers such as ethylene vinylene acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.Methods for preparing such formulations are clear to those skilled in the art.Liposomal suspensions can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0167] PCSK9 RNAi agent can be formulated into dosage unit form compositions for ease of administration and uniformity of dosage.Dosage unit form refers to a physically separate unit that is suitable as a unit dose for the subject to be treated, and each unit contains a predetermined amount of active compound that is calculated to produce desired therapeutic effect in combination with required pharmaceutical carrier.The specification of dosage unit form of the present disclosure is determined and directly depends on the inherent properties of active compound and the therapeutic effect that is achieved, and the inherent limitation of the technology that formulates this active compound for individual treatment.

[0168] The pharmaceutical composition may contain other additional ingredients commonly included in pharmaceutical compositions. Such additional ingredients include, but are not limited to, antipruritics, astringents, local anesthetics, analgesics, antihistamines, or anti-inflammatory agents (e.g., acetaminophen, NSAIDs, diphenhydramine). It is also contemplated that cells, tissues, or isolated organs expressing or containing the RNAi agent defined herein may be used as a "pharmaceutical composition." As used herein, the terms "pharmacologically effective amount," "therapeutically effective amount," or simply "effective amount" refer to the amount of an RNAi agent that produces a pharmacological, therapeutic, or preventative result.

[0169] In some embodiments, the methods disclosed herein further comprise administering a second therapeutic agent or agent in addition to administering an RNAi agent disclosed herein. In some embodiments, the second therapeutic agent is another PCSK9 RNAi agent (e.g., a PCSK9 RNAi agent that targets a different sequence within the PCSK9 target). In other embodiments, the second therapeutic agent can be a small molecule drug, an antibody, an antibody fragment, or an aptamer.

[0170] In some embodiments, the described PCSK9 RNAi agent is optionally combined with one or more additional therapeutic agents. The PCSK9 RNAi agent and the additional therapeutic agents can be administered as a single composition or separately. In some embodiments, the one or more additional therapeutic agents are administered separately in a dosage form separate from the RNAi agent (e.g., the PCSK9 RNAi agent is administered by subcutaneous injection, and the additional therapeutic agents used in the method of therapeutic administration regimen are administered orally). In some embodiments, the described PCSK9 RNAi agent is administered to a subject in need thereof by subcutaneous injection, and one or more optional additional therapeutic agents are administered orally, which together provide a treatment regimen for diseases and conditions related to or including hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD), and / or other PCSK9-related diseases. In some embodiments, the PCSK9 RNAi agent described is administered to a subject in need thereof by subcutaneous injection, and one or more optional additional therapeutic agents are administered by separate subcutaneous injection.In some embodiments, the PCSK9 RNAi agent and one or more additional therapeutic agents are combined in a single dosage form (for example, a "cocktail" formulated into a single composition for subcutaneous injection).The PCSK9 RNAi agent, with or without one or more additional therapeutic agents, can be combined with one or more excipients to form a pharmaceutical composition.

[0171] Generally, an effective amount of a PCSK9 RNAi agent ranges from about 0.1 to about 100 mg / kg body weight / dose, e.g., from about 1.0 to about 50 mg / kg body weight / dose. In some embodiments, an effective amount of active compound ranges from about 0.25 to about 5 mg / kg body weight per dose. In some embodiments, an effective amount of active ingredient ranges from about 0.5 to about 4 mg / kg body weight per dose. In some embodiments, an effective amount of a PCSK9 RNAi agent may be a fixed dose. In some embodiments, the fixed dose ranges from about 5 mg to about 1,000 mg of PCSK9 RNAi agent. In some embodiments, the fixed dose ranges from 50 to 400 mg of PCSK9 RNAi agent. Administration may be weekly, biweekly, monthly, quarterly, or any other interval, depending on the dose of PCSK9 RNAi agent administered, the activity level of the particular PCSK9 RNAi agent, and the desired level of inhibition for a particular subject. The examples herein demonstrate suitable levels of inhibition in particular animal species. The dosage will depend on variables such as the overall health of the patient or subject, the relative biological potency of the compound being delivered, the formulation of the drug, the presence and type of excipients in the formulation, the route of administration, etc. It is also understood that the initial dosage administered may be increased beyond the upper levels set forth above in order to rapidly achieve the desired blood or tissue levels, or the initial dosage may be less than optimal.

[0172] The pharmaceutical compositions described herein comprising a PCSK9 RNAi agent can be combined with an excipient or a second therapeutic agent or agent (including, but not limited to, a second or other RNAi agent, a small molecule agent, an antibody, an antibody fragment, a peptide, and / or an aptamer) to treat a disease or to form a medicament or composition for treating a disease.

[0173] The described PCSK9 RNAi agents, when added to pharmaceutically acceptable excipients or adjuvants, can be packaged in kits, containers, packs, or dispensers. The pharmaceutical compositions described herein can be packaged in pre-filled syringes, pen syringes, auto-injectors, infusion bags / devices, or vials.

[0174] Drug Substances and Formulations of PCSK9 RNAi Agents In some embodiments, a PCSK9 RNAi agent disclosed herein has a PCSK9 RNAi drug substance nucleotide sequence as set forth below in Table 8. The PCSK9 RNAi agent nucleotide sequence found in a PCSK9 RNAi drug substance includes the antisense strand nucleotide sequence as set forth below in Table 7A and the sense strand nucleotide sequence as set forth below in Table 7B.

[0175] [Table 7A]

[0176] [Table 7B]

[0177] As used in Tables 7A, 7B, and 8 herein, the following notation is used to denote modified nucleotides, targeting groups, and linking groups: A, C, G, and U represent adenosine, cytidine, guanosine, and uridine, respectively; a, c, g, and u represent 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoroadenosine, cytidine, guanosine, and uridine, respectively; s represents a phosphorothioate linkage; (invAb) represents an inverted abasic deoxyribose residue (see Table 6); (NAG37)s represents the following structure (shown as both the sodium salt and the free acid): [ka] (shown in the form of the sodium salt), or [ka] (shown in free acid form).

[0178] Each sense and / or antisense strand can have any of the targeting or linking groups listed above and other targeting or linking groups conjugated to the 5' and / or 3' ends of the sequence.

[0179] The antisense strand sequence of the PCSK9 RNAi agent is designed to target mRNA transcripts from the PCSK9 gene in a human subject, thereby silencing translation of the PCSK9 protein using an RNA interference mechanism in a human subject that has PCSK9.

[0180] In some embodiments, the methods disclosed herein use the PCSK9 RNAi drug substances shown in Table 8 below.

[0181] [Table 8]

[0182] In some embodiments, the PCSK9 RNAi drug substance is prepared or provided as a salt, mixed salt, or free acid, hi some embodiments, the form is the sodium salt.

[0183] In some embodiments, the PCSK9 RNAi drug substances provided in Table 8 are formulated with one or more pharmaceutically acceptable excipients to form pharmaceutical compositions suitable for administration to a human subject. In some embodiments, the PCSK9 RNAi drug substances listed in Table 8 are formulated at 200 mg / mL in aqueous sodium phosphate buffer (0.5 mM monosodium phosphate, 0.5 mM disodium phosphate) suitable for subcutaneous administration in humans.

[0184] Methods of Treatment and Inhibition of Expression The PCSK9 RNAi agents disclosed herein can be used to treat subjects (e.g., humans or other mammals) with a disease or disorder that would benefit from administration of the RNAi agent. In some embodiments, the RNAi agents disclosed herein can be used to treat subjects (e.g., humans) that would benefit from a reduction and / or inhibition of PCSK9 mRNA expression and / or PCSK9 protein levels, such as subjects diagnosed with or suffering from a condition related to or including hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD), and / or other PCSK9-related diseases.

[0185] In some embodiments, a therapeutically effective amount of one or more PCSK9 RNAi agents is administered to a subject. Treating a subject includes therapeutic and / or prophylactic treatment. A therapeutically effective amount of one or more PCSK9 RNAi agents described herein is administered to a subject. The subject may be an adult, adolescent, child, or infant. The pharmaceutical compositions described herein may be for humans or animals.

