Targeting ligands for therapeutic compounds

Abstract

To provide novel targeting ligands that may be linked to compounds, for example therapeutic compounds, that are useful in directing the compounds to the target in vivo.SOLUTION: The targeting ligands disclosed herein serve to target expression-inhibiting oligomeric compounds, such as RNAi agents, to liver cells to modulate gene expression. The targeting ligands disclosed herein, when conjugated to an expression-inhibiting oligomeric compound, may be used in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Compositions including the targeting ligands disclosed herein, when linked to expression-inhibiting oligomeric compounds, are capable of mediating expression of target nucleic acid sequences in liver cells, such as hepatocytes, which may be useful in the treatment of diseases or conditions that respond to inhibition of gene expression or activity in a cell, tissue, or organism.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 304,652, filed March 7, 2016, U.S. Provisional Patent Application No. 62 / 370,754, filed August 4, 2016, and U.S. Provisional Patent Application No. 62 / 426,916, filed November 28, 2016, the contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] background Many compounds need to be delivered to specific location (for example, to desired cell(s)) in order to have therapeutic effect or be useful for diagnostic purposes.This is often the case when trying to deliver therapeutic compounds in vivo.In addition, being able to efficiently deliver compounds to specific location limits or potentially eliminates the unintended consequences (such as off-target effects) that may be caused by administering the compound.One way to facilitate the delivery of compounds such as therapeutic compounds to desired locations in vivo is to link or attach the compound to targeting ligand.

[0003] One of the classes of therapeutic compounds that can be targeted by using targeting ligands is oligomeric compounds.Oligomeric compounds that comprise nucleotide sequences that are at least partially complementary to target nucleic acids have been shown to change the function and activity of the target both in vitro and in vivo.When delivered to cells that contain target nucleic acids (such as mRNA), oligomeric compounds have been shown to regulate the expression of the target, resulting in the altered transcription or translation of target nucleic acids.In certain cases, the oligomeric compounds can reduce gene expression by inhibiting nucleic acid targets and / or causing the degradation of target nucleic acids.

[0004] If the target nucleic acid is mRNA, one mechanism by which expression-inhibiting oligomeric compounds can regulate the expression of mRNA targets is through RNA interference. RNA interference is a biological process in which RNA or RNA-like molecules (such as chemically modified RNA molecules) can suppress gene expression by degradation. The process of post-transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes.

[0005] Synthetic RNA and RNA-like molecules have been shown to induce RNA interference in vivo.For example, Elbashir et al. (Nature 2000, 411, 494-98) describe the RNAi caused by the introduction of a double strand of 21-nucleotide synthetic RNA molecules in cultured mammalian cells.The type of synthetic RNA or RNA-like molecule that can trigger the RNAi response mechanism can be composed of modified nucleotides and / or one or more non-phosphodiester bonds.

[0006] Additionally, single-stranded RNA and RNA-like molecules (which may contain modified nucleotides and have one or more non-phosphodiester bonds) may also alter the expression of a target nucleic acid, such as a target mRNA. Summary of the Invention

[0007] overview Disclosed herein are targeting ligands that may facilitate delivery of therapeutic compounds to specific organs or tissues, e.g., specific target sites, within the body of a subject, such as a human patient or animal. In some embodiments, the targeting ligands described herein may facilitate targeted delivery of expression-inhibiting oligomeric compounds. In some embodiments, the targeting ligands facilitate delivery of expression-inhibiting oligomeric compounds to the liver.

[0008] The targeting ligands disclosed herein comprise or consist of one or more targeting moieties, one or more tethers, one or more branch point groups, and one or more linkers.

[0009] Disclosed herein are targeting ligands comprising, consisting of, or consisting essentially of the general structure of Formula A: [ka] {wherein n is an integer from 1 to 4 (e.g., 1, 2, 3, or 4)}.

[0010] In some embodiments, the targeting ligands disclosed herein comprise, consist of, or consist essentially of the structure of Formula B: [ka] wherein n is an integer between 1 and 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); X is O, S, or NH; and the targeting moiety is selected from the group consisting of: N-acetyl-galactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionyl-galactosamine, Nn-butanoylgalactosamine, and N-iso-butanoylgalactosamine.

[0011] In some embodiments, the targeting ligands disclosed herein comprise, consist of, or consist essentially of the following structure: [ka] {wherein n is an integer of 1 to 20} (Structure 1).

[0012] In some embodiments, the disclosed targeting ligands comprise, consist of, or consist essentially of a structure selected from the following structures: [ka] (Structure 101); [ka] (Structure 102); and [ka] (Structure 103).

[0013] The targeting ligands disclosed herein comprise one or more targeting moieties. In some embodiments, the targeting ligands disclosed herein comprise N-acetyl-galactosamine as the targeting moiety.

[0014] The targeting ligands disclosed herein can be linked directly or indirectly to a compound, such as a therapeutic compound, for example, an expression-inhibiting oligomeric compound, for example, to the 3' or 5' end of the expression-inhibiting oligomeric compound. In some embodiments, the expression-inhibiting oligomeric compound includes one or more modified nucleotides. In some embodiments, the expression-inhibiting oligomeric compound is an RNAi agent, such as a double-stranded RNAi agent. In some embodiments, the targeting ligands disclosed herein are linked to the 5' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the targeting ligands disclosed herein are linked to the RNAi agent at the 5' end of the sense strand of the double-stranded RNAi agent via a phosphate group, a phosphorothioate group, or a phosphonate group.

[0015] Disclosed herein are compositions comprising a targeting ligand and an expression-inhibiting oligomeric compound.Disclosed herein are compositions comprising a targeting ligand and an RNAi agent.

[0016] In some embodiments, a composition disclosed herein comprising a targeting ligand and an RNAi agent has a structure represented by the following structural formula: [ka] {wherein Z comprises or consists of an expression-inhibiting oligomeric compound} (structure 101a); [ka] {wherein Z comprises or consists of an expression-inhibiting oligomeric compound} (structure 102a); and [ka] wherein Z comprises or consists of an expression-inhibiting oligomeric compound (structure 103a). Disclosed herein are phosphoramidite compounds that contain targeting ligands.

[0017] In some embodiments, phosphoramidite compounds comprising targeting ligands disclosed herein have a structure represented by the following structural formula: [ka] {wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)} (structure 1d); [ka] (Structure 101d); [ka] (Structure 102d); or [ka] (Structure 103d). Also disclosed are pharmaceutical compositions comprising the targeting ligands disclosed herein.

[0018] Disclosed are methods for treating a disease or disorder that would benefit from the administration of a therapeutic oligomeric compound, the method comprising administering to a subject a therapeutic oligomeric compound linked to a targeting ligand disclosed herein. Disclosed herein are methods for inhibiting expression of a target nucleic acid in a subject, the methods comprising administering a therapeutic amount of an expression-inhibiting oligomeric compound linked to a targeting ligand disclosed herein. Disclosed herein is a method for delivering an expression-inhibiting oligomeric compound to the liver in vivo, the method comprising administering to a subject an expression-inhibiting oligomeric compound linked to a targeting ligand disclosed herein.

[0019] As used herein, the term "linked," when referring to a connection between two molecules, means that the two molecules are joined together by a covalent bond or that the two molecules are linked through a non-covalent bond (e.g., a hydrogen bond or an ionic bond). In some instances where the term "linked" refers to a bond between two molecules through a non-covalent bond, the bond between the two different molecules is such that the bond is at least 1×10 in a physiologically acceptable buffer (e.g., phosphate-buffered saline). -4 Less than M (e.g., 1 × 10 -5 Less than M, 1 x 10 -6 Less than M or 1 x 10 -7 K (less than M) D It has.

[0020] As used herein, the term "directly linked" refers to a first compound or group that is linked to a second compound or group without any intervening atom or group of atoms. As used herein, the term "indirectly linked" refers to a first compound that is linked to a second compound or group by an intervening group, compound, or molecule, such as, for example, a linking group. Unless otherwise specified, the term "linked" as used herein includes both "directly linked" and "indirectly linked," as those terms are defined herein.

[0021] As used herein, an "oligomeric compound" is a nucleotide sequence containing approximately 10 to 50 nucleotides or nucleotide base pairs. In some embodiments, an oligomeric compound has a nucleobase sequence that is at least partially complementary to a coding sequence within a target nucleic acid or target gene expressed in a cell. In some embodiments, upon delivery to a gene-expressing cell, the oligomeric compound can inhibit expression of the responsible gene and is therefore referred to herein as an "expression-inhibiting oligomeric compound." Gene expression can be inhibited in vitro or in vivo. "Oligomeric compounds" include, but are not limited to, oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and Dicer substrates.

[0022] As used herein, the term "oligonucleotide" means a polymer of linked nucleosides, each of which may or may not be independently modified. As used herein, the term "single-stranded oligonucleotide" refers to a single-stranded oligomeric compound having a sequence that is at least partially complementary to a target mRNA, i.e., that can hybridize to a target mRNA by hydrogen bond formation under physiological conditions in mammals (or comparable conditions in vitro). In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.

[0023] As used herein, "RNAi agent" refers to an agent comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can reduce or inhibit translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence-specific manner. As used herein, an RNAi agent functions through the RNA interference mechanism (i.e., causes RNA interference by interacting with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) in mammalian cells) or by any alternative mechanism or pathway(s). As the term is used herein, it is believed that RNAi agents primarily function through the RNA interference mechanism, but the disclosed RNAi agents are not tied to or limited to any particular pathway or mechanism of action. RNAi agents include, but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates. The RNAi agents described herein are composed of oligonucleotides having a strand at least partially complementary to the targeted mRNA. In some embodiments, the RNAi agents described herein are double-stranded and comprise an antisense strand and a sense strand that is at least partially complementary to the antisense strand. The RNAi agents may comprise modified nucleotides and / or one or more non-phosphodiester linkages. In some embodiments, the RNAi agents described herein are single-stranded.

[0024] As used herein, the terms "suppression," "reduction," "inhibition," "downregulation," 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 polypeptide, protein, or protein subunit translated from the mRNA, in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with an oligomeric compound linked to a targeting ligand as described herein, compared to a second cell, group of cells, tissue, organ, or subject that has not been so treated. As used herein, the term "sequence" or "nucleotide sequence" means a sequence or order of nucleic acid bases or nucleotides written using a sequence of letters using standard nucleotide nomenclature.

[0025] As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence (e.g., the sense strand of an RNAi agent or a target mRNA) related to a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or the antisense strand of a double-stranded RNAi agent), refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base-pair hydrogen bonds under physiological conditions in a mammal (or comparable conditions in vitro)) with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specific conditions, and form a double-stranded or double-helical structure. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides, or nucleotide mimetics, at least to the extent that they fulfill the above requirements for ability to hybridize.

[0026] As used herein, "perfectly complementary" or "fully complementary" means that all (100%) of the bases in a contiguous sequence of a first polynucleotide hybridize to the same number of bases in a contiguous sequence of a second polynucleotide, which may include all or part of the first or second nucleotide sequence. As used herein, "partially complementary" means that in a pair of hybridized nucleobase sequences, at least 70%, but not all, of the bases in the contiguous sequence of a first polynucleotide hybridize to the same number of bases in the contiguous sequence of a second polynucleotide.

[0027] As used herein, "substantially complementary" means that in a pair of hybridized nucleic acid base sequences, at least 85%, but not all, of the bases in the contiguous sequence of a first polynucleotide hybridize to the same number of bases in the contiguous sequence of a second polynucleotide. As used herein, the terms "complementary," "fully complementary," and "substantially complementary" may be used in reference to matching bases between the sense and antisense strands of a double-stranded RNAi agent, between the antisense strand of a double-stranded RNAi agent and the sequence of a target mRNA, or between a single-stranded antisense oligonucleotide and the sequence of a target mRNA.

[0028] As used herein, the terms "treat," "treatment," and the like refer to methods or steps taken to provide relief from or alleviation of the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, the phrase "introducing into a cell," when referring to an oligomeric compound, refers to functionally delivering the oligomeric compound into a cell. The phrase "functional delivery" refers to delivering the oligomeric compound to a cell in a manner that enables the oligomeric compound to have its desired biological activity, e.g., sequence-specific inhibition of gene expression.