[0186] The PCSK9 RNAi agents described herein can be used to treat at least one symptom in a subject having a PCSK9-related disease or disorder or a disease or disorder mediated at least in part by expression of the PCSK9 gene. In some embodiments, the PCSK9 RNAi agents are used to treat or manage clinical symptoms in a subject who would benefit from a reduction in PCSK9 mRNA or PCSK9 protein levels or who has a disease or disorder mediated at least in part by a reduction in PCSK9 mRNA or PCSK9 protein levels. A therapeutically effective amount of one or more of the PCSK9 RNAi agents or PCSK9 RNAi agent-containing compositions described herein is administered to the subject. In some embodiments, the methods disclosed herein comprise administering a composition comprising a PCSK9 RNAi agent described herein to the subject to be treated. In some embodiments, a prophylactically effective amount of any one or more of the described PCSK9 RNAi agents is administered to the subject, thereby treating the subject by preventing or inhibiting at least one symptom.

[0187] In certain embodiments, the present disclosure provides methods for treating a disease, disorder, symptom, or condition mediated at least in part by PCSK9 gene expression in a patient in need thereof, the method comprising administering to the patient any of the PCSK9 RNAi agents described herein.

[0188] In some embodiments, the gene expression level and / or mRNA level of the PCSK9 gene in a subject administered a described PCSK9 RNAi agent is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or more than 99% compared to the subject before administration of the PCSK9 RNAi agent or a subject not receiving the PCSK9 RNAi agent. The PCSK9 mRNA level in a subject may be reduced in cells, cell groups, and / or tissues of the subject. In some embodiments, PCSK9 gene expression is inhibited by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or more than 65% in liver cells compared to the subject before administration of the PCSK9 RNAi agent or a subject not receiving the PCSK9 RNAi agent.

[0189] In some embodiments, PCSK9 protein levels in a subject administered a described PCSK9 RNAi agent are reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% compared to the subject before administration of the PCSK9 RNAi agent or compared to a subject not receiving the PCSK9 RNAi agent. Protein levels in a subject may be reduced in the subject's cells, cell populations, tissues, blood, and / or other bodily fluids.

[0190] The reduction of PCSK9 mRNA level and PCSK9 protein level can be evaluated by any method known in the art. As used herein, the reduction or decrease of PCSK9 mRNA level and / or protein level is collectively referred to herein as the reduction or decrease of PCSK9 or the inhibition or decrease of PCSK9 gene expression. The examples provided herein illustrate known methods for evaluating the inhibition of PCSK9 gene expression. Those skilled in the art will further know suitable methods for evaluating the inhibition of PCSK9 gene expression in vivo and / or in vitro.

[0191] In some embodiments, disclosed herein are methods for treating (including prophylactic or preventative treatment) a disease, disorder, or condition comprising hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD), and / or other PCSK9-related diseases, the method comprising administering to a subject in need thereof a therapeutically effective amount of a PCSK9 RNAi agent comprising an antisense strand that is at least partially complementary to a portion of PCSK9 mRNA having a sequence in Table 1. In some embodiments, disclosed herein are methods for treating (including prophylactic or preventative treatment) a disease or condition comprising hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD), and / or other PCSK9-related diseases, the method comprising administering to a subject in need thereof a therapeutically effective amount of a PCSK9 RNAi agent comprising an antisense strand comprising any of the sequences in Table 2, Table 3, Table 5C, Table 7A, or Table 8, and a sense strand comprising any of the sequences in Table 2, Table 4, Table 5C, Table 7A, or Table 8 that is at least partially complementary to the antisense strand.In some embodiments, disclosed herein are treatments (including prophylactic or preventative treatments) of diseases or conditions including hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD), and / or other PCSK9-related diseases, the methods comprising administering to a subject in need thereof a therapeutically effective amount of a PCSK9 RNAi agent comprising a sense strand comprising any of the sequences in Table 2, Table 4, Table 5C, Table 7B, or Table 8 and an antisense strand comprising any of the sequences in Table 2, Table 3, Table 5C, Table 7A, or Table 8 that is at least partially complementary to the sense strand.

[0192] In some embodiments, disclosed herein are methods for inhibiting expression of the PCSK9 gene in a cell, the method comprising administering to the cell a PCSK9 RNAi agent comprising an antisense strand that is at least partially complementary to a portion of the PCSK9 mRNA having a sequence in Table 1. In some embodiments, disclosed herein are methods for inhibiting expression of the PCSK9 gene in a cell, the method comprising administering to the cell a PCSK9 RNAi agent comprising an antisense strand that comprises any of the sequences in Table 2, Table 3, Table 5C, Table 7A, or Table 8, and a sense strand that comprises any of the sequences in Table 2, Table 4, Table 5C, Table 7B, or Table 8 that is at least partially complementary to the antisense strand. In some embodiments, disclosed herein are methods for inhibiting expression of the PCSK9 gene in a cell, the method comprising administering to the cell a PCSK9 RNAi agent comprising a sense strand that comprises any of the sequences in Table 2, Table 4, Table 5C, Table 7B, or Table 8, and an antisense strand that comprises any of the sequences in Table 2, Table 3, Table 5C, Table 7A, or Table 8 that is at least partially complementary to the sense strand.

[0193] The use of PCSK9 RNAi agents provides methods for therapeutic treatment (including prophylactic treatment) of diseases / disorders related to or including hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD), and / or other PCSK9-related diseases. The described PCSK9 RNAi agents mediate RNA interference to inhibit the expression of one or more genes required for the production of PCSK9 protein. PCSK9 RNAi agents can also be used to treat or prevent various diseases, disorders, or conditions, including hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD), and / or other PCSK9-related diseases. Additionally, compositions for delivering PCSK9 RNAi agents to liver cells, specifically hepatocytes, in vivo are described.

[0194] Cells, tissues, organs, and non-human organisms Cells, tissues, organs, and non-human organisms are contemplated that comprise at least one of the PCSK9 RNAi agents described herein. The cells, tissues, organs, or non-human organisms are produced by delivering the RNAi agent to the cells, tissues, organs, or non-human organisms.

[0195] Additional Exemplary Embodiments Certain additional exemplary embodiments of the disclosed invention are provided herein, which are merely illustrative and do not limit the scope of the disclosure or the claims appended hereto.

[0196] Embodiment 1. An RNAi agent for inhibiting expression of the PCSK9 gene, comprising: an antisense strand, wherein nucleotides 1-21 of the antisense strand comprise nucleotides 1-21 of the antisense strand sequence of Table 2, Table 3, Table 5C, Table 7A, or Table 8; and a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand wherein all or substantially all of the nucleotides in the antisense strand and / or the sense strand are modified nucleotides, and the RNAi agent is linked to a targeting ligand that comprises an N-acetylgalactosamine.

[0197] Embodiment 2. An RNAi agent for inhibiting expression of a PCSK9 gene, wherein the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides that differs by 0 or 1 nucleotide from 15 contiguous nucleotides of any of the sense strand sequences of Table 2, Table 4, Table 5C, Table 7B, or Table 8, and wherein the sense strand has a region of at least 85% complementarity with the antisense strand over at least 15 contiguous nucleotides.

[0198] Embodiment 3. The RNAi agent of embodiment 1 or 2, wherein at least one nucleotide of the RNAi agent comprises a modified internucleoside linkage.

[0199] Embodiment 4. The RNAi agent of any one of embodiments 1-3, wherein the modified nucleotides are independently selected from the group consisting of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-seconucleotide mimics, locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted 2'-O-methyl nucleotides, inverted 2'-deoxy nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, vinylphosphonate-containing nucleotides, cyclopropylphosphonate-containing nucleotides, and 3'-O-methyl nucleotides;

[0200] Embodiment 5. The RNAi agent of embodiment 4, wherein all or substantially all of the modified nucleotides are 2'-O-methyl nucleotides, 2'-fluoro nucleotides, or a combination thereof.

[0201] Embodiment 6. The RNAi agent of any one of embodiments 1-5, wherein the antisense strand consists of, or consists essentially of, the nucleotide sequence of any one of the modified antisense strand sequences in Table 3, Table 5C, Table 7A, or Table 8.

[0202] Embodiment 7. The RNAi agent of any one of embodiments 1-6, wherein the sense strand consists of, consists essentially of, or comprises a nucleotide sequence of any of the modified sense strand sequences in Table 4, Table 5C, Table 7B, or Table 8.

[0203] Embodiment 8. The RNAi agent of embodiment 1, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences in Table 3, Table 5C, Table 7A, or Table 8, and the sense strand comprises the nucleotide sequence of any one of the modified sequences in Table 4, Table 5C, Table 7B, or Table 8.

[0204] Embodiment 9. The targeting ligand is: [ka] 9. The RNAi agent of any one of embodiments 1 to 8, comprising:

[0205] Embodiment 10. The RNAi agent of any one of embodiments 1 to 8, wherein the targeting ligand is linked to the sense strand.

[0206] Embodiment 11 The RNAi agent of embodiment 10, wherein the targeting ligand is linked to the 5' end of the sense strand.

[0207] Embodiment 12. The RNAi agent of any one of embodiments 1 to 11, wherein the sense strand is 15 to 30 nucleotides in length and the antisense strand is 21 to 30 nucleotides in length.

[0208] Embodiment 13. The RNAi agent of embodiment 12, wherein the sense strand and the antisense strand are each 21 to 27 nucleotides in length.

[0209] Embodiment 14. The RNAi agent of embodiment 13, wherein the sense strand and the antisense strand are each 21 to 24 nucleotides in length.

[0210] Embodiment 15. The RNAi agent of embodiment 14, wherein the sense strand and the antisense strand are each 21 nucleotides in length.

[0211] Embodiment 16. The RNAi agent of any one of embodiments 1 to 15, wherein the RNAi agent has two blunt ends.

[0212] Embodiment 17. The RNAi agent of any one of embodiments 1 to 16, wherein the sense strand comprises one or two terminal caps.

[0213] Embodiment 18. The RNAi agent of any one of embodiments 1 to 17, wherein the sense strand comprises one or two inverted abasic residues.

[0214] Embodiment 19. The RNAi agent of embodiment 1, wherein the RNAi agent is composed of a sense strand and an antisense strand that form a duplex sequence of any of the duplexes listed in Table 5A, Table 5B, Table 5C, or Table 8.

[0215] Embodiment 20. The RNAi agent of any one of embodiments 1 to 19, wherein the RNAi agent is a pharmaceutically acceptable salt.

[0216] Embodiment 21 The RNAi agent of embodiment 20, wherein the RNAi agent is a sodium salt.

[0217] Embodiment 22. A composition comprising the RNAi agent of any one of embodiments 1 to 21, wherein the composition comprises a pharmaceutically acceptable excipient.

[0218] Embodiment 23. The composition of embodiment 22, wherein the pharmaceutically acceptable excipient is sodium phosphate buffer.

[0219] Embodiment 24. The composition of embodiment 22, wherein the pharmaceutically acceptable excipient is isotonic saline or water for injection.

[0220] Embodiment 25. A method for inhibiting expression of the PCSK9 gene in liver cells, comprising introducing into the cells an effective amount of an RNAi agent according to any one of embodiments 1 to 21 or a composition according to any one of embodiments 22 to 24.

[0221] Embodiment 26 The method of embodiment 25, wherein the subject is a human subject.

[0222] Embodiment 27. The method of embodiment 25 or 26, wherein PCSK9 mRNA levels are reduced by at least about 50% in the hepatocytes or in the subject.

[0223] Embodiment 28. The method of any one of embodiments 25-27, wherein PCSK9 protein levels are reduced by at least about 50% in the liver cells or in the subject.

[0224] Embodiment 29. A method for treating a PCSK9-associated disease, disorder, or condition, comprising administering to a human subject in need thereof a therapeutically effective amount of a composition described in any one of embodiments 22-24.

[0225] Embodiment 30. The method of embodiment 29, wherein the disease comprises hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD), and / or other PCSK9-related diseases.

[0226] Embodiment 31. The method of any one of embodiments 25-30, wherein the level of serum PCSK9 protein in the subject is reduced.

[0227] Embodiment 32. The method of any one of embodiments 25-31, wherein the RNAi agent is administered to the human subject at a dose of about 0.05 mg / kg to about 5.0 mg / kg of body weight of the human subject.

[0228] Embodiment 33. Use of an RNAi agent according to any one of embodiments 1 to 21 or a composition according to any one of embodiments 22 to 24 for the treatment of a disease, disorder, or condition mediated at least in part by decreased expression of the PCSK9 gene.

[0229] Embodiment 34. The use of embodiment 33, wherein the disease comprises hypercholesterolemia, familial hypercholesterolemia, including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD), including clinical atherosclerotic cardiovascular disease (ASCVD), and / or other PCSK9-related diseases.

[0230] Embodiment 35. Use of an RNAi agent according to any one of embodiments 1 to 21 or a composition according to any one of embodiments 22 to 24 for the preparation of a pharmaceutical composition for treating a disease, disorder, or condition mediated at least in part by decreased expression of the PCSK9 gene.

[0231] Embodiment 36. The use of any one of embodiments 33 to 35, wherein the RNAi agent is administered to a human subject at a dose of about 0.05 mg to about 5.0 mg per kg of body weight of the human subject.

[0232] The embodiments and clauses provided above are illustrated in the following non-limiting examples. [Example]

[0233] Example 1. Synthesis of PCSK9 RNAi Agents The PCSK9 RNAi agent duplexes shown above in Tables 5A, 5B, 5C, and 8 were synthesized according to the following general procedure.

[0234] A.Synthesis The sense and antisense strands of the RNAi agents were synthesized according to the solid-phase phosphoramidite technique used in oligonucleotide synthesis. Such standard syntheses are generally known in the art. Depending on the scale, either a MerMade96E® (Bioautomation), a MerMade12® (Bioautomation), or an OP Pilot100 (GE Healthcare) was used. Synthesis was performed on a solid support made of controlled pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis (Aston, PA, USA)). The monomer located at the 3' end of each strand was attached to the solid support as the synthesis initiation point. All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA) or Hongene Biotech (Shanghai, China). 2'-O-methyl phosphoramidites included (5'-O-dimethoxytrityl-N 6 -(Benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, 5'-O-dimethoxy-trityl-N 4 -(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphoramidite, (5'-O-dimethoxytrityl-N 2The protecting groups included 5'-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite. The 2'-deoxy-2'-fluorophosphoramidite had the same protecting groups as the 2'-O-methylamidite. 5'-(4,4'-dimethoxytrityl)-2',3'-seco-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite were also purchased from Thermo Fisher Scientific or Hongene Biotech. 5'-Dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from Glen Research (VA) or Hongene Biotech. Cyclopropylphosphonate phosphoramidite was synthesized according to International Patent Application Publication No. WO 2017 / 214112 (see also Altenhofer et al., Chem. Communications (Royal Soc. Chem.), 57(55):6808-6811 (July 2021)). Inverted abasic (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from ChemGenes (Wilmington, MA, USA) or SAFC (St. Louis, MO, USA). 5'-O-dimethoxytrityl-N 2 ,N 6 -(Phenoxyacetic acid)-2'-O-methyl-diaminopurine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was obtained from ChemGenes or Hongene Biotech.

[0235] Phosphoramidites containing targeting ligands were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM). All other amidites were dissolved in anhydrous acetonitrile (50 mM) or in anhydrous dimethylformamide and molecular sieves (3 Å). 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile), 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile), or 4,5-dicyanoimidazole (DCI) were used as activator solutions. Coupling times were 12 min (RNA), 15 min (targeting ligand), 90 s (2'OMe), and 60 s (2'F). To introduce phosphorothioate linkages, a 100 mM solution of 3-phenyl-1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, Massachusetts, USA) in anhydrous acetonitrile was used. Each PCSK9 RNAi agent duplex synthesized and tested in the following examples utilized N-acetylgalactosamine as "NAG" in the targeting ligand chemical structures shown in Table 6. The (NAG37) and (NAG37)s targeting ligand phosphoramidite compounds can be synthesized according to Arrowhead Pharmaceuticals, Inc., International Patent Application Publication No. WO 2018 / 044350.