[0029] Unless otherwise specified, symbols used in this specification [ka] The use of means that any group(s) can be linked according to the scope of the invention described herein.

[0030] As used herein, the term "isomers" refers to compounds that have identical molecular formulae but differ in the nature or sequence of bonds of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are called "stereoisomers." Stereoisomers that are not mirror images of one another are called "diastereoisomers," and stereoisomers that are non-superimposable mirror images are called "enantiomers," or sometimes optical isomers. A carbon atom attached to four non-identical substituents is called a "chiral center." As used herein, for each structure in which an asymmetric center exists and thereby exhibits an enantiomer, diastereomer, or other stereoisomeric configuration, without the structure being specifically identified as having a particular configuration, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure and racemic forms. For example, the structures disclosed herein are intended to encompass mixtures of diastereomers as well as single stereoisomers.

[0031] The term "substituted," as used herein, means that any one or more hydrogens on the indicated atom, usually a carbon, oxygen, or nitrogen atom, may be replaced with any group defined herein, provided that the replacement does not exceed the standard ionic valence of the indicated atom, and that the replacement results in a stable compound. Non-limiting examples of substituents include C-C alkyl, C-C alkenyl, C-C alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, aryl, keto, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halo (e.g., F, Cl, Br, I). When a substituent is keto or oxo (i.e., =0), two (2) hydrogens on the atom are replaced. A ring double bond, as used herein, is a double bond formed between two adjacent ring atoms (e.g., C=C, C=N, N=N, etc.).

[0032] Some compounds of the present disclosure may exist in tautomeric forms, which are also intended to be included within the scope of the present disclosure. "Tautomers" are compounds whose structures differ significantly in the arrangement of atoms, but which exist in easy and rapid equilibrium. It should be understood that compounds of the present disclosure may be represented as different tautomers. When a compound has tautomeric forms, it should also be understood that all tautomeric forms are within the scope of the disclosure, and the name of the compound does not intend to exclude any tautomeric forms.

[0033] The compounds and pharmaceutically acceptable salts of the present disclosure may exist in one or more tautomeric forms, including ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomers of heterocyclic rings (e.g., the nucleobases guanine, thymine, and cytosine), amine-enamine and enamine-enamine, and geometric isomers, as well as mixtures thereof. The cyclic chain tautomerism exhibited by glucose and other sugars results from an aldehyde group (-CHO) in the sugar chain molecule reacting with a hydroxyl group (-OH) in the same molecule to form a ring (ring shape). All such tautomeric forms are included within the scope of the present disclosure. Tautomers exist as mixtures of tautomeric sets in solution. In solids, one tautomer typically predominates. Even if one tautomer is described, the present disclosure includes all tautomers of the compounds disclosed herein. The concept of tautomers that are interconvertible by tautomerization is referred to as tautomerism. In tautomerism, a simultaneous shift of an electron and a hydrogen atom occurs.

[0034] Tautomerization is catalyzed by: bases: 1. deprotonation; 2. formation of a delocalized anion (e.g., enolate); 3. protonation at a different position on the anion; acids: 1. protonation; 2. formation of a delocalized cation; 3. deprotonation at a different position adjacent to the cation.

[0035] As used herein, the term "alkyl," unless otherwise specified, refers to a linear or branched, saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms. For example, "C1-C6 alkyl" includes alkyl groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement. As used herein, the term "aminoalkyl" refers to an alkyl group, as defined above, substituted at any position with one or more amino groups, as allowed by the standard ionic valence. The amino groups may be unsubstituted, monosubstituted, or disubstituted.

[0036] As used herein, unless otherwise specified, the term "cycloalkyl" means a saturated or unsaturated non-aromatic hydrocarbon ring group having 3 to 14 carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, methylcyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and the like. Cycloalkyls may contain multiple spiro or fused rings. Cycloalkyl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted in any position allowed by the standard ionic valence.

[0037] As used herein, unless otherwise specified, the term "alkenyl" refers to a linear or branched non-aromatic hydrocarbon group containing at least one carbon-carbon double bond and having 2 to 10 carbon atoms. Up to five carbon-carbon double bonds may be present in such a group. For example, a "C2-C6" alkenyl is defined as an alkenyl radical having 2 to 6 carbon atoms. Examples of alkenyl include, but are not limited to, ethenyl, propenyl, butenyl, and cyclohexenyl. The linear, branched, or cyclic portion of the alkenyl group may contain double bonds and may be optionally mono-, di-, tri-, tetra-, or penta-substituted, or penta-substituted, at any position allowed by the standard ionic valence. The term "cycloalkenyl" refers to a monocyclic hydrocarbon group having the specified number of carbon atoms and at least one carbon-carbon double bond.

[0038] As used herein, unless otherwise specified, the term "alkynyl" refers to a linear or branched hydrocarbon group containing 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Up to five carbon-carbon triple bonds may be present. Thus, "C2-C6 alkynyl" refers to an alkynyl radical having 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl. The linear or branched portion of the alkynyl group may contain triple bonds allowed by the standard ionic valence and is optionally mono-, di-, tri-, tetra-, or penta-substituted, or penta-substituted, at any position allowed by the standard ionic valence.

[0039] As used herein, "alkoxyl" or "alkoxy" refers to an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. 1-6 Alkoxy is intended to include C1, C2, C3, C4, C5, and C6 alkoxy groups. 1-8Alkoxy is intended to include C1, C2, C3, C4, C5, C6, C7, and C8 alkoxy groups. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, s-pentoxy, n-heptoxy, and n-octoxy. As used herein, "keto" refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl group, as defined herein, attached through a carbonyl bridge. Examples of keto groups include, but are not limited to, alkanoyl (e.g., acetyl, propionyl, butanoyl, pentanoyl, hexanoyl), alkenoyl (e.g., acryloyl), alkynoyl (e.g., ethinoyl, propynoyl, butynoyl, pentinoyl, hexynoyl), aryloyl (e.g., benzoyl), and heteroaryloyl (e.g., imidazoloyl, quinolinoyl, pyridinoyl, pyrroloyl).

[0040] As used herein, "alkoxycarbonyl" refers to any alkoxy group, as defined above, attached by a carbonyl bridge (i.e., -C(O)O-alkyl-). Examples of alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, iso-propoxycarbonyl, n-propoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, or n-pentoxycarbonyl.

[0041] As used herein, "aryloxycarbonyl" refers to any aryl group, as defined herein, attached by an oxycarbonyl bridge (i.e., -C(O)O-aryl-). Examples of aryloxycarbonyl groups include, but are not limited to, phenoxycarbonyl and naphthyloxycarbonyl.

[0042] As used herein, "heteroaryloxycarbonyl" refers to any heteroaryl group, as defined herein, attached by an oxycarbonyl bridge (i.e., -C(O)O-heteroaryl-). Examples of heteroaryloxycarbonyl groups include, but are not limited to, 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

[0043] As used herein, "aryl" or "aromatic" refers to any stable monocyclic or polycyclic carbon ring of up to seven atoms in each ring, in which at least one ring is aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl, and biphenyl. When the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring. Aryl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted in any position allowed by the standard ionic valence.

[0044] As used herein, the term "heteroaryl" refers to a stable monocyclic or polycyclic ring of up to seven atoms in each ring, in which at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N, and S. Examples of heteroaryl groups include, but are not limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrolazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl, isoindolonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. "Heteroaryl" is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl. When a heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom-containing ring. Heteroaryl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted in any position allowed by standard ionic valences.

[0045] As used herein, the term "heterocycle," "heterocyclic," or "heterocyclyl" means a 3- to 14-membered aromatic or non-aromatic heterocycle containing 1 to 4 heteroatoms selected from the group consisting of O, N, and S, including polycyclic groups. As used herein, the term "heterocycle" is also considered synonymous with the terms "heterocycle" and "heterocyclyl," and is understood to have the same definitions as described herein. "Heterocyclyl" includes the heteroaryls described above as well as dihydro and tetrahydro analogs thereof.Examples of heterocyclyl groups include, but are not limited to, azetidinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, and naphthopyridinyl. , oxadiazolyl, oxooxazolidinyl, oxazolyl, oxazoline, oxopiperazinyl, oxopiperidinyl, oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyridinonyl, pyrimidyl, pyrimidinonyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl aryl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl Heterocyclyl groups include azolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxidethiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, and N-oxide. The attachment of heterocyclyl substituents can occur via a carbon atom or via a heteroatom. Heterocyclyl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted at any position allowed by the standard ionic valence.

[0046] Those skilled in the art will readily understand or recognize that the compounds and compositions disclosed herein 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. Thus, as used herein, the structures disclosed herein assume that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to encompass the disclosed compounds and compositions regardless of their protonation state based on the pH of the environment, as will be readily understood by those skilled in the art.

[0047] When used in the claims herein, the phrase "consisting of" excludes any component, step, or ingredient not specified in the claim. As used in the claims herein, the phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of 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 case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. [Brief explanation of the drawings]

[0049] [Figure 1] FIG. 1 is the 1H NMR spectrum of compound 3 (described below in Example 1). [Figure 2] FIG. 2 is the 1H NMR spectrum of compound 4 (described below in Example 1). [Figure 3] FIG. 3 is the 1H NMR spectrum of compound 6 (described below in Example 1). [Figure 4] FIG. 4 is the 1H NMR spectrum of compound 7 (described below in Example 1). [Figure 5] FIG. 5 is the 1H NMR spectrum of compound 9 (described below in Example 1). [Figure 6] FIG. 6 is the 1H NMR spectrum of compound 10 (structure 101d herein, described below in Example 1). [Figure 7] FIG. 7 is the 1H NMR spectrum of compound 13 (structure 103d herein, described below in Example 2). [Figure 8] FIG. 8 is the 1H NMR spectrum of compound 16 (structure 102d herein, described below in Example 3). [Figure 9] FIG. 9 is an HPLC chromatograph of AM03704 conjugated to structure 103d (described below in Example 5). [Figure 10] FIG. 10 is an HPLC chromatograph of AM03704 conjugated to structure 101d (described below in Example 5). [Figure 11] FIG. 11 is an HPLC chromatograph of AM03704 conjugated to structure 102d (described below in Example 5). [Figure 12] FIG. 12 is a graph illustrating normalized mouse Factor 12 (mF12) protein levels in wild-type mice (described below in Example 6). [Figure 13]FIG. 13 is a graph illustrating normalized mouse Factor 12 (F12) protein levels in wild-type mice (described below in Example 7). [Figure 14] FIG. 14 is a graph illustrating normalized lipoprotein(a) (Lp(a)) particle levels in Lp(a) transgenic (Tg) mice (described below in Example 8). [Figure 15] FIG. 15 is a graph illustrating normalized lipoprotein(a) (Lp(a)) particle levels in Lp(a) Tg mice (described below in Example 9). [Figure 16] FIG. 16 is a graph illustrating normalized apo(a) levels in apo(a) transgenic (Tg) mice (described below in Example 10). [Figure 17] FIG. 17 is a graph illustrating normalized cF12 protein levels in cynomolgus monkeys (described below in Example 12). [Figure 18] FIG. 18 is a graph illustrating normalized AAT (Z-AAT) protein levels in PiZ transgenic mice (described below in Example 13). [Figure 19] FIG. 19 is a graph illustrating normalized mouse Factor 12 (F12) protein levels in wild-type mice (described below in Example 14). DETAILED DESCRIPTION OF THE INVENTION

[0050] Detailed Description Described herein are novel targeting ligands linked to compounds, such as therapeutic or diagnostic expression-inhibiting oligomeric compounds. In some embodiments, the compounds linked to the targeting ligands described herein comprise or consist of therapeutic compounds that are RNAi agents. The targeting ligands can be used to target therapeutic compounds to a desired location of a target nucleic acid or target gene. Also described herein are compositions comprising targeting ligands and therapeutic compounds, such as compositions comprising or consisting of targeting ligands and expression-inhibiting oligomeric compounds.