[0236] B. Cleavage and Deprotection of Support-Bound Oligomers After completion of the solid-phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt% methylamine and 28% ammonium hydroxide solution (Aldrich) in water for 1.5 hours at 30° C. The solution was evaporated and the solid residue was reconstituted in water (see below).

[0237] C. Purification The crude oligomer was purified by anion-exchange HPLC using a TSKgel SuperQ-5PW 13 μm 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 supplemented with 1.5 M sodium chloride. UV traces were recorded at 260 nm. Appropriate fractions were pooled and then run on a size-exclusion HPLC column packed with Sephadex G-25fine using filtered DI water or a running buffer of 100 mM ammonium bicarbonate (pH 6.7) and 20% acetonitrile.

[0238] D. Annealing Complementary strands were mixed to form RNAi agents by combining equimolar RNA solutions (sense and antisense) in 1x phosphate-buffered saline (Corning, Cellgro). A portion of the RNAi agent was lyophilized and stored at -15 to -25°C. The concentration of the duplex was determined by measuring the absorbance of the solution in 1x phosphate-buffered saline with a UV-Vis spectrometer. The absorbance of the solution at 260 nm was then multiplied by a conversion factor and a dilution factor to determine the concentration of the duplex. The conversion factor used was 0.050 mg / (mL cm) or calculated from the experimentally determined extinction coefficient.

[0239] Example 2. hPCSK9 AAV Mouse Model To evaluate specific PCSK9 RNAi agents, we used a human PCSK9 (hPCSK9) AAV (adeno-associated virus) mouse model. Six- to eight-week-old male C57BL / 6 mice were transduced with hPCSK9 AAV serotype 8 (administered at least 14 days before administration of the PCSK9 RNAi agent or control). The hPCSK9 AAV genome contains the 291-3637 region (open reading frame and 3'-UTR region) of the human PCSK9 cDNA sequence (GenBank NM_174936.4 (SEQ ID NO: 1)) driven by a liver-specific promoter. Mice were injected via the tail vein with 4E12-1E13 GC / kg of virus in PBS in a total volume of 10 mL / kg animal body weight to generate the hPCSK9 AAV model mice. Inhibition of hepatic hPCSK9 expression by the PCSK9 RNAi agent resulted in a decrease in measured serum levels of hPCSK9 protein. Before treatment (day −7 to day 1), serum hPCSK9 protein levels were measured using the Human Proprotein Convertase 9 / PCSK9 Quantikine ELISA kit (Biotechne), and mice were grouped according to their mean hPCSK9 protein levels.

[0240] Mice were anesthetized with 2–3% isoflurane, and blood samples were collected from the submandibular region into serum separator tubes (Sarstedt AG & Co., Numbrecht, Germany). Blood was allowed to clot for 20 minutes at ambient temperature. The tubes were centrifuged at 8,000 × g for 3 minutes to separate serum and stored at 4°C. Serum was collected and measured using a Human Proprotein Convertase 9 / PCSK9 Quantikine ELISA kit according to the manufacturer's instructions. To account for changes in hPCSK9 expression not related to treatment, each animal's serum hPCSK9 level can be normalized to a control group of mice injected with vehicle control. To do so, each animal's hPCSK9 level at a given time point was first divided by that animal's pretreatment expression level (day 1) to determine the "normalized to pretreatment" expression ratio. Expression at a given time point was then normalized to the control group by dividing the "normalized to pretreatment" ratio for each individual animal by the average "normalized to pretreatment" ratio for all mice in the normal vehicle control group. Alternatively, serum hPCSK9 levels for each animal were assessed simply by normalizing to pre-treatment levels.

[0241] Example 3. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, each mouse received a single subcutaneous dose of 250 μl / 25 g animal body weight containing 1.0 mg / kg (mpk) of a PCSK9 RNAi agent formulated in isotonic saline or a vehicle control (isotonic saline without an RNAi agent) according to Table 9 below. [Table 9]

[0242] Each PCSK9 RNAi agent comprised a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising three N-acetylgalactosamine groups (tridentate ligand) having the modified sequence shown in the duplex structure herein (see Table 3, Table 4, Table 5A, Table 5B, Table 5C, and Table 6 for specific modification and structural information related to PCSK9 RNAi agents comprising the (NAG37)s ligand).PCSK9 RNAi agent AD07404 (Group 2) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 614 of the gene, PCSK9 RNAi agent AD07690 (Group 3) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 604 of the gene, PCSK9 RNAi agent AD07691 (Group 4) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 605 of the gene, PCSK9 RNAi agent AD07692 (Group 5) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 612 of the gene, PCSK9 RNAi agent AD07418 (Group 6) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3468 of the gene, and PCSK9 RNAi agent AD07419 (Group 7) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3501 of the gene. RNAi agent AD07420 (Group 8) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3502 of the gene, PCSK9 RNAi agent AD07421 (Group 9) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3531 of the gene, PCSK9 RNAi agent AD07422 (Group 10) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3532 of the gene, PCSK9 RNAi agent AD07423 (Group 11) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene, and PCSK9 RNAi agent AD07710 (Group 12) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3301 of the gene (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 genes).

[0243] Injections were administered into the loose skin of the neck and shoulder area, between the skin and the muscle (i.e., subcutaneous injection). Four mice (n=4) were tested in each group. Serum was collected on days 1 (before treatment), 8, 15, 22, and 29, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. The experimental data are shown in Table 10 below. [Table 10]

[0244] As shown in Table 10, although some of the PCSK9 RNAi agents tested showed little or no inhibitory activity compared to saline controls, certain PCSK9 RNAi agents potently silenced PCSK9 gene expression. In particular, a single dose of 1.0 mg / kg AD07423 (targeting position 3536 of the PCSK9 gene) showed a reduction in PCSK9 protein of approximately 59% (0.411) at 15 days.

[0245] Example 4. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, each mouse received a single subcutaneous dose of 250 μl / 25 g animal body weight containing 1.5 mg / kg (mpk) of a PCSK9 RNAi agent formulated in isotonic saline or a vehicle control (isotonic saline without an RNAi agent) according to Table 11 below. [Table 11]

[0246] Each PCSK9 RNAi agent comprised a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising three N-acetylgalactosamine groups (tridentate ligand) having the modified sequence shown in the duplex structure herein (see Table 3, Table 4, Table 5A, Table 5B, Table 5C, and Table 6 for specific modification and structural information related to PCSK9 RNAi agents comprising the (NAG37)s ligand). PCSK9 RNAi agent AD07423 (Group 2) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene, PCSK9 RNAi agent AD07711 (Group 3) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3466 of the gene, PCSK9 RNAi agent AD07712 (Group 4) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3466 of the gene, PCSK9 RNAi agent AD07713 (Group 5) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3500 of the gene, and PCSK9 RNAi agent AD07714 (Group 6) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3534 of the gene (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 genes).

[0247] Injections were administered into the loose skin of the neck and shoulder area, between the skin and the muscle (i.e., subcutaneous injection). Four mice (n=4) were tested in each group. Serum was collected on days 1 (before treatment), 8, and 15, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. The experimental data are shown in Table 12 below. [Table 12]

[0248] As shown in Table 12, each of the PCSK9 RNAi agents tested demonstrated silencing of PCSK9 protein expression in the hPCSK9 AAV mouse model. In particular, a single dose of 1.5 mg / kg of the PCSK9 RNAi agent AD07423 demonstrated approximately 72% (0.281) inhibition at day 15.

[0249] Example 5. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, each mouse received a single subcutaneous dose of 250 μl / 25 g animal body weight containing 1.5 mg / kg (mpk) of a PCSK9 RNAi agent formulated in isotonic saline or a vehicle control (isotonic saline without an RNAi agent) according to Table 13 below. [Table 13]

[0250] Each PCSK9 RNAi agent comprised a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising three N-acetylgalactosamine groups (tridentate ligand) having the modified sequence shown in the duplex structure herein (see Tables 3, 4, 5A, 5B, 5C, and 6 for specific modification and structural information related to PCSK9 RNAi agents comprising the (NAG37)s ligand). The PCSK9 RNAi agents in each of Groups 2-11 comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene, and PCSK9 RNAi agent AD07802 (Group 12) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3540 of the gene (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 genes).