[0051] The new targeting ligands disclosed herein are suitable for synthesis as phosphoramidites, providing effective targeting or biodistribution, sufficient stability in vivo or in vitro, and reducing the cost and burden of manufacturing, and may have enhanced efficacy over previously considered targeting ligands linked to expression-inhibiting oligomeric compounds such as RNAi agents.

[0052] Targeting Ligands Targeting ligands consist of one or more targeting groups or targeting moieties, which may enhance the pharmacokinetic or biodistribution properties of the compound to which they are linked, and may help improve cell- or tissue-specific distribution or cell-specific uptake of the conjugated composition. Generally, targeting ligands help guide the delivery of the therapeutic compound linked to them to the desired target site. In some cases, the targeting moiety can bind to a cell or cell receptor and initiate endocytosis to facilitate the entry of the therapeutic compound into the cell. Targeting moieties can include compounds with affinity for cell receptors, cell surface molecules, or antibodies. Various targeting ligands, including targeting moieties, can be linked to therapeutic agents and other compounds to target the agent to cells and specific cell receptors. Types of targeting moieties include carbohydrates, cholesterol and cholesteryl groups, and steroids. Targeting moieties capable of binding to cell receptors include saccharides such as galactose, galactose derivatives (such as N-acetyl-galactosamine), mannose, and mannose derivatives; other carbohydrates; glycans; haptens; vitamins; folates; biotin; aptamers; and peptides such as RGD-containing peptides, insulin, EGF, and transferrin.

[0053] Targeting moieties known to bind to the asialoglycoprotein receptor (ASGPR) are particularly useful in directing delivery of oligomeric compounds to the liver. The asialoglycoprotein receptor is abundantly expressed in liver cells, including hepatocytes. Cell receptor targeting moieties that target ASGPR include galactose and galactose derivatives. In particular, clusters of galactose derivatives containing clusters of two, three, or four N-acetyl-galactosamine (GalNAc or NAG) can facilitate the uptake of specific compounds into liver cells. The GalNAc clusters attached to the oligomeric compounds serve to target the compositions to the liver, where the N-acetyl-galactosamine sugars can bind to the asialoglycoprotein receptor on the surface of liver cells. Binding to the asialoglycoprotein receptor is thought to initiate receptor-mediated endocytosis, thereby facilitating the compound's entry into the cells.

[0054] The targeting ligands disclosed herein may comprise one, two, three, four, or more than four targeting moieties. In some embodiments, the targeting ligands disclosed herein may comprise one, two, three, four, or more than four targeting moieties linked to a branch point group. In some embodiments, the targeting ligands disclosed herein may comprise one, two, three, four, or more than four targeting moieties linked to a branch point group, wherein each targeting moiety is linked to the branch point group via a tether.

[0055] In some embodiments, the targeting ligands disclosed herein may comprise one, two, three, four, or more than four asialoglycoprotein receptor (ASGPR) targeting moieties linked to a branch point group. It may include 1, 2, 3, 4, or more than 4 ASGPR targeting moieties linked to a branch point group, where each ASGPR targeting moiety is linked to the branch point group via a tether.

[0056] The targeting ligands described herein are represented by Formula I below: [ka] wherein n is an integer from 1 to 4 (e.g., 1, 2, 3, or 4) (Formula I). In some embodiments, n in Formula I is an integer from 1 to 3, 1 to 2, 2 to 4, 2 to 3, or 3 to 4.

[0057] The targeting ligands disclosed herein can be linked to a therapeutic compound, such as an oligomeric compound. In some embodiments, the targeting ligand is linked to the therapeutic compound via an additional linker and / or a cleavable moiety, which is then linked to the therapeutic compound. In some embodiments, the targeting ligand is attached to the therapeutic compound itself.

[0058] In some embodiments, the therapeutic compound is an expression-inhibiting oligomeric compound. In some embodiments, the expression-inhibiting oligomeric compound is an RNAi agent. In some embodiments, the expression-inhibiting oligomeric compound is a double-stranded RNAi agent.

[0059] In some embodiments, the targeting ligand is directly or indirectly linked to the 5'-end of the sense strand of the double-stranded RNAi agent. In some embodiments, the targeting ligand is directly or indirectly linked to the 3'-end of the sense strand of the double-stranded RNAi agent. In some embodiments, the targeting ligand is directly or indirectly linked to the 5'-end or 3'-end of the antisense strand of the double-stranded RNAi agent. In some embodiments, the targeting ligand is directly or indirectly linked to the 5'-end or 3'-end of the single-stranded RNAi agent.

[0060] In some embodiments, the targeting ligand is linked to the double-stranded RNAi agent via a phosphate group, a phosphonate group, a phosphorothioate group, or other internucleoside linking group at the 5' end of the terminal nucleoside of the sense strand of the double-stranded RNAi agent.

[0061] In some embodiments, the targeting ligands disclosed herein comprise a cleavable moiety. In some embodiments, the cleavable moiety comprises or consists of a phosphate group or other internucleoside linking group that can be cleaved. In some embodiments, the targeting ligand is linked to a therapeutic compound via the cleavable moiety.

[0062] In some embodiments, the targeting ligands disclosed herein are linked to an additional group or a group that includes a cleavable moiety, hi some embodiments, the targeting ligand is linked to a cleavable moiety, which is in turn linked to an expression-inhibiting oligomeric compound.

[0063] In some embodiments, the targeting ligand is a phosphoramidite compound (also referred to herein as a "phosphoramidite-containing compound"). Phosphoramidite compounds containing the targeting ligands described herein can be useful for easily attaching the targeting ligand to a therapeutic compound or other group using methods commonly known in the art for phosphoramidite synthesis. In some embodiments, the phosphoramidite compound containing the targeting ligand is linked to an expression-inhibiting oligomeric compound using methods commonly known in the art. In some embodiments, the targeting ligand-containing phosphoramidite is linked to the 5' end of the sense strand of a double-stranded RNAi agent.

[0064] In some embodiments, the expression-inhibiting oligomeric compound linked to a targeting ligand comprises a single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide. In some embodiments, the targeting ligand is directly linked to the single-stranded antisense oligonucleotide. In some embodiments, an additional group is inserted between the targeting ligand and the single-stranded oligonucleotide. In some embodiments, the targeting ligand linked to the RNAi agent comprises a targeting moiety or one or more N-acetyl-galactosamine sugars as the targeting moiety.

[0065] In some embodiments, the targeting ligand linked to the expression-inhibiting oligomeric compound comprises a tether comprising polyethylene glycol (PEG). In some embodiments, the tether consists of PEG. In some embodiments, the tether comprises PEG having 1 to 10 ethylene glycol units. In some embodiments, the tether comprises PEG having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ethylene glycol units.

[0066] In some embodiments, the targeting ligand linked to the RNAi agent comprises polyethylene glycol (PEG) as a linker. In some embodiments, the linker comprises PEG. In some embodiments, the linker consists of PEG. In some embodiments, the linker comprises PEG having 1-20 ethylene glycol units. In some embodiments, the tether comprises PEG having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ethylene glycol units. In some embodiments, the expression-inhibiting oligomeric compound linked to any targeting ligand disclosed herein comprises an RNAi agent. In some embodiments, the targeting ligand disclosed herein is linked directly or indirectly to an RNAi agent.

[0067] In some embodiments, the targeting ligand disclosed herein is directly linked to the RNAi agent. In some embodiments, the targeting ligand disclosed herein is indirectly linked to the RNAi agent when additional group(s) are inserted between the RNAi agent and the linker of the targeting ligand. In some embodiments, a second linker is included between the linker and the therapeutic compound. A targeting ligand structure and a phosphoramidite compound comprising the targeting ligand. The targeting ligands disclosed herein can consist of one or more targeting moieties, tethers, branch point groups, and linkers. The targeting ligands disclosed herein can include one, two, three, four, or more than four targeting moieties.

[0068] In some embodiments, the targeting ligand disclosed herein is synthesized in the form of phosphoramidite compound.Phosphoramidite is widely used in the chemical synthesis of RNA and DNA.In some embodiments, the phosphoramidite-containing targeting ligand disclosed herein is added to the 5'-end of the sense strand of double-stranded RNAi agent.When said targeting ligand is to be linked to the 5'-end of expression-inhibiting oligomeric compound, preparing said targeting ligand as phosphoramidite can be particularly advantageous.Without wishing to be bound by theory, it is understood that preparing said targeting ligand as phosphoramidite not only allows the linking of the targeting ligand as the last component when said targeting ligand is linked to the 5'-end of expression-inhibiting oligomeric compound (thereby reducing production costs), but also allows the targeting ligand to potentially block the addition of the sense strand into RISC when said targeting ligand is attached to the 5'-end of the sense strand of double-stranded RNAi agent. When the expression-inhibiting oligomeric compound is a double-stranded RNAi agent, the targeting ligand can be prepared as a phosphoramidite compound when the targeting ligand is to be linked to the 5' end of the sense strand of the RNAi agent.

[0069] In some embodiments, the targeting ligand is represented by the following formula B: [ka] wherein n is an integer from 1 to 20; X is O, S, or NH; and the targeting moiety is selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, Nn-butanoylgalactosamine, or N-iso-butanoylgalactosamine (Formula B). In some embodiments, n is 6. In some embodiments, n is 8. In some embodiments, n is 4.

[0070] In some embodiments, the targeting ligand has a structure represented by the following structural formula: [ka] wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (Structure 1).

[0071] In some embodiments, the targeting ligand has a structure represented by Structure 1 {wherein n=6}. In some embodiments, the targeting ligand has a structure represented by Structure 1 {wherein n=8}. In some embodiments, the targeting ligand has a structure represented by Structure 1 {wherein n=4}.

[0072] In some embodiments, the targeting ligand is linked to the expression-inhibiting oligomeric compound and has a structure represented by the following structural formula: [ka] wherein Z comprises or consists of an expression-inhibiting oligomeric compound (structure 1a).

[0073] In some embodiments, the targeting ligand is linked to the expression-inhibiting oligomeric compound and has a structure represented by the following structural formula: [ka] wherein Z consists of or comprises an expression-inhibiting oligomeric compound (structure 1b).

[0074] In some embodiments, the targeting ligand is linked to the expression-inhibiting oligomeric compound and has a structure represented by the following structural formula: [ka] wherein Z consists of or comprises an expression-inhibiting oligomeric compound (structure 1c).

[0075] In some embodiments, the targeting ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (structure 1d).

[0076] In some embodiments, the targeting ligand comprises or consists of a structure represented by the following structural formula: [ka] (Structure 101).

[0077] In some embodiments, the targeting ligand is linked to the expression-inhibiting oligomeric compound and has a structure represented by the following structural formula: [ka] wherein Z comprises or consists of an expression-inhibiting oligomeric compound (structure 101a).

[0078] In some embodiments, the targeting ligand is linked to the expression-inhibiting oligomeric compound and has a structure represented by the following structural formula: [ka] wherein Z consists of or comprises an expression-inhibiting oligomeric compound (structure 101b).

[0079] In some embodiments, the targeting ligand is linked to the expression-inhibiting oligomeric compound and has a structure represented by the following structural formula: [ka] wherein Z consists of or comprises an expression-inhibiting oligomeric compound (structure 101c).

[0080] In some embodiments, the targeting ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 101d).

[0081] In some embodiments, the targeting ligand comprises or consists of a structure represented by the following structural formula: [ka] (Structure 102).

[0082] In some embodiments, the targeting ligand is linked to the expression-inhibiting oligomeric compound and has a structure represented by the following structural formula: [ka] wherein Z comprises or consists of an expression-inhibiting oligomeric compound (structure 102a).

[0083] In some embodiments, the targeting ligand is linked to the expression-inhibiting oligomeric compound and has a structure represented by the following structural formula: [ka] where Z consists of or comprises an expression-inhibiting oligomeric compound (structure 102b).

[0084] In some embodiments, the targeting ligand is linked to the expression-inhibiting oligomeric compound and has a structure represented by the following structural formula: [ka] wherein Z consists of or comprises an expression-inhibiting oligomeric compound (structure 102c).

[0085] In some embodiments, the targeting ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 102d).

[0086] In some embodiments, the targeting ligand comprises or consists of a structure represented by the following structural formula: [ka] (Structure 103).

[0087] In some embodiments, the targeting ligand is linked to the expression-inhibiting oligomeric compound and has a structure represented by the following structural formula: [ka] wherein Z comprises or consists of an expression-inhibiting oligomeric compound (structure 103a).

[0088] In some embodiments, the targeting ligand is linked to the expression-inhibiting oligomeric compound and has a structure represented by the following structural formula: [ka] wherein Z consists of or comprises an expression-inhibiting oligomeric compound (structure 103b).

[0089] In some embodiments, the targeting ligand is linked to the expression-inhibiting oligomeric compound and has a structure represented by the following structural formula: [ka] wherein Z consists of or comprises an expression-inhibiting oligomeric compound (structure 103c).

[0090] In some embodiments, the targeting ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 103d).

[0091] In some embodiments, the targeting ligand has a structure represented by the following structural formula: [ka] (Structure 2).

[0092] In some embodiments, the expression-inhibiting oligomeric compound is linked to a targeting ligand and has a structure represented by the following structural formula: [ka] wherein Z consists of or comprises an expression-inhibiting oligomeric compound; A is O or S; and A' is O - , S - , or N.H.- {Structure 2b).

[0093] In some embodiments, the targeting ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 2d).

[0094] In some embodiments, the targeting ligand has a structure represented by the following structural formula: [ka] (Structure 3).

[0095] In some embodiments, the expression-inhibiting oligomeric compound is linked to a targeting ligand and has a structure represented by the following structural formula: [ka] wherein Z consists of or comprises an expression-inhibiting oligomeric compound; A is O or S; and A' is O - , S - , or N.H. - {Structure 3b).

[0096] In some embodiments, the targeting ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 3d).

[0097] In some embodiments, the targeting ligand has a structure represented by the following structural formula: [ka] (Structure 4).

[0098] In some embodiments, the expression-inhibiting oligomeric compound is linked to a targeting ligand and has a structure represented by the following structural formula: [ka] wherein Z consists of or comprises an expression-inhibiting oligomeric compound; A is O or S; and A' is O - , S - , or N.H. - {Structure 4b).

[0099] In some embodiments, the targeting ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 4d).

[0100] In some embodiments, the targeting ligand has a structure represented by the following structural formula: [ka] (Structure 5).

[0101] In some embodiments, the expression-inhibiting oligomeric compound is linked to a targeting ligand and has a structure represented by the following structural formula: [ka] wherein Z consists of or comprises an expression-inhibiting oligomeric compound; A is O or S; and A' is O - , S - , or N.H. - {Structure 5b).

[0102] In some embodiments, the targeting ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 5d).

[0103] In some embodiments, the targeting ligand has a structure represented by the following structural formula: [ka] (Structure 6).

[0104] In some embodiments, the expression-inhibiting oligomeric compound is linked to a targeting ligand and has a structure represented by the following structural formula: [ka] wherein Z consists of or comprises an expression-inhibiting oligomeric compound; A is O or S; and A' is O - , S - , or N.H. - {Structure 6b).

[0105] In some embodiments, the targeting ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 6d).

[0106] In some embodiments, the targeting ligand is in the form of a galactose cluster. As used herein, galactose clustering includes targeting ligands having two to four terminal galactose derivatives. As used herein, the term galactose derivative includes both galactose and galactose derivatives that have affinity for the asialoglycoprotein receptor that is equal to or exceeds that of galactose. A galactose derivative is a saccharide sugar that is a type of targeting moiety. The terminal galactose derivative may be linked to a tether through the C-1 carbon of the saccharide.

[0107] In some embodiments, the targeting ligand comprises three terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for the asialoglycoprotein receptor, hi some embodiments, the targeting ligand comprises three terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting moieties.

[0108] In some embodiments, the targeting ligand is comprised of four terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for the asialoglycoprotein receptor, hi some embodiments, the targeting ligand comprises four terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting moieties.

[0109] In some embodiments, each targeting moiety comprises a galactosamine derivative, i.e., N-acetyl-galactosamine. Other saccharides with affinity for the asialoglycoprotein receptor that can be used as targeting moieties can be selected from the list including: galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, Nn-butanoylgalactosamine, and N-iso-butanoylgalactosamine. The affinity of many galactose derivatives for the asialoglycoprotein receptor has been studied (see, for example, Iobst, ST and Drickamer, KJBC 1996, 271, 6686) or can be easily measured using commonly used methods well known in the art.

[0110] When referring to three terminal N-acetyl-galactosamines, terms commonly used in the art include tri-antennary, trivalent, and trimer.

[0111] Linker The targeting ligands disclosed herein include a linker. The linker is a group of atoms that is linked to a branch point group at one end and to a therapeutic compound at the other end (or to the phosphorus atom of the phosphoramidite by a subphosphorylation reaction using a phosphoramidite-forming reagent when the targeting ligand is synthesized as a phosphoramidite compound). In some embodiments, the linker is linked to a branch point group at one end and to a group or groups that are then attached to the expression-inhibiting oligomeric compound at the other end. In some embodiments, the linker is directly attached to the oligomeric compound. In some embodiments, the linker is linked to a cleavable moiety, which is then linked to the oligomeric compound. Examples of cleavable moieties include, but are not limited to, groups including phosphate groups, disulfide moieties, and / or other internucleoside linkages that can be cleaved. In some embodiments, the linker is not linked to a cleavable moiety. In some embodiments, the linker is linked to a phosphorothioate group or a phosphonate group.

[0112] In some embodiments, the linker consists of or includes a polyethylene glycol ("PEG") moiety. The incorporation of a PEG moiety into the linker confers advantageous properties over certain other linkers, such as linkers consisting of or including substituted or unsubstituted alkyl chains. For example, the incorporation of a PEG moiety into the linker enhances the solubility of the targeting ligand-containing phosphoramidite compound in solvents commonly used in nucleotide synthesis, compared to compounds containing alkyl chain linkers, which can result in a simplified manufacturing process.

[0113] In some embodiments, the targeting ligand comprises a linker having the following structure: [ka] {wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)} (Structure 1001).

[0114] In some embodiments, the targeting ligand comprises a linker attached to a phosphate group having the following structure: [ka] {wherein n is an integer selected from 1 to 20} (Structure 1002).

[0115] In some embodiments, the targeting ligand comprises a linker linked to a phosphorothioate group having the following structure: [ka] {wherein n is an integer selected from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)} (structure 1003).

[0116] In some embodiments, the targeting ligand comprises a linker having the following structure: [ka] (Structure 1004).

[0117] In some embodiments, the targeting ligand comprises a linker attached to a phosphate group having the following structure: [ka] (Structure 1005).

[0118] In some embodiments, the targeting ligand comprises a linker linked to a phosphorothioate group having the following structure: [ka] (Structure 1006).

[0119] In some embodiments, the linker is linked to the expression-inhibiting oligomeric compound, i.e., the double-stranded RNAi agent. In some embodiments, the linker is linked to the 5'-end of the sense strand of the double-stranded RNAi agent. In some embodiments, the linker is linked to the 3'-end of the sense strand of the double-stranded RNAi agent. In some embodiments, the linker is linked to the 3'-end of the antisense strand of the double-stranded RNAi agent. In some embodiments, the linker is linked to the 5'-end of the antisense strand of the double-stranded RNAi agent.

[0120] In some embodiments, the linker is linked to a cleavable moiety. In some embodiments, a terminal phosphate group of an expression-inhibiting oligomeric compound can serve as the cleavable moiety. In some embodiments, an independently selected cleavable moiety is linked to the linker. As used herein, a cleavable moiety is a group that is stable outside a cell but is cleaved upon entry into a target cell. A cleavable moiety is susceptible to cleavage under certain conditions, such as pH, or by a specific cleaving agent, such as a molecule or redox agent that promotes degradation.

[0121] In some embodiments, the cleavable moiety may be pH sensitive. For example, endosomes and lysosomes are known to generally have a more acidic pH (pH of about 4.5 to 6.5) than human blood (pH of about 7.35 to 7.45), which may facilitate cleavage of the cleavable moiety. In some embodiments, the cleavable moiety is a phosphate group, which can be cleaved by agents known to decompose or hydrolyze phosphate groups.

[0122] Branch point group The targeting ligands disclosed herein comprise at least one branch point group. The branch point group of the targeting ligands disclosed herein is attached to a linker. In some embodiments, the branch point group of the targeting ligands disclosed herein is linked to a linker at one end, and the branch point group is linked to one or more tethers at the other end(s). In some embodiments, the branch point group is attached to a linker and one or more tethers. In some embodiments, the branch point group is indirectly attached to the expression-inhibiting oligomeric compound (e.g., via a linker). In some embodiments, the branch point group is linked to the expression-inhibiting oligomeric compound via an additional group(s).

[0123] A branch point group as disclosed herein is any group that allows for the attachment of one or more targeting moieties, which in turn allows for attachment to a linker. The branch point groups disclosed herein are any groups that allow for the attachment of two, three, or four galactose derivatives and further allow for the attachment of the branch point to a linker.

[0124] In some embodiments, the targeting ligand comprises a branch point having the following structure: [ka] (Structure 2001), [ka] (Structure 2002).

[0125] Tether The targeting ligands disclosed herein comprise one or more tethers. The tethers are connected between the branch point group and the respective targeting moieties. In some embodiments, the tether is connected directly to the targeting ligand at one end and directly to the branch point group at the other end. In some embodiments, the tether is connected directly to the targeting ligand at one end and indirectly to the branch point group at the other end. In some embodiments, the tether is connected indirectly to the targeting ligand at one end and indirectly to the branch point group at the other end. In some embodiments, the targeting ligands described herein comprise three tethers and three targeting moieties. In some embodiments, the targeting ligands described herein comprise four tethers and four targeting moieties. In some embodiments, the targeting ligands described herein comprise one tether and one targeting moiety. In some embodiments, the targeting ligands described herein comprise multiple tethers and multiple targeting moieties.

[0126] In some embodiments, an additional tether or other group is inserted between the tether and the targeting moiety. In some embodiments, a second tether is inserted between the tether and the targeting moiety. In some embodiments, a second tether and a third tether are inserted between the tether and the targeting moiety. In some embodiments, a second, third, and fourth tether are inserted between the tether and the targeting moiety. As disclosed herein, there is at least one tether for every targeting moiety. In some embodiments, there are two or more tethers for each targeting moiety. The targeting ligands disclosed herein are intended to encompass such compositions.

[0127] In some embodiments, additional groups can be inserted between the tether and the branch point group. As disclosed herein, the tether serves as a spacer that can add additional flexibility and / or length to the link between the targeting moiety and the branch point group, linker, and therapeutic compound. In some embodiments, the tether includes an alkyl group (including a cycloalkyl group), an alkenyl group (including a cycloalkenyl group), an alkynyl group, an aryl group, an aralkyl group, an aralkenyl group, or an aralkynyl group. In some embodiments, the tether includes one or more heteroatoms, heterocycles, heteroaryls, amino acids, nucleotides, or saccharides.

[0128] In some embodiments, the targeting ligand comprises a tether having the following structure: [ka] wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and X is O, S, or NH (Structure 301).

[0129] In some embodiments, the targeting ligand comprises a tether having the following structure: [ka] wherein X is O, S, or NH - is} (structure 302).

[0130] In some embodiments, the targeting ligand comprises a tether having the following structure: [ka] (Structure 302a).

[0131] In some embodiments, the targeting ligand comprises a tether having the following structure: [ka] wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and X is O, S, or NH (Structure 303).

[0132] In some embodiments, the targeting ligand comprises a tether having the following structure: [ka] wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and X is O, S, or NH (Structure 304).

[0133] In some embodiments, the targeting ligand comprises a tether having the following structure: [ka] {wherein X is O, S, or NH} (structure 305).

[0134] In some embodiments, the targeting ligand comprises a tether having the following structure: [ka] {wherein X is O, S, or NH} (structure 306).