[0251] Injections were administered into the loose skin of the neck and shoulder area, between the skin and the muscle (i.e., subcutaneous injection). Four mice (n=4) were tested in each group. Serum was collected on days 1 (before treatment), 8, and 15, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. Data from the experiment are shown in Table 14 below. [Table 14]

[0252] As shown in Table 14, each PCSK9 RNAi agent potently silenced PCSK9 gene expression. All PCSK9 RNAi agents targeting position 3536 of the PCSK9 gene (i.e., Groups 2-11) performed better than the PCSK9 RNAi agent in Group 12 (AD07802) targeting position 3540. Groups 2-11 each showed a 70%-80% reduction in PCSK9 protein normalized to pretreatment and control at day 15.

[0253] Example 6. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, each mouse received a single subcutaneous dose of 250 μl / 25 g animal body weight containing 1.0 mg / kg (mpk) of a PCSK9 RNAi agent formulated in isotonic saline or a vehicle control (isotonic saline without an RNAi agent) according to Table 15 below. [Table 15]

[0254] Each PCSK9 RNAi agent contained a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand containing three N-acetylgalactosamine groups (tridentate ligand) with the modified sequence shown in the duplex structure herein (see Tables 3, 4, 5A, 5B, 5C, and 6 for specific modification and structural information related to PCSK9 RNAi agents containing the (NAG37)s ligand). All PCSK9 RNAi agents tested contained a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 gene).

[0255] Injections were administered into loose skin in the neck and shoulder area, between the skin and muscle (i.e., subcutaneous injection). Four mice (n=4) were studied in each group for groups 1-8, and three mice (n=3) were studied for group 9. Serum was collected on days 1 (pre-treatment), 8, 15, and 22, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. The experimental data are shown in Table 16 below. [Table 16]

[0256] As shown in Table 16, each of the PCSK9 RNAi agents silenced expression of the PCSK9 gene.

[0257] Example 7. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, each mouse received a single subcutaneous dose of 250 μl / 25 g animal body weight containing 0.3 mg / kg (mpk) of a PCSK9 RNAi agent formulated in isotonic saline or a vehicle control (isotonic saline without an RNAi agent) according to Table 17 below. [Table 17]

[0258] Each PCSK9 RNAi agent contained a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand containing three N-acetylgalactosamine groups (tridentate ligand) having the modified sequence shown in the duplex structure herein (see Tables 3, 4, 5A, 5B, 5C, and 6 for specific modification and structural information related to PCSK9 RNAi agents containing the (NAG37)s ligand). Each PCSK9 RNAi agent tested contained a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 gene).

[0259] Injections were administered into loose skin in the neck and shoulder area, between the skin and muscle (i.e., subcutaneous injection). Four mice (n=4) were studied in each group for groups 1-10, and three mice (n=3) were studied for group 11. Serum was collected on days 1 (pre-treatment), 8, 15, 22, 29, and 36, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. The experimental data are shown in Table 18 below. [Table 18]

[0260] As shown in Table 18, each PCSK9 RNAi agent silenced expression of the PCSK9 gene and reduced PCSK9 protein over 36 days compared to controls.

[0261] Example 8. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, each mouse received a single subcutaneous dose of 250 μl / 25 g animal body weight containing 0.3 mg / kg, 1.0 mg / kg (mpk) of a PCSK9 RNAi agent formulated in isotonic saline or a vehicle control (isotonic saline without an RNAi agent) according to Table 19 below. [Table 19]

[0262] Each PCSK9 RNAi agent comprised a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising three N-acetylgalactosamine groups (tridentate ligand) having the modified sequence shown in the duplex structure herein (see Table 3, Table 4, Table 5A, Table 5B, Table 5C, and Table 6 for specific modification and structural information related to PCSK9 RNAi agents comprising the (NAG37)s ligand).PCSK9 RNAi agent AD08110 (Group 2) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene, PCSK9 RNAi agent AD08575 (Group 3) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene, PCSK9 RNAi agent AD08581 (Group 4) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene, PCSK9 RNAi agent AD08878 (Group 5) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene, PCSK9 RNAi agent AD08879 (Group 6) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene, and PCSK9 RNAi agent AD08880 (Group 7) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene. RNAi agent AD07421 (Group 8) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3531 of the gene, PCSK9 RNAi agent AD08834 (Group 9) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3474 of the gene, PCSK9 RNAi agent AD08835 (Group 10) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3486 of the gene, PCSK9 RNAi agent AD08836 (Group 11) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3546 of the gene, and PCSK9 RNAi agent AD08837 (Group 12) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3547 of the gene (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 genes).

[0263] Injections were administered into the loose skin of the neck and shoulder area, between the skin and the muscle (i.e., subcutaneous injection). Four mice (n=4) were tested in each group. Serum was collected on days 1 (before treatment), 8, 15, and 22, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. The experimental data are shown in Table 20 below. [Table 20]

[0264] As shown in Table 20, at day 22, Groups 2-7, each containing a nucleotide sequence designed to inhibit expression at position 3536 of the PCSK9 gene, performed best. As noted above, Groups 2-7 were dosed at only 0.3 mg / kg, while Groups 8-12 were dosed at 1.0 mg / kg, indicating that the PCSK9 RNAi agents in Groups 2-7 inhibited PCSK9 gene expression substantially more potently than the RNAi agents in Groups 8-12.

[0265] Example 9. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, each mouse received a single subcutaneous dose of 250 μl / 25 g animal body weight containing 1.0 mg / kg (mpk) of a PCSK9 RNAi agent formulated in isotonic saline or a vehicle control (isotonic saline without an RNAi agent) according to Table 21 below. [Table 21]

[0266] Each PCSK9 RNAi agent comprised a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising three N-acetylgalactosamine groups (tridentate ligand) having the modified sequence shown in the duplex structure herein (see Table 3, Table 4, Table 5A, Table 5B, Table 5C, and Table 6 for specific modification and structural information related to PCSK9 RNAi agents comprising the (NAG37)s ligand). PCSK9 RNAi agent AD07421 (Group 2) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3531 of the gene, PCSK9 RNAi agent AD08796 (Group 3) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 1393 of the gene, PCSK9 RNAi agent AD08838 (Group 4) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 1859 of the gene, PCSK9 RNAi agent AD08839 (Group 5) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3482 of the gene, PCSK9 RNAi agent AD08840 (Group 6) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3465 of the gene, and PCSK9 RNAi agent AD08841 (Group 7) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 887 of the gene. RNAi agent AD08842 (Group 8) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3523 of the gene, PCSK9 RNAi agent AD08843 (Group 9) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 1543 of the gene, PCSK9 RNAi agent AD08844 (Group 10) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 1090 of the gene, and PCSK9 RNAi agent AD08845 (Group 11) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3548 of the gene (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 genes).

[0267] Injections were administered into the loose skin of the neck and shoulder area, between the skin and the muscle (i.e., subcutaneous injection). Four mice (n=4) were tested in each group. Serum was collected on days 1 (before treatment), 8, 15, and 22, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. The experimental data are shown in Table 22 below. [Table 22]

[0268] Example 10. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, each mouse received a single subcutaneous dose of 250 μl / 25 g animal body weight containing 1.0 mg / kg (mpk) of a PCSK9 RNAi agent formulated in isotonic saline or control vehicle (isotonic saline without the RNAi agent) according to Table 23 below. [Table 23]

[0269] Each PCSK9 RNAi agent comprised a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising three N-acetylgalactosamine groups (tridentate ligand) having the modified sequence shown in the duplex structure herein (see Table 3, Table 4, Table 5A, Table 5B, Table 5C, and Table 6 for specific modification and structural information related to PCSK9 RNAi agents comprising the (NAG37)s ligand). PCSK9 RNAi agent AD07421 (Group 2) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3531 of the gene, PCSK9 RNAi agent AD08631 (Group 3) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3501 of the gene, PCSK9 RNAi agent AD09027 (Group 4) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3501 of the gene, PCSK9 RNAi agent AD09028 (Group 5) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3501 of the gene, PCSK9 RNAi agent AD09029 (Group 6) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3501 of the gene, and PCSK9 RNAi agent AD09030 (Group 7) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3501 of the gene. RNAi agent AD09031 (Group 8) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3501 of the gene, PCSK9 RNAi agent AD09032 (Group 9) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3501 of the gene, PCSK9 RNAi agent AD09033 (Group 10) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3501 of the gene, and PCSK9 RNAi agent AD09034 (Group 11) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3501 of the gene (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 genes).