[0135] In some embodiments, the targeting ligand comprises two or more types of tethers. In some embodiments, the tether functions as a flexible hydrophilic spacer (e.g., US 5,885,968; and Biessen et al. J. Med. Chem. 1995, 39, 1538-1546, both of which are incorporated herein by reference in their entireties) and comprises a PEG spacer. In other embodiments, the PEG spacer comprises 1 to 20 ethylene units (PEG1 to PEG2).20 For example, the PEG spacer has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ethylene units.

[0136] Targeting part: The targeting ligands disclosed herein can comprise one to four, or more than four, targeting moieties. In some embodiments, the targeting ligand may be a galactose cluster. As used herein, galactose clustering includes targeting ligands having two to four terminal galactose derivatives. As used herein, the term galactose derivative includes both galactose and galactose derivatives that have affinity for the asialoglycoprotein receptor that is equal to or exceeds that of galactose. A galactose derivative is a saccharide sugar that is a type of targeting moiety. The terminal galactose derivative is linked to a tether through the C-1 carbon of the saccharide.

[0137] In some embodiments, the targeting ligand comprises three terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for the asialoglycoprotein receptor, hi some embodiments, the targeting ligand comprises three terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting moieties.

[0138] For example, each of structures 1, 101, 102, and 103 is a targeting ligand having three terminal N-acetyl-galactosamines as the targeting moiety. In some embodiments, each targeting moiety comprises a galactosamine derivative, i.e., N-acetyl-galactosamine. Other saccharides with affinity for the asialoglycoprotein receptor that can be used as targeting moieties can be selected from the list including: galactose, galactosamine, N-formyl-galactosamine, N-propionyl-galactosamine, Nn-butanoylgalactosamine, and N-iso-butanoylgalactosamine. The affinity of many galactose derivatives for the asialoglycoprotein receptor has been studied (see, e.g., Iobst, ST and Drickamer, KJBC 1996, 271, 6686, which is incorporated herein by reference in its entirety) or can be easily measured using commonly used methods well known in the art.

[0139] In some embodiments, the targeting moiety is a cell-targeting moiety. In some embodiments, the targeting moiety comprises N-acetyl-galactosamine: [ka] / [ka] .

[0140] In some embodiments, the targeting ligand comprises three targeting moieties. In some embodiments, the targeting ligand comprises four targeting moieties. In some embodiments, the targeting ligand comprises one targeting moiety. In some embodiments, the targeting ligand comprises two targeting moieties. In some embodiments, the targeting ligand comprises four or more targeting moieties.

[0141] In some embodiments, the targeting moiety comprises one or more of galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, Nn-butanoylgalactosamine, or N-iso-butanoylgalactosamine.

[0142] For example, in some embodiments, the N-acetyl-galactosamine targeting moiety of any of structures 1-6 can be replaced with an alternative targeting moiety. In some embodiments, the N-acetyl-galactosamine targeting moiety of any of structures 101, 102, or 103 can be replaced with an alternative targeting moiety. Such alternative targeting moieties include, for example, galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, Nn-butanoylgalactosamine, or N-iso-butanoylgalactosamine.

[0143] Additionally, in some embodiments, the targeting moieties of structures 1-6 may be substituted with, for example, other carbohydrates; glycans; haptens; vitamins; folates; biotin; aptamers; and / or peptides, such as, for example, RGD-containing peptides, insulin, EGF, and / or transferrin.

[0144] In some embodiments, the targeting moiety of structure 101, 102, or 103 can be substituted with, for example, other carbohydrates; glycans; haptens; vitamins; folates; biotin; aptamers; and / or peptides, such as, for example, RGD-containing peptides, insulin, EGF, and / or transferrin.

[0145] In some embodiments, the targeting ligand is in the form of an N-acetyl-galactosamine trimer. In some embodiments, the targeting ligand is in the form of an N-acetyl-galactosamine tetramer.

[0146] oligomeric compounds The targeting ligands disclosed herein can be linked to oligomeric compounds. In some embodiments, the oligomeric compounds are expression-inhibiting oligomeric compounds. In some embodiments, the expression-inhibiting oligomeric compounds are RNAi agents. In some embodiments, the expression-inhibiting oligomeric compounds are double-stranded RNAi agents. In some embodiments, the expression-inhibiting oligomeric compounds are single-stranded oligonucleotides. The expression-inhibiting oligomeric compounds can be synthesized using methods commonly used in the art.

[0147] The expression-inhibiting oligomeric compound may contain one or more modified nucleotides. A nucleotide base (or nucleobase) is a heterocyclic pyrimidine or purine compound that is a constituent of all nucleic acids and includes adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). As used herein, the term "nucleotide" may include a modified nucleotide or nucleotide mimic, an abasic site, or a surrogate replacement moiety. As used herein, a "modified nucleotide" is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide), a nucleotide mimic, an abasic site, or a surrogate replacement moiety. In some embodiments, modified nucleotides include 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of the five-membered sugar ring). Modified nucleotides include, but are not limited to, 2'-modified nucleotides, 2'-O-methyl nucleotides (also represented herein as a lowercase "n" in a nucleotide sequence), 2'-deoxy-2'-fluoro nucleotides (represented herein as Nf, also represented herein as 2'-fluoro nucleotides), 2'-deoxy nucleotides (also represented herein as dN), 2'-methoxyethyl (2'-O-2-methoxylethyl) nucleotides (also represented herein as NM or 2'-MOE), 2'-amino nucleotides, 2'-alkyl nucleotides, 3'-3' linked (inverted) nucleotides (also represented herein as invdN, invN, invn, invX), unnatural base containing nucleotides, locked nucleotides, bridged nucleotides, peptide nucleic acids, 2',3'-seconucleotide mimics (unlocked nucleobase analogs, referred to herein as N UNA or NUNA), locked nucleotides (referred to herein as N LNA or NLNA), 3'-O-methoxy (2'-internucleotide linkage) nucleotides (also referred to herein as 3'-OMen), 2'-F-arabinonucleotides (also referred to herein as NfANA or Nf ANAExamples of suitable nucleotides include nucleotides such as nucleotides of the nucleotide sequence (also referred to herein as X, Ab, A, B, C, D, E, E, F, G, G, H ...

[0148] Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines, N-2-, N-6-, and O-6-substituted purines (e.g., 2-aminopropyladenine), 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyluracil, 5-propynylcytosine, These include 6-azo-uracil, 6-azo-cytosine, 6-azo-thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-substituted uracils and cytosines (e.g., 5-halouracil and cytosine (e.g., 5-bromouracil and 5-bromocytosine), 5-trifluoromethyluracil, 5-trifluoromethylcytosine), 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-azaguanine, 3-deazaguanine, and 3-azaguanine.

[0149] In the expression-inhibiting oligomeric compounds described herein, any modified nucleotide may be linked by a phosphate-containing or non-phosphate-containing covalent internucleoside linkage. Modified internucleoside linkages or backbones include, but are not limited to, 5'-phosphorothioates (also represented herein by a lowercase "s" before the nucleotide, also represented as sN, sn, sNf, or sdN), chiral phosphorothioates, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, 3'-alkylene phospho ... Examples of modified internucleoside linkages include phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl-phosphonates, thionoalkyl-phosphotriesters, morpholino linkages, boranophosphonates typically having a 3'-5' linkage, 2'-5'-linked analogs of boranophosphonates, and boranophosphonates with reverse polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 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 hybrid N, O, S, and CH2 moieties.

[0150] In some embodiments, the expression-inhibiting oligomeric compound is a double-stranded RNAi agent, comprising a sense strand and an antisense strand that are at least partially complementary to each other (at least 70% complementary). The antisense strand comprises a region having a sequence that is fully complementary (100% complementary) or at least substantially complementary (at least 85% complementary) to the sequence of the target mRNA. The sense and antisense strands of the double-stranded RNAi agent can each be 16 to 30 nucleotides in length. The sense and antisense strands can be the same length or different lengths. In some embodiments, the sense strand is approximately 19 nucleotides in length, while the antisense strand is approximately 21 nucleotides in length. In some embodiments, the sense strand is approximately 21 nucleotides in length, while the antisense strand is approximately 23 nucleotides in length. In other embodiments, the sense and antisense strands are each independently 17 to 21 nucleotides in length. In some embodiments, both the sense and antisense strands are each 21-26 nucleotides in length. In some embodiments, both the sense and antisense strands are each 26 nucleotides in length. In some embodiments, the sense and antisense strands are each independently 17-26 nucleotides in length. In some embodiments, the double-stranded RNAi agent has a duplex length of about 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. This region of complete or substantial complementarity between the sense and antisense strands is typically 15-25 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length) and occurs at or near the 5' end of the antisense strand.

[0151] Expression-inhibiting oligomeric compounds conjugated to the ligands disclosed herein optionally and independently contain (as extensions) an additional 1, 2, 3, 4, 5, or 6 nucleotides at the 3' end, 5' end, or both the 3' and 5' ends of the core sequence, which, if present, may or may not be complementary to the corresponding sequence in the targeted mRNA.

[0152] In some embodiments, when a double-stranded RNAi agent is bound to a targeting ligand disclosed herein, additional nucleotides in the sense strand, if present, may or may not be identical to the corresponding sequence in the targeted mRNA. Additional nucleotides in the antisense strand, if present, may or may not be complementary to the corresponding additional nucleotides in the sense strand. A double-stranded RNAi agent can be formed by annealing an antisense strand with a sense strand.

[0153] In some embodiments, the targeting ligand is linked to the RNAi agent at the 3' or 5' end of either the sense strand or the antisense strand of the RNAi agent. In some embodiments, the targeting ligand is linked to the 5' end of the sense strand. In some embodiments, the targeting ligand is linked to the 3' end of the sense strand. In some embodiments, the targeting ligand is linked to the RNAi agent via a labile, cleavable, or reversible bond. In some embodiments, the labile, cleavable, or reversible bond is comprised in a cleavable moiety added between the RNAi agent and the targeting ligand.

[0154] In some embodiments, the expression-inhibiting oligomeric compound is a single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide utilizes the RNA interference mechanism to inhibit expression of a target mRNA. In some embodiments, the single-stranded oligonucleotide is active in reducing target nucleic acid expression through a mechanism other than RNA interference.

[0155] In some embodiments, the gene expression level and / or mRNA level of the target in a subject administered the targeting ligand conjugated to an expression-inhibiting oligomeric compound is reduced by at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% compared to the subject before administration or to a subject not receiving the targeting ligand complex. The gene expression level and / or mRNA level of the subject may be reduced in cells, cell groups, and / or tissues of the subject. In some embodiments, the protein level of a subject administered the targeting ligand conjugated to an expression-inhibiting oligomeric compound is reduced by at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, compared to the level of the subject before administration of the targeting ligand complex or to a subject not receiving the targeting ligand complex. The protein level of the subject may be reduced in the subject's cells, cell populations, tissues, blood, and / or other bodily fluids. Reductions in gene expression, mRNA, or protein levels can be assessed by any method known in the art. Reduction or decrease in mRNA and / or protein levels is also considered herein to be inhibiting, reducing, or reducing the expression of the targeted gene.

[0156] Specific expression-inhibiting oligomeric compounds that can be used with the disclosed targeting ligands are known in the art.In particular, numerous references disclose expression-inhibiting oligomeric compounds that can be linked to the targeting ligands disclosed herein for delivery of compositions to the liver.Non-limiting examples include U.S. Patent Application No. 15 / 281,309, entitled "Compositions and Methods for Inhibiting Gene Expression of LPA" (the above document is incorporated herein by reference in its entirety), which discloses various double-stranded expression-inhibiting oligomeric compounds that target the human apolipoprotein (a) gene [LPA] and are suitable for use with the targeting ligands disclosed herein (to inhibit the expression of apo(a) protein, which is a part of lipoprotein (a) particles, thereby lipoprotein (a) particle (Lp(a))).The apo(a) gene [LPA] is primarily expressed in the liver of humans and non-human primates. Similarly, for example, U.S. Patent Application No. 15 / 229,314, entitled "RNAi Therapy for Hepatitis B Virus Infection," which is also incorporated herein by reference in its entirety, discloses various double-stranded expression-inhibiting oligomeric compounds targeting hepatitis B virus, which are suitable for use with the targeting ligands disclosed herein. Hepatitis B virus is a strictly hepatotropic, double-stranded DNA-containing virus, and is classified as a member of the hepadnavirus family, which belongs to the Hepadnaviridae family. Furthermore, as another example, U.S. Patent Application No. 15 / 229,314, entitled "Compositions and Methods for Inhibiting Gene Expression of Factor XII," which is also incorporated herein by reference in its entirety, discloses various double-stranded expression-inhibiting oligomeric compounds targeting the Factor XII (or Factor X2, F12) gene, which are suitable for use with the targeting ligands disclosed herein. Factor XII is a serine protease expressed primarily in the liver and found in the blood.As another example, U.S. Patent Application No. 14 / 740,307, entitled "Compositions and Methods for Inhibiting Gene Expression of Alpha-1 Antitrypsin," (herein incorporated by reference in its entirety), discloses various double-stranded expression-inhibiting oligomeric compounds targeting the alpha-1 antitrypsin (or AAT) gene, suitable for use with the targeting ligands disclosed herein. AAT is a protease inhibitor belonging to the serpin superfamily, and AAT protein is typically synthesized primarily in the liver by hepatocytes and secreted into the blood. Furthermore, WO 2016 / 01123, entitled "Organic Compositions to Treat APOC3-Related Diseases," (herein incorporated by reference in its entirety), discloses various double-stranded expression-inhibiting oligomeric compounds targeting human apolipoprotein III (APOC3), suitable for use with the targeting ligands disclosed herein. Apolipoprotein C-III is a component of lipoprotein that is thought to inhibit the hepatic uptake of triglyceride-rich particles.Further references can also be found in the art, disclosing various therapeutic compounds, including expression-inhibiting oligomeric compounds, that can be suitable for use with the targeting ligand disclosed herein.These include but are not limited to, the composition that is desired to target to the liver.

[0157] Pharmaceutical Compositions and Formulations The targeting ligands disclosed herein, when linked to an oligomeric compound, can be used to treat a subject (e.g., a human or mammal) suffering from a disease or disorder that would benefit from administration of the compound. In some embodiments, the targeting ligands disclosed herein, when linked to an expression-inhibiting oligomeric compound, can be used to treat a subject (e.g., a human) suffering from a disease or disorder that would benefit from reduction or inhibition of target mRNA expression. The subject is administered a therapeutically effective amount of any one or more expression-inhibiting oligomeric compounds, such as RNAi agents, linked to a targeting ligand disclosed herein. The subject can be a human, a patient, or a human patient. The subject can be an adult, adolescent, child, or infant. The aforementioned pharmaceutical compositions comprising a targeting ligand linked to an expression-inhibiting oligomeric compound can be used to provide a method for the therapeutic therapy of a disease. Such a method comprises administering to a human or animal a pharmaceutical composition described herein.

[0158] The pharmaceutical compositions and methods disclosed herein can reduce the level of a target mRNA in a cell, a group of cells, a tissue, or a subject by administering to the subject a therapeutically effective amount of an expression-inhibiting oligomeric compound described herein linked to a targeting ligand, thereby inhibiting expression of the target mRNA in the subject. In some embodiments, the subject has previously been identified as having pathogenic upregulation of a target gene in the target cell or tissue.

[0159] In some embodiments, pharmaceutical compositions comprise at least one expression-inhibiting oligomeric compound linked to a targeting ligand. These pharmaceutical compositions are particularly useful for inhibiting expression of the target mRNA in a target cell, cell population, tissue, or organism. The pharmaceutical compositions are used to treat a subject suffering from a disease or disorder that would benefit from reduced target mRNA levels or inhibited target gene expression. The pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reduced target mRNA levels or inhibited target gene expression. In one embodiment, the method comprises administering to the subject to be treated a composition comprising a targeting ligand described herein linked to an expression-inhibiting oligomeric compound, such as an RNAi agent. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) are added to the pharmaceutical composition comprising a targeting ligand linked to an expression-inhibiting oligomeric compound, thereby forming a pharmaceutical formulation suitable for in vivo delivery to humans.

[0160] In some embodiments, the aforementioned pharmaceutical compositions comprising a targeting ligand linked to an expression-inhibiting oligomeric compound are used to treat or manage a clinical condition associated with expression of the target mRNA. Alternatively, in some embodiments, a therapeutically or prophylactically effective amount of one or more pharmaceutical compositions is administered to a subject in need of such treatment, prevention, or management. In some embodiments, administration of any conjugated ligand covalently linked to an oligomeric compound can be used to reduce the number, severity, and / or frequency of disease symptoms in a subject.

[0161] The aforementioned pharmaceutical compositions comprising a targeting ligand linked to an expression-inhibiting oligomeric compound can be used to treat at least one symptom in a subject suffering from a disease or disorder that would benefit from reducing or inhibiting the expression of a target mRNA. In some embodiments, the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions comprising an expression-inhibiting oligomeric compound, such as an RNAi agent linked to a targeting ligand as described herein, thereby treating the symptom. In other embodiments, the subject is administered a prophylactically effective amount of one or more expression-inhibiting oligomeric compounds, thereby preventing at least one symptom.

[0162] In some embodiments, the expression or level of target mRNA in a subject administered an expression-inhibiting oligomeric compound linked to a targeting ligand disclosed herein is reduced by at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, compared to a subject not receiving the pharmaceutical composition. The gene expression level in the subject can be reduced in the subject's cells, cell populations, and / or tissues. In some embodiments, mRNA levels are reduced. In other embodiments, expressed protein levels are reduced. In some embodiments, the protein level of a subject administered an expression-inhibiting oligomeric compound linked to a targeting ligand disclosed herein is reduced by at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, compared to a subject not receiving the pharmaceutical composition. Reductions in expression, mRNA levels, or protein levels can be assessed by any method known in the art. Reduction or reduction in mRNA levels and / or protein levels is referred to herein as reduction or reduction of target RNA or inhibiting or reducing expression of target mRNA.

[0163] The administration route is the route by which the expression-inhibiting oligomeric compound comes into contact with the body. Generally, methods for administering drugs and nucleic acids for the treatment of mammals are well known in the art and can be applied to the administration of the compositions described herein. The expression-inhibiting oligomeric compound linked to a targeting ligand described herein can be administered via any suitable route with a preparation appropriately adapted for the particular route. Thus, the pharmaceutical compositions described herein can be administered, for example, by intravenous, intramuscular, intradermal, subcutaneous, intraarticular, or intraperitoneal injection. In some embodiments, the pharmaceutical compositions described herein are administered via inhalation.

[0164] Pharmaceutical compositions comprising expression-inhibiting oligomeric compounds linked to targeting ligands described herein can be delivered to cells, cell groups, tumors, 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 regional administration), systemic administration, or by subcutaneous, intravenous, peritoneal, 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 subcutaneously or by intravenous infusion or injection.

[0165] Thus, in some embodiments, the pharmaceutical compositions described herein may include one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions described herein may be formulated for administration to a subject.

[0166] As used herein, a pharmaceutical composition or drug comprises a pharmacologically effective amount of one or more of the aforementioned therapeutic compounds and at least one pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient (excipient) is a substance other than an active pharmaceutical ingredient (API, therapeutic agent, e.g., F12 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 may function to a) aid in the processing of the drug delivery system during manufacturing; b) protect, assist, or enhance the stability, bioavailability, or patient acceptability of the API; c) aid in product identification; and / or d) enhance the overall safety, efficacy, or other attributes of the delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0167] Excipients include, but are not limited to: absorption enhancers, anti-caking agents, anti-foaming agents, antioxidants, binders, buffering agents, carriers, coatings, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, chain extenders, bulking agents, flavorings, glidants, wetting agents, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, water repellents, and wetting agents.

[0168] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where soluble in water) 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). It 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, including, for example, water, ethanol, polyols (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 the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0169] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in suitable solvent with one or combination of the above-listed components as needed, and then sterilize by filtering.Generally, dispersion is prepared by incorporating active compound into the sterile vehicle that contains basic dispersion medium and other components that are required from the above-listed components.For the sterile powder that prepares sterile injectable solution, preparation method comprises vacuum drying and freeze-drying, thereby obtaining the powder of active compound and any other required components from the solution that has been previously sterilized and filtered.

[0170] 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, e.g., in the form of an aqueous suspension of microcrystals. Liposomal preparations or biodegradable polymer systems may also be used to present drugs for both intra-articular and ocular administration.

[0171] Formulations suitable for topical administration, including ophthalmic treatment, include liquid or semi-liquid preparations, such as liniments, lotions, gels, topicals, oil-in-water or water-in-oil emulsions (e.g., creams, ointments, or pastes); or solutions or suspensions, such as eye drops. Formulations for topical administration to the skin surface can be prepared by dispersing the agent in a dermatologically acceptable carrier, such as a lotion, cream, ointment, or soap. Carriers that can form a film or layer over the skin to localize application and prevent removal are useful. For topical administration to the surface of internal tissues, the agent can be dispersed in a liquid tissue adhesive or other substance known to promote adhesion to tissue surfaces. For example, hydroxypropyl cellulose or fibrinogen / thrombin solutions can be used to advantage. Alternatively, tissue-coating solutions, such as pectin-containing formulations, can be used.

[0172] For inhalation therapy, inhalation of powders (self-propelling or spray formulations) dispensed using a spray can, nebulizer, or atomizer can be used. Such formulations can be in the form of fine powders for pulmonary administration from a powder inhalation device or self-propelling powder dispensing formulations. For self-propelling solution and spray formulations, the effect can be achieved by selecting a valve with the desired spray characteristics (i.e., capable of producing a spray with the desired particle size) or by incorporating the active ingredient as a powder suspended to a controlled particle size. For administration by inhalation, the compound can also be delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be via transmucosal or transdermal means. For transmucosal or transdermal administration, wetting agents appropriate for the barrier to be permeated can be used in the formulation. Such wetting agents are generally known in the art and include, for example, detergents and bile salts for transmucosal administration. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are typically formulated into ointments, salves, gels, or creams as generally known in the art.

[0173] The active compound can be prepared with a carrier that protects the compound from rapid elimination from the body, such as sustained-release formulations, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can also 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.

[0174] Oral or parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.As used herein, dosage unit form refers to a physically discrete unit suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.The details of the dosage unit form of the present disclosure are determined and directly depend on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the inherent limitations in the art of compounding such active compounds for individual treatment.In addition, administration can be by periodic injection of a bolus, or can be more continuously administered by intravenous, intramuscular, or intraperitoneal administration from an external reservoir (for example, an intravenous bag).

[0175] In connection with the methods of the present disclosure, pharmacogenomics (i.e., the study of the correlation between an individual's genotype and that individual's response to a foreign compound or drug) can be considered. Differences in the metabolism of therapeutic drugs can lead to severe toxicity or therapeutic failure by altering the relationship between the dose and blood concentration of a pharmacologically active drug. Thus, a physician or clinician can consider applying knowledge gained from relevant pharmacogenomic studies when deciding whether to administer a drug and adjusting the dosage and / or treatment regimen of treatment with the drug.

[0176] Pharmaceutical compositions can contain other additional components that are commonly found in pharmaceutical compositions.Such additional components include, but are not limited to, antipruritics, astringents, local anesthetics, or anti-inflammatory drugs (for example, antihistamines, diphenhydramine, etc.).It is also conceivable that cells, tissues, or isolated organs that express or contain the RNAi agent defined herein can be used as "pharmaceutical compositions".As used herein, "pharmacologically effective amount," "therapeutically effective amount," or simply "effective amount" refers to the amount of RNAi agent that produces pharmacological, therapeutic, or preventive results.

[0177] Generally, an effective amount of active compound is in the range of about 0.1 to about 100 mg / kg body weight / day, e.g., about 1.0 to about 50 mg / kg body weight / day. In some embodiments, an effective amount of active compound is in the range of about 0.25 to about 5 mg / kg body weight per dose. In some embodiments, an effective amount of active ingredient is in the range of about 0.5 to about 3 mg / kg body weight per dose. The amount administered may also depend on variables such as the patient's overall health, the relative biological efficacy of the compound being delivered, the drug formulation, the presence and type of excipients in the formulation, and the route of administration. It should also be understood that the initial dose administered may be increased beyond a high level to rapidly achieve the desired blood or tissue concentration, or the initial dose may be less than optimal.