[0270] Injections were administered into the loose skin of the neck and shoulder area, between the skin and the muscle (i.e., subcutaneous injection). Four mice (n=4) were tested in each group. Serum was collected on days 1 (before treatment), 8, 15, and 22, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. The experimental data are shown in Table 24 below. [Table 24]

[0271] Example 11. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, each mouse received a single subcutaneous dose of 250 μl / 25 g animal body weight containing 1.0 mg / kg (mpk) of a PCSK9 RNAi agent formulated in isotonic saline or control vehicle (isotonic saline without the RNAi agent) according to Table 25 below. [Table 25]

[0272] Each PCSK9 RNAi agent comprised a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising three N-acetylgalactosamine groups (tridentate ligand) having the modified sequence shown in the duplex structure herein (see Table 3, Table 4, Table 5A, Table 5B, Table 5C, and Table 6 for specific modification and structural information related to PCSK9 RNAi agents comprising the (NAG37)s ligand). PCSK9 RNAi agent AD07421 (Group 2) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3531 of the gene, PCSK9 RNAi agent AD08837 (Group 3) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3547 of the gene, PCSK9 RNAi agent AD09038 (Group 4) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3547 of the gene, PCSK9 RNAi agent AD09039 (Group 5) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3547 of the gene, and PCSK9 RNAi agent AD09040 (Group 6) comprises a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3547 of the gene. RNAi agent AD09041 (Group 7) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3547 of the gene, PCSK9 RNAi agent AD08835 (Group 8) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3486 of the gene, PCSK9 RNAi agent AD09042 (Group 9) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3486 of the gene, and PCSK9 RNAi agent AD09043 (Group 10) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3486 of the gene (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 genes).

[0273] Injections were administered into the loose skin of the neck and shoulder area, between the skin and the muscle (i.e., subcutaneous injection). Four mice (n=4) were tested in each group. Serum was collected on days 1 (before treatment), 8, 15, and 22, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. The experimental data are shown in Table 26 below. [Table 26]

[0274] Example 12. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, each mouse received a single subcutaneous dose of 250 μl / 25 g animal body weight containing 0.3 mg / kg, 1.0 mg / kg (mpk) PCSK9 RNAi agent formulated in isotonic saline or vehicle control (isotonic saline without RNAi agent) according to Table 27 below. [Table 27]

[0275] Each PCSK9 RNAi agent comprised a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising three N-acetylgalactosamine groups (tridentate ligand) having the modified sequence shown in the duplex structure herein (see Table 3, Table 4, Table 5A, Table 5B, Table 5C, and Table 6 for specific modification and structural information related to PCSK9 RNAi agents comprising the (NAG37)s ligand). PCSK9 RNAi agent AD08106 (groups 2 and 7) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene, PCSK9 RNAi agent AD07918 (groups 3 and 8) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene, PCSK9 RNAi agent AD09131 (groups 4 and 9) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene, PCSK9 RNAi agent AD09132 (groups 5 and 10) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene, and PCSK9 RNAi agent AD08879 (groups 6 and 11) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 genes).

[0276] Injections were administered into the loose skin of the neck and shoulder area, between the skin and the muscle (i.e., subcutaneous injection). Four mice (n=4) were tested in each group. Serum was collected on days 1 (before treatment), 8, 15, and 22, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. The experimental data are shown in Table 28 below. [Table 28]

[0277] As shown in Table 28, each PCSK9 RNAi agent silenced expression of the PCSK9 gene. Notably, at day 22, PCSK9 RNAi agent AD08870 was the most potent of the PCSK9 RNAi agents tested, demonstrating a reduction in PCSK9 protein of approximately 63% (0.369) when administered as a single dose at 0.3 mg / kg and an approximately 80% (0.199) reduction when administered as a single dose at 1.0 mg / kg, thus demonstrating a dose-response.

[0278] Example 13. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, each mouse received a single subcutaneous dose of 250 μl / 25 g animal body weight containing 0.3 mg / kg, 1.0 mg / kg, or 3.0 mg / kg (mpk) of a PCSK9 RNAi agent formulated in isotonic saline or a vehicle control (isotonic saline without an RNAi agent) according to Table 29 below. [Table 29]

[0279] Each PCSK9 RNAi agent comprised a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising three N-acetylgalactosamine groups (tridentate ligand) having the modified sequence shown in the duplex structure herein (see Table 3, Table 4, Table 5A, Table 5B, Table 5C, and Table 6 for specific modification and structural information related to PCSK9 RNAi agents comprising the (NAG37)s ligand). PCSK9 RNAi agent AD09059 (groups 2, 3, and 4) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3523 of the gene, PCSK9 RNAi agent AD09042 (groups 5, 6, and 7) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3486 of the gene, PCSK9 RNAi agent AD09131 (groups 8, 9, and 10) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3531 of the gene, and PCSK9 RNAi agent AD08842 (groups 11 and 12) comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3523 of the gene (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 genes).

[0280] Injections were administered into loose skin in the neck and shoulder area, between the skin and muscle (i.e., subcutaneous injection). Four mice (n=4) were studied in each group for groups 1-11, and three mice (n=3) were studied for group 12. Serum was collected on days 1 (pre-treatment), 9, 15, and 22, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. The experimental data are shown in Table 30 below. [Table 30]

[0281] Example 14. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, each mouse received a single subcutaneous dose of 250 μl / 25 g animal body weight containing 0.3 mg / kg, 1.0 mg / kg, or 3.0 mg / kg (mpk) of a PCSK9 RNAi agent formulated in isotonic saline or a vehicle control (isotonic saline without the RNAi agent) according to Table 31 below. [Table 31]

[0282] The PCSK9 RNAi agent AD08110 comprised a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand containing three N-acetylgalactosamine groups (tridentate ligand) with the modified sequence shown in the duplex structure herein (see Table 3, Table 4, Table 5A, Table 5B, Table 5C, and Table 6 for specific modification and structural information, including the (NAG37)s ligand). PCSK9 RNAi agent AD08110 (groups 2, 3, and 4) contained a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene, duplex AD09549 (groups 5, 6, and 7) contained a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3535 of the gene, and duplex AC001566 (groups 8, 9, and 10) contained a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3535 of the gene (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 genes).

[0283] Duplex AD09549 contained the following modified nucleotide sequence: Antisense strand (5'→3'): asGfsuuaCfaaaagcaAfaAfcagsgsu (SEQ ID NO: 466) Sense strand (5'→3'): (NAG37)csusguuuUfGfCfuuuuguaacu (SEQ ID NO: 467) (see Table 6 for (NAG37) targeting ligand structure).

[0284] Duplex AC001566 contained the following modified nucleotide sequence: Antisense strand (5'→3'): asGfsuuaCfaaaagcaAfaAfcagsgsu (SEQ ID NO: 468) Sense strand (5'→3'): csusguuuUfGfCfuuuuguaacu(C6-NH)-Tri-NAG-PEG4 (SEQ ID NO: 469). The sense strand was synthesized using a commercially available C6-amino linker at the 3' end to facilitate linkage to a trivalent N-acetylgalactosamine targeting ligand constructed with a terminal azide, resulting in a molecule with the following structure: [ka] (tri-NAG-PEG4)

[0285] Injections were administered into the loose skin of the neck and shoulder area, between the skin and the muscle (i.e., subcutaneous injection). Four mice (n=4) were tested in each group. Serum was collected on days 1 (before treatment), 8, 15, and 22, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. The experimental data are shown in Table 32 below. [Table 32]

[0286] As shown in Table 32 above, the PCSK9 RNAi agent AD8110, which targets position 3536 of the PCSK9 gene, was shown to be superior to AD09549 and AC001566 in inhibiting PCSK9 gene expression at all dosage levels. For example, a 3.0 mg / kg dose of AD08110 reduced PCSK9 protein expression by more than 90% on days 8 (approximately 91% reduction), 15 (approximately 93% reduction), and 22 (approximately 93% reduction).