[0178] For the treatment of a disease or for forming a medicament or composition for the treatment of a disease, the pharmaceutical compositions described herein, comprising an expression-inhibiting oligomeric compound, such as an RNAi agent, linked to a targeting ligand, may be combined with an excipient or a second therapeutic agent or treatment, including, but not limited to: a second or other expression-inhibiting oligomeric compound, a small molecule drug, an antibody, an antibody fragment, and / or a vaccine.

[0179] The aforementioned targeting ligands, when linked to expression-inhibiting oligomeric compounds and 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 or vials. The embodiments provided above will now be illustrated with the following non-limiting examples. [Example]

[0180] The following examples are intended to illustrate, but not limit, the specific embodiments disclosed herein. Some of the abbreviations used in the experimental details for the synthesis of the following examples are defined below: h or hr = hour; min = minute; mol = mole; mmol = millimole; M = mole; μM = micromole; g = gram; μg = microgram; rt or RT = room temperature; L = liter; mL = milliliter; wt = weight; Et2O = diethyl ether; THF = tetrahydrofuran; DMSO = dimethyl sulfoxide; EtOAc = ethyl acetate; Et3N or TEa = triethylamine; i-Pr2NEt, DIPEA or DIEA = diisopropylethylamine; CH2Cl2 or DCM = methylene chloride; CHCl3 = chloroform; CDCl3 = deuterated chloroform; CCl4 = carbon tetrachloride; MeOH = methanol; EtOH = ethanol; DMF = dimethylformamide; BOC = t-butoxycarbonyl; CBZ = benzyloxycarbonyl ;TBS = t-butyldimethylsilyl; TBSCl = t-butyldimethylsilyl chloride; TFA = trifluoroacetic acid; DMAP = 4-dimethylaminopyridine; NaN3 = sodium azide; Na2SO4 = sodium sulfate; NaHCO3 = sodium bicarbonate; NaOH = sodium hydroxide; MgSO4 = magnesium sulfate; K2CO3 = potassium carbonate; KOH = potassium hydroxide; NH4OH = ammonium hydroxide; NHCl = ammonium chloride; SiO2 = silica; Pd-C = palladium on carbon; HCl = hydrogen chloride or hydrochloric acid; NMM = N-methylmorpholine; H2 = hydrogen gas; KF = potassium fluoride; EDC-HCl = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; MTBE = methyl tert-butyl ether; MeOH = methanol; Ar = argon; SiO2 = silica; RT = retention time.

[0181] Additionally, representative expression-inhibiting oligomeric compounds suitable for use with the targeting ligands disclosed herein are set forth in various tables within the Examples below. The following notation is used to indicate modified nucleotides with respect to the sequences set forth in the tables disclosed herein:

[0182] N = 2'-OH (unmodified) ribonucleotide (uppercase without f or d designation) n = 2'-OMe modified nucleotide nf = 2'-fluoro-modified nucleotide dN = 2'-deoxynucleotide N UNA = 2',3'-seconucleotide mimics (unlocked nucleobase analogues) N LNA = locked nucleotide Nf ANA = 2'-F-arabinonucleotide NM = 2'-methoxyethyl nucleotide X or Ab = abasic ribose R = Ribitol (invdN) = inverted deoxyribonucleotide (3'-3' linked nucleotide) (invAb) = inverted abasic nucleotide (invX) = inverted abasic nucleotide (invn) = inverted 2'-OMe nucleotide s = phosphorothioate-linked nucleotide vpdN = vinylphosphonate deoxyribonucleotide (3'OMen) = 3'-OMe nucleotide (5Me-Nf) = 5'-Me,2'-fluoronucleotide cPrp = cyclopropylphosphonate The compounds of the present disclosure can be made using synthetic chemistry techniques known to those skilled in the art.

[0183] Example 1. Synthesis of targeting ligand, phosphoramidite compound, structure 101b 1) Preparation of tri-tert-butyl N-[N-(benzyloxycarbonyl)-L-γ-glutamyl]-L-glutamate (3) [ka]

[0184] To a nitrogen-flushed, 250 mL, three-neck, flat-bottom flask equipped with a thermocouple, a magnetic stir bar, a nitrogen inlet, and a powder funnel was added 1 (10.00 g, 29.64 mmol), followed by THF (100 mL). The resulting solution was stirred, and N-methylmorpholine (7.82 mL, 71.15 mmol) was added.

[0185] The powder funnel was replaced with a rubber septum, and the mixture was cooled to 0°C using an ice bath. Isobutyl chloroformate (iBuCOCl, 3.85 mL, 29.64 mmol, 1.0 equiv.) was added dropwise to the reaction mixture over 10 min, maintaining the pot temperature below 4.0°C. Following the addition, the mixture was stirred for an additional 40 min, and the septum was replaced with a powder funnel. 2 (8.767 g, 29.64 mmol, 1.0 equiv.) was added portionwise to the reaction mixture over 15 min, maintaining the pot temperature below 4.0°C. Following the addition of 2, the ice bath and powder funnel were removed, and the reaction was allowed to warm to ambient temperature during the remaining steps. Following the addition of 2, the clear, colorless solution was aged for 25 min.

[0186] A sample of the reaction (98 μL diluted in 5.0 mL of ACN in a 5 mL volumetric flask) was taken 40 min after the start of the addition of 2 and analyzed for percent conversion by RP-HPLC. 23% of 1 was found to remain, so after 60 min of reaction, additional iBuCOCl (1.16 mL, 30 mol%) and 2 (2.63 g, 30 mol%) were added sequentially. The solution was aged for an additional 60 min until a sample showed >99% conversion by HPLC. The total reaction time was 2.5 h from the start of the first addition of 2.

[0187] The reaction mixture was cooled to 3°C in an ice bath and diluted with 0.5M HCl. (aq)The quenched reaction mixture was poured into a stirred solution of 1,000 ml of ethanol and stirred for approximately 5 minutes. The quenched reaction mixture was transferred to a 500 ml separatory funnel, and ethyl acetate (100 ml) was added. The layers were separated, and the organic phase was washed with brine (100 ml), dried over MgSO, filtered into a 500 ml flat-bottom flask, and concentrated in vacuo to again give a viscous, colorless oil. The oil was dissolved in MTBE (100 ml) and concentrated in vacuo to again give a viscous, colorless oil.

[0188] To the stirred oil was added hexane (100 mL). A white haze appeared in the solution, which then disappeared with further stirring. Seed crystals were added and the mixture was stirred for 40 minutes, during which time white crystals slowly formed.

[0189] Within 20 minutes, the slurry was thick enough to interfere with stirring, and additional hexane (50 mL) was added. After 40 minutes, the slurry was filtered through a coarse fritted funnel, washed three times with hexane (~10 mL each), and air-dried in the funnel for 1 hour to give 3 as a fine white powder (15.64 g, 91%). 1 The H NMR is shown in Figure 1. At the 75 gram scale, the yield was 917% with 99% purity.

[0190] 2) Preparation of N-[N-(benzyloxycarbonyl)-L-γ-glutamyl]-L-glutamic acid (4) [ka]

[0191] To a 3000 mL, three-neck, flat-bottom flask equipped with an overhead stirrer, powder funnel, thermocouple, and heating mantle was added 3 (72.57 g, 125.4 mmol) and formic acid (reagent grade, >95%, 1.45 L, 20 volume equivalents). The powder funnel was replaced with a stopper / N2, and the resulting solution was heated to 45 °C and stirred for 1 h while monitored by RP-HPLC. The reaction was considered complete when less than 2.0 area % of the mono-t-butyl ester remained.

[0192] A sample of the reaction (50 μL diluted in 950 μL of HO) was taken 60 minutes after the addition of formic acid and analyzed by RP-HPLC for the percent mono-t-butyl ester remaining. The analysis showed that 1.8% of the mono-t-Bu ester remained; therefore, at 90 minutes, the heat was removed.

[0193] The reaction was diluted with toluene and acetonitrile (ACN, 1500 mL each), and the mixture was concentrated in vacuo. Formic acid was azeotropically removed with 1:1 ACN:toluene (~600 mL) and twice with ACN (~500 mL each). The material was dried overnight under high vacuum to give compound 4 (54.3 g, quantitative yield) as a white foamy solid. Compound 4 (L / N 1321-063B) 1 The H NMR is shown in Figure 2.

[0194] 3) Preparation of N-[N-(benzyloxycarbonyl)-L-γ-glutamyl]-L-glutamic acid, tri-[NAG-PEG2]-amide (6) [ka]

[0195] A 1-liter flat-bottom flask was charged with NAG-amine p-tosylate salt (5, 59.19 g, 97.6 mmol, 4.13 equiv.) and Z-bis-Glu triacid salt (4, 10.01 g, 23.6 mmol, 1.0 equiv.). The mixture was dissolved in acetonitrile (500 mL) and concentrated in vacuo to azeotropically remove water. The residue was dissolved in fresh acetonitrile (400 mL) and transferred to a nitrogen-flushed, 1-liter, three-neck flat-bottom flask equipped with a stir bar and a thermocouple. The water content was measured by KF (257 ppm).

[0196] To the stirred solution under nitrogen, TBTU (28.20 g, 87.8 mmol, 3.7 equiv) was added via powder funnel. The remaining TBTU on the funnel was rinsed from the reaction using additional acetonitrile (100 mL). DIPEA (34.0 mL, 25.2 g, 8.0 equiv) was added dropwise via syringe over 20 minutes, maintaining the reaction temperature below 25°C. The mixture was stirred for 2 hours from the start of the DIPEA addition while being monitored by HPLC. Analysis at 78 minutes showed complete consumption of the starting material.

[0197] After 2 hours, the solvent was removed in vacuo and the resulting viscous oil was dissolved in dichloromethane (1000 mL) and 1.0 N HCl (aq) (3 × 500 mL) and washed with NaHCO 3(aq) (3 x 500 mL) The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give an off-white waxy solid (33.5 g).

[0198] Flash column chromatography was performed on an ISCO CombiFlash automated purification system using chloroform and methanol as eluents. All fractions that were suspected to contain the product based on the UV chromatogram (220 nm) were analyzed by HPLC, and all fractions containing at least 97.0% AUC of the product were pooled and concentrated to give 18.75 g (97.0% purity) of 6. Less pure fractions were pooled to give an additional 12.2 g (78.8% purity) of 6. The overall yield of 6 was 70.9%. 1 The H NMR is shown in Figure 3.

[0199] 4) Preparation of tri-NAG-bis-Glu-NH2 tosylate salt (7) [ka]

[0200] Compound 6 (5.737 g, 3.46 mmol) in MeOH (155 mL) with p-TsOH-HO (0.657 g, 3.46 mmol) was hydrogenated in the presence of Pd / C 10% (688 mg) for 6 hours. TLC (CHCl:MeOH = 8.5:1.5) confirmed that the reaction was complete by then. The reaction flask was filled with Ar, EtOH was added (200 mL), and the solution was filtered through a celite cake. The product was concentrated and dried in vacuo. Yield 4.81 g of the product, tosylate salt 7. Compound 7 1 The H NMR is shown in Figure 4.

[0201] 5) Preparation of tri-NAG-bis-Glu-NH-PEG-OH (9): [ka]

[0202] Procedure A (when tri-NAG amine salt 7 is less than 96% pure): NAG amine salt 7 (∼90% pure, 18.50 g, 10.90 mmol) and HO-PEG6-CO2TFP ester 8 (6.57 g, 13.08 mmol) were dissolved in dichloromethane (185 mL) and cooled to 0 °C. To this solution was added triethylamine (6.10 mL, 43.59 mmol). The solution was warmed to room temperature and stirred for 18 h with HPLC monitoring. The reaction was quenched with saturated aqueous NaHCO3 and brine (1:1, 140 mL), stirred at RT for 30 min, and the layers were separated. The organic layer was washed with saturated aqueous NaHCO3 (3 × 140 mL) and brine (1:1) and dried over Na2SO4. The drying agent was filtered off and the solution was concentrated and purified by flash chromatography to give 9 (13.56 g, 67%) as a white solid. 1 The H NMR is shown in Figure 5.