[0287] Example 15. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, each mouse received a single subcutaneous dose of 250 μl / 25 g animal body weight containing 0.3 mg / kg, 1.0 mg / kg (mpk) of a PCSK9 RNAi agent formulated in isotonic saline or a vehicle control (isotonic saline without an RNAi agent) according to Table 33 below. [Table 33]

[0288] Each PCSK9 RNAi agent comprised a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising three N-acetylgalactosamine groups (tridentate ligand) having the modified sequence shown in the duplex structure herein (see Table 3, Table 4, Table 5A, Table 5B, Table 5C, and Table 6 for specific modification and structural information, including the (NAG37)s ligand). Each PCSK9 RNAi agent tested in this example comprised a nucleotide sequence designed to inhibit expression of the PCSK9 gene at position 3536 of the gene and was tested as a single injection at either 0.3 mg / kg or 1.0 mg / kg (AD09327 (Groups 2 and 8), AD09851 (Groups 3 and 9), AD09852 (Groups 4 and 10), AD09853 (Groups 5 and 11), AD09854 (Groups 6 and 12), except for AD09855 in Group 7, which was administered at 0.3 mg / kg only) (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 genes).

[0289] Injections were administered into the loose skin of the neck and shoulder area, between the skin and the muscle (i.e., subcutaneous injection). Four mice (n=4) were tested in each group. Serum was collected on days 1 (before treatment), 8, 15, and 22, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. The experimental data are shown in Table 34 below. [Table 34]

[0290] As shown in Table 34, each PCSK9 RNAi agent silenced expression of the PCSK9 gene, with AD09327 showing the greatest reduction in PCSK9 protein at the lowest dose level (0.3 mg / kg).

[0291] Example 16. In vivo testing of PCSK9 RNAi agents in hPCSK9 AAV mice The hPCSK9 AAV mouse model described in Example 2 above, harboring an hPCSK9 AAV containing the 291-3637 region of the human PCSK9 cDNA sequence, was used. On day 1, animals were given a single subcutaneous dose of 250 μl / 25 g of body weight containing 0.3 mg / kg, 1.0 mg / kg, or 3.0 mg / kg (mpk) of a PCSK9 RNAi agent formulated in isotonic saline, Leqvio® (Novartis Pharmaceuticals Corporation), or a vehicle control (isotonic saline without an RNAi agent) according to Table 35 below. [Table 35]

[0292] Leqvio® was obtained commercially. Leqvio®, or inclisiran, is a long-acting RNA interference (RNAi) therapeutic that inhibits the synthesis of PCSK9, a target that lowers low-density lipoprotein (LDL) cholesterol (Fitzgerald et al, A Highly Durable RNAi Therapeutic Inhibitor of PCSK9, N Engl J Med. 2017 Jan 5;376(1):41-51).

[0293] Each PCSK9 RNAi agent tested contained a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand containing three N-acetylgalactosamine groups (tridentate ligand) with the modified sequence shown in the duplex structure herein (see Tables 3, 4, 5A, 5B, 5C, and 6 for specific modification and structural information, including the (NAG37)s ligand). PCSK9 RNAi agent AD09327 (Groups 2-7) and PCSK9 RNAi agent AD08879 (Groups 11 and 12) both contained nucleotide sequences designed to inhibit expression of the PCSK9 gene at position 3536 of the gene (see, e.g., SEQ ID NO: 1 and Table 2 for the referenced PCSK9 gene). Groups designated "ETT" and "DCI" indicate PCSK9 RNAi agents synthesized using ETT and DCI activator reagents, respectively.

[0294] Injections were administered into the loose skin of the neck and shoulder area, between the skin and the muscle (i.e., subcutaneous injection). Four mice (n=4) were tested in each group. Serum was collected on days 1 (before treatment), 8, 15, and 22, and PCSK9 expression levels were measured according to the procedure set forth in Example 2 above. The experimental data are shown in Table 36 below. [Table 36]

[0295] As shown in Table 36, in the hPCSK9 AAV mouse model used in this particular study, the PCSK9 RNAi agent AD09327 (regardless of ETT or DCI) demonstrated improved PCSK9 inhibition at all dose levels compared to the commercially available Leqvio® product.

[0296] Example 17. In vivo testing of PCSK9 RNAi agents in cynomolgus monkeys The PCSK9 RNAi agents AD07918 and AD09327 were evaluated in cynomolgus monkeys (cynos). On day 1, three female cynomolgus monkeys (n=3) in each group were injected subcutaneously with 0.3 mL / kg of 3.0 mg / kg of PCSK9 RNAi agent formulated in isotonic saline. The dose was administered subcutaneously at the mid-scapular region via syringe and needle. [Table 37]

[0297] The RNAi agents comprised modified nucleotides conjugated at the 5' end of the sense strand to a targeting ligand comprising three N-acetylgalactosamine groups (tridentate ligand) with the modified sequence shown in the duplex structure herein (see Tables 3, 4, 5A, 5B, 5C, 6, 7A, 7B, and 8 for specific modification and structural information, including the (NAG37)s ligand). The PCSK9 RNAi agent comprised a nucleotide sequence designed to inhibit expression of the human PCSK9 gene at position 3536 (see, e.g., SEQ ID NO: 1).

[0298] Cynomolgus monkeys were fasted overnight before each blood draw. Blood (approximately 5 mL) from each animal was collected into anticoagulant-free tubes (serum separator tubes) from the femoral vein (or alternate veins) on days -14 (pre-dose), -7 (pre-dose), 1 (pre-dose), 8, 15, 22, 29, 43, 57, 71, and 85. A separate vein not used for dosing may be used as an alternative blood collection site. Pre-dose blood samples were collected from any available surplus animals.

[0299] Serum collected from cynomolgus monkeys was quantified for human PCSK9 protein levels by ELISA using a Human Proprotein Convertase 9 / PCSK9 Quantikine Elisa kit (Catalog No. DPC900, R&D Systems, Inc.). PCSK9 expression levels were normalized to the mean pre-dose PCSK9 levels and are shown in Table 38 below. [Table 38]

[0300] Low density lipoprotein (LDL) and non-high density lipoprotein (HDL) cholesterol levels were quantified and normalized to mean pre-dose levels, and the data are presented in Table 39 below. [Table 39]

[0301] As shown in Table 38, both PCSK9 RNAi agents potently silenced PCSK9 protein expression, but AD09327 was more potent, showing about 82% inhibition at day 22 with a single dose of 3.0 mg / kg, and further showing a significant reduction of about 50% at day 85 after dosing. Additionally, as shown in Table 39, both PCSK9 RNAi agents potently reduced LDL cholesterol and non-HDL cholesterol levels, but AD09327 was more potent, showing a reduction of LDL by about 61% (0.387) at day 29 and a reduction of non-HDL cholesterol by about 60% (0.398) at day 22.

[0302] Example 18. In vivo testing of PCSK9 RNAi agents in cynomolgus monkeys The PCSK9 RNAi agents AD08879 and AD09327, Leqvio®, and AC001566 were evaluated in cynomolgus monkeys. On days 1 and 29, three male cynomolgus monkeys (n=3) in each group were injected subcutaneously with 0.2 mL / kg of 2.0 mg / kg of PCSK9 RNAi agent formulated in isotonic saline. Administration was subcutaneously administered via syringe and needle at the mid-scapular region. [Table 40]

[0303] The PCSK9 RNAi agents comprised modified nucleotides conjugated at the 5' end of the sense strand to a targeting ligand comprising three N-acetylgalactosamine groups (tridentate ligand) having the modified sequence shown in the duplex structure herein (see Tables 3, 4, 5A, 5B, 5C, 6, 7A, 7B, and 8 for specific modification and structural information related to PCSK9 RNAi agents comprising the (NAG37)s ligand). The PCSK9 RNAi agents AD08879 and AD09327 comprised nucleotide sequences designed to inhibit expression of the human PCSK9 gene at position 3536 (see, e.g., SEQ ID NO: 1).

[0304] AC001566 contained the structure described in Example 14 above.

[0305] Cynomolgus monkeys were fasted overnight before each blood collection. Blood (approximately 5 mL) from each animal was collected into anticoagulant-free tubes (serum separator tubes) from the femoral vein (or alternate veins) on days -8 (pre-dose), 1 (pre-dose), 8, 15, 22, 29, 36, 43, 50, 57, 71, 85, 99, and 113. A separate vein not used for dosing may be used as an alternative blood collection site. Pre-dose blood samples were collected from any available surplus animals.

[0306] Serum collected from cynomolgus monkeys was quantified for human PCSK9 protein levels by ELISA using a Human Proprotein Convertase 9 / PCSK9 Quantikine Elisa kit (Catalog No. DPC900, R&D Systems, Inc.). PCSK9 expression levels were normalized to the mean pre-dose PCSK9 levels and are shown in Table 41 below. [Table 41]

[0307] Low density lipoprotein (LDL) and non-high density lipoprotein (HDL) cholesterol levels were quantified and normalized to mean pre-dose levels, and the data are presented in Table 42 below. [Table 42]

[0308] Other embodiments While the present invention has been described in conjunction with its detailed description, it is to be understood that the above description is intended to illustrate, but not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An RNAi agent for inhibiting expression of the PCSK9 gene, comprising: an antisense strand, wherein nucleotides 1-21 of the antisense strand comprise nucleotides 1-21 of the antisense strand sequence of Table 2, Table 3, Table 5C, Table 7A, or Table 8; and a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand; Including, An RNAi agent, wherein all or substantially all of the nucleotides of the antisense strand and / or the sense strand are modified nucleotides, and wherein the RNAi agent is linked to a targeting ligand that comprises an N-acetylgalactosamine.

2. 2. The RNAi agent of claim 1, wherein the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides that differs by 0 or 1 nucleotide from 15 contiguous nucleotides of any one of the sense strand sequences of Table 2, Table 4, Table 5C, Table 7B, or Table 8, and the sense strand has a region of at least 85% complementarity with the antisense strand over at least 15 contiguous nucleotides.

3. 3. The RNAi agent of claim 1 or 2, wherein at least one nucleotide of the RNAi agent comprises a modified internucleoside linkage.

4. 4. The RNAi agent of any one of claims 1 to 3, wherein the modified nucleotides are independently selected from the group consisting of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-seconucleotide mimics, locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted 2'-O-methyl nucleotides, inverted 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, vinyl phosphonate-containing nucleotides, cyclopropyl phosphonate-containing nucleotides, and 3'-O-methyl nucleotides.

5. 5. The RNAi agent of claim 4, wherein all or substantially all of the modified nucleotides are 2'-O-methyl nucleotides, 2'-fluoro nucleotides, or a combination thereof.

6. 6. The RNAi agent of any one of claims 1-5, wherein the antisense strand consists of, or consists essentially of, the nucleotide sequence of any one of the modified antisense strand sequences in Table 3, Table 5C, Table 7A, or Table 8.

7. 7. The RNAi agent of any one of claims 1-6, wherein the sense strand consists of, consists essentially of, or comprises the nucleotide sequence of any one of the modified sense strand sequences in Table 4, Table 5C, Table 7B, or Table 8.

8. 2. The RNAi agent of claim 1, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences in Table 3, Table 5C, Table 7A, or Table 8, and the sense strand comprises the nucleotide sequence of any one of the modified sequences in Table 4, Table 5C, Table 7B, or Table 8.

9. The targeting ligand has the following structure: 【Chemistry 1】 or a pharmaceutically acceptable salt thereof.

10. The RNAi agent of any one of claims 1 to 9, wherein the targeting ligand is linked to the sense strand.

11. The RNAi agent of claim 10, wherein the targeting ligand is linked to the 5' end of the sense strand.

12. The RNAi agent of any one of claims 1 to 11, wherein the sense strand is 15 to 30 nucleotides in length and the antisense strand is 21 to 30 nucleotides in length.

13. The RNAi agent of claim 12, wherein the sense strand and the antisense strand are each 21 to 27 nucleotides in length.

14. The RNAi agent of claim 13, wherein the sense strand and the antisense strand are each 21 to 24 nucleotides in length.

15. The RNAi agent of claim 14, wherein the sense strand and the antisense strand are each 21 nucleotides in length.

16. The RNAi agent of any one of claims 1 to 15, wherein the RNAi agent has two blunt ends.

17. The RNAi agent of any one of claims 1 to 16, wherein the sense strand comprises one or two terminal caps.

18. The RNAi agent of any one of claims 1 to 17, wherein the sense strand comprises one or two inverted abasic residues.

19. 2. The RNAi agent of claim 1, wherein the RNAi agent is composed of a sense strand and an antisense strand that form a duplex sequence of any of the duplexes set forth in Table 5A, Table 5B, Table 5C, or Table 8.

20. The RNAi agent of any one of claims 1 to 19, wherein the RNAi agent is a pharmaceutically acceptable salt.

21. 21. The RNAi agent of claim 20, wherein the RNAi agent is a sodium salt.

22. 22. The RNAi agent of any one of claims 1 to 21, comprising an antisense strand comprising or consisting of the nucleotide sequence of SEQ ID NO: 401, and a sense strand comprising or consisting of the nucleotide sequence of SEQ ID NO:

430.

23. 23. The RNAi agent of claim 22, comprising a modified antisense strand comprising, or consisting of, the nucleotide sequence of SEQ ID NO: 309, and a modified sense strand comprising, or consisting of, the nucleotide sequence of SEQ ID NO:

368.

24. 24. A composition comprising the RNAi agent of any one of claims 1 to 23, wherein the composition comprises a pharmaceutically acceptable excipient.

25. 25. The composition of claim 24, wherein the pharmaceutically acceptable excipient is sodium phosphate buffer.

26. 26. The composition of claim 25, wherein the pharmaceutically acceptable excipient is isotonic saline or water for injection.

27. 27. A method for inhibiting expression of the PCSK9 gene in liver cells, comprising introducing into the cells an effective amount of an RNAi agent according to any one of claims 1 to 23 or a composition according to any one of claims 24 to 26.

28. 28. The method of claim 27, wherein the subject is a human subject.

29. 29. The method of claim 27 or 28, wherein PCSK9 mRNA levels are reduced by at least about 50% in the liver cells or the subject.

30. 30. The method of any one of claims 27 to 29, wherein PCSK9 protein levels are reduced by at least about 50% in the liver cells or in the subject.

31. 27. A method for treating a PCSK9-related disease, disorder, or condition, comprising administering to a human subject in need thereof a therapeutically effective amount of the composition of any one of claims 24-26.

32. 32. The method of claim 31 , wherein the disease is hypercholesterolemia, familial hypercholesterolemia including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD) including clinical atherosclerotic cardiovascular disease (ASCVD).

33. 33. The method of any one of claims 27 to 32, wherein the level of serum PCSK9 protein is reduced in the subject.

34. 34. The method of any one of claims 27-33, wherein the RNAi agent is administered to the human subject at a dose of about 0.05 mg / kg to about 5.0 mg / kg of body weight of the human subject.

35. Use of the RNAi agent of any one of claims 1 to 23 or the composition of any one of claims 24 to 26 for the treatment of a disease, disorder, or condition mediated at least in part by decreased PCSK9 gene expression.

36. 36. The use of claim 35, wherein the disease is hypercholesterolemia, familial hypercholesterolemia including heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), familial hypobetalipoproteinemia, hyperlipidemia, coronary artery disease, polygenic dyslipidemia, heart disease, cardiovascular disease (CVD) including clinical atherosclerotic cardiovascular disease (ASCVD), and / or other PCSK9-related diseases.

37. Use of the RNAi agent of any one of claims 1 to 23 or the composition of any one of claims 24 to 26 in the preparation of a pharmaceutical composition for treating a disease, disorder, or condition mediated at least in part by decreased PCSK9 gene expression.

38. 38. The use of any one of claims 35 to 37, wherein the RNAi agent is administered to a human subject at a dose of about 0.05 mg to about 5.0 mg per kg of body weight of the human subject.

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