[0203] Flash column chromatography was performed on an ISCO CombiFlash automated purification system using dichloromethane and methanol as eluents. Pure fractions were pooled and concentrated to give 13.56 g of 9 (99% purity). Less pure fractions were pooled and concentrated to give 13.56 g of 9 (99% purity). 4.9 g of 9 (~95% purity) was obtained.

[0204] Procedure B (when tri-NAG amine salt 7 is >96% pure): Product 7 (1.94 g, 1.272 mmol) in DCM (40 mL) was stirred with HO-PEG-COTFP ester 8 (767 mg, 1.526 mmol) and DIPEA (443 μL, 2.544 mmol) under Ar for 16 h. The reaction mixture was concentrated in vacuo, dissolved in CHCl, and added dropwise to stirring EtO (90 mL). The precipitate was separated, rinsed with EtO (3 × 35 mL), and dried in vacuo. Yield 2.275 g (96%).

[0205] 6) Preparation of tri-NAG-bis-Glu-NH-PEG6 phosphoramidite (10): Compound 9 (6.62 g, 3.56 mmol) and 4,5-dicyanoimidazole (0.11 g, 0.89 mmol) were dissolved in anhydrous dichloromethane (230 mL) and placed under a nitrogen atmosphere. To this mixture was added a solution of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite ("Phos Reagent", 1.46 mL, 4.62 mmol) in anhydrous dichloromethane (5 mL) dropwise over 5 min. The reaction mixture was stirred at room temperature for 3 h with HPLC monitoring (<1% SM remaining).

[0206] The reaction mixture was washed with saturated aqueous NaHCO3 (2 x 150 mL), 3% DMF in HO (v / v, 2 x 150 mL), HO (3 x 150 mL), and brine (1 x 150 mL), and the organic layer was dried over Na2SO4. The drying agent was filtered off, and the solution was concentrated in vacuo to give the crude product. The crude product was suspended in 5% toluene-hexane (50 mL) and stirred for 5 minutes, after which the solvent was decanted. The process was repeated with 5% toluene-hexane (1 x 50 mL) and hexane (2 x 50 mL). The solid was dried in vacuo to give 6.69 g (91%) of 10 as a white solid (compound 10). Compound 10 (structure 101d herein) 1 The H NMR is shown in Figure 6.

[0207] Example 2. Synthesis of targeting ligand, phosphoramidite compound, structure 103d 1) Preparation of tri-NAG-bis-Glu-NH-PEG-OH (12): [ka]

[0208] The product 7 (2.44 g, 1.44 mmol) from Example 1 above was dissolved in DCM (30 mL) and placed under an argon atmosphere. To the solution was added HO-PEG-COTFP ester 11 (717 mg, 1.73 mmol) and DIPEA (502 μL, 2.88 mmol). The resulting mixture was stirred for 16 h. The reaction mixture was concentrated in vacuo and redissolved in CHCl. The solution was then added dropwise to stirring EtO (90 mL). The precipitate was separated, rinsed with EtO, and dried in vacuo to give 2.60 g (102%) of product 12, which was used without further purification.

[0209] 2) Preparation of tri-NAG-bis-Glu-NH-PEG4 phosphoramidite (13): The product 12 (1.80 g, 1.01 mmol) was co-evaporated twice with pyridine and then dissolved in anhydrous dichloromethane (25 mL) and placed under an argon atmosphere. To the solution were added diisopropylammonium tetrazolide (87 mg, 0.51 mmol) and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (458 mg, 1.52 mmol). The reaction mixture was stirred at room temperature for 5 h while being monitored by TLC (CHCl3:MeOH:Et3N 95:5:2). When all the starting material was consumed, the reaction mixture was diluted with DCM (250 mL) and washed with saturated aqueous NaHCO3 (100 mL) and saturated aqueous brine (100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated. The crude material was purified by column chromatography (DCM:MeOH:EtN 97:3:2) to give 1.04 g (53%) of compound 13. Compound 13 (structure 103d herein) 1 The H NMR is shown in Figure 7.

[0210] Example 3. Synthesis of targeting ligand, phosphoramidite compound, structure 102d 1) Preparation of tri-NAG-bis-Glu-NH-PEG-OH (15): [ka]

[0211] Product 7 (3.09 g, 1.82 mmol) from Example 1 above was dissolved in DCM (30 mL) and placed under an argon atmosphere. To the solution was added HO-PEG-COTFP ester 14 (1.29 g, 2.18 mmol) and DIPEA (634 μL, 3.64 mmol). The resulting mixture was stirred for 16 h. The reaction mixture was concentrated in vacuo and redissolved in CHCl. The solution was then added dropwise to stirring EtO (180 mL). The precipitate was separated, rinsed with EtO, and dried in vacuo to give 3.54 g (99%) of product 15, which was used without further purification.

[0212] 2) Preparation of tri-NAG-bis-Glu-NH-PEG phosphoramidite (16): Product 15 (1.79 g, 0.92 mmol) was co-evaporated twice with pyridine and then dissolved in anhydrous dichloromethane (25 mL) and placed under an argon atmosphere. To the solution were added diisopropylammonium tetrazolide (79 mg, 0.46 mmol) and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (416 mg, 1.38 mmol). The reaction mixture was stirred at room temperature for 3 h while monitored by TLC (CHCl3:MeOH:Et3N 95:5:2). When all the starting material was consumed, the reaction mixture was concentrated in vacuo and redissolved in DCM. The solution was then added dropwise to stirring Et2O (90 mL). The precipitate was separated, rinsed with Et2O, and dried. The crude material was purified by column chromatography (CHCl:MeOH:EtN 97:3:2) to give 950 mg (48%) of compound 16. Compound 16 (structure 102d herein) 1 The H NMR is shown in Figure 8.

[0213] Example 4. Oligonucleotide composition synthesis A.Synthesis RNAi agents were synthesized using the solid-phase phosphoramidite technique used in oligonucleotide synthesis. Depending on the scale, either MerMade96E (Bioautomation) or MerMade2 (Bioautomation) was used. Synthesis was performed on a solid support consisting of controlled pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston, PA, USA). All RNAs, 2'-modified RNAs, and UNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the following 2'-O-methyl phosphoramidites were used: (5'-O-dimethoxytrityl-N6-(benzyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5'-O-dimethoxy-trityl-N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, (5'-O-dimethoxytrityl-N2-(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. (amino) phosphoramidites. Phosphoramidites containing targeting ligands were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidites were dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3 Å) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as the activator solution. Coupling times were 10 min (RNA), 15 min (targeting ligand), 90 s (2'OMe), and 60 s (2'F). To introduce phosphorothioate bonds, a 100 mM solution of 3-phenyl-1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used.

[0214] 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 2 h at 30° C. The solution was evaporated, and the solid residue was reconstituted in water (see below).

[0215] C. Purification The crude oligomer was purified by anion-exchange HPLC using a TKSgel SuperQ-5PW1 3u 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 but with the addition of 1.5 M sodium chloride. The UV trace at 260 nm was recorded. Appropriate fractions were pooled and then run on a size-exclusion HPLC column using a GE Healthcare XK16 / 40 column packed with Sephadex G-25 media, with a running buffer consisting of 100 mM ammonium bicarbonate, pH 6.7, and 20% acetonitrile.

[0216] D. Annealing Complementary strands were mixed to form RNAi agents by combining equimolar RNA solutions (sense and antisense) in 0.2x PBS (phosphate-buffered saline, 1x, Corning, Cellgro). This solution was placed in a 70°C thermomixer, heated to 95°C, held at 95°C for 5 minutes, and slowly cooled to room temperature. Some RNAi agents were lyophilized and stored at -15 to -25°C. The duplex concentration was determined by measuring the absorbance of the solution in 0.2x PBS with a UV-visible spectrometer. The duplex concentration was then determined by multiplying the solution's absorbance at 260 nm by the conversion factor and dilution factor. Unless otherwise noted, all conversion factors were 0.037 mg / (mL·cm). In some experiments, the conversion factor was calculated from experimentally determined extinction coefficients.

[0217] Example 5. Properties of phosphoramidite-containing compounds containing targeting ligands with PEG linkers of different lengths The following targeting ligand phosphoramidite compounds were synthesized according to the methods previously disclosed in Examples 1-4: [ka] (Structure 101d); [ka] (Structure 102d); [ka] (Structure 103d).

[0218] Each of the phosphoramidite compounds of structures 101d, 102d, and 103d carries 16 equivalents for attachment to the 5' end of the single-stranded oligonucleotide AM03704-SS, which is the sense strand that can be used to synthesize a double-stranded RNAi agent targeting F12. AM03704 has the nucleotide sequence shown in the following table:

[0219] Table 1. Sense strand sequences for Example 5 [Table 1]

[0220] The composition was solubilized in dichloromethane (DCM) and dried over sieves. The phosphoramidite compound of structure 103d (i.e., with a PEG-4 linker) exhibited gelation problems at both 0.05 M and 0.25 M. As shown in Figure 9, under these conditions, only a very small amount of targeting ligand structure 103d can be attached to the 5' end of oligonucleotide AM03704-SS.

[0221] Both structures 101d and 102d showed the binding of a targeting ligand to the oligonucleotide. Figure 9 shows the HPLC chromatogram of AM03704 bound to structure 101d. For the targeting ligand of structure 101, approximately 78% of the targeting ligand-complexed oligonucleotide (FLP = full-length product) was formed. Figure 10 shows the HPLC chromatogram of AM03704 bound to structure 102d. Approximately 40% of the targeting ligand-complexed oligonucleotide was formed, while approximately 60% of the oligonucleotide remained unbound.

[0222] Surprisingly and unexpectedly, at 16 equivalents, structure 101d substantially outperforms both structure 102d and structure 103d for conjugation to oligonucleotides at the 5' end of the sequence. Furthermore, both structures 101d and 102d exhibited superior solubility compared to structure 103d. As previously noted, structure 103d was difficult to dissolve using standard concentrations and solvent conditions typical for oligonucleotide synthesis. Preparation of a targeting ligand linked to an expression-inhibiting oligomeric compound having a targeting ligand of structure 103 (by using the phosphoramidite compound of structure 103d) required the addition of a more aggressive polar solvent.

[0223] Example 6. Comparison of 3' and 5' Sense Strand Attachment Sites of GalNAc-Targeted Ligands Using F12 Expression Inhibition Oligomeric Compounds in Wild-Type Mice To assess the difference in the attachment site of the GalNAc ligand between the 3' and 5' ends of the sense strand, expression-inhibitory oligomeric compounds (double-stranded RNAi agents) directed against F12 (also referred to herein as F12 RNAi agents) were prepared having the sequences shown in Table 2 below:

[0224] Table 2. F12 Expression Inhibitory Oligomeric Compounds (RNAi Agent Duplexes) of Example 6 [Table 2]

[0225] In Table 2 above, the following notation is used: (NAG15)= [ka] (NAG18)= [ka]

[0226] (NAG18) has the chemical structure represented herein by Structure 2. Each strand of the F12 RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strands were mixed to form a duplex by combining equimolar RNA solutions (sense and antisense) in 0.2x PBS (phosphate-buffered saline, 1x, Corning, Cellgro) according to the method generally described in Example 4 herein.

[0227] The F12 RNAi agent linked to its respective GalNAc ligand (i.e., (NAG15) or (NAG18)) was combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection.

[0228] The F12 RNAi agents linked to each GalNAc ligand were delivered by SC injection. On day 1, 200 μl of solution per 20 g mouse containing either saline or one of the two F12 RNAi agents (AD02803 or AD02807) at a dose of 3 mg / kg (mpk) in buffered saline was injected SC into the loose skin on the back between the shoulders. Three (3) wild-type mice were present per treatment group. As previously noted, AD02803 contains (NAG15) attached to the 3' end of the sense strand, whereas AD2807 contains (NAG18) attached to the 5' end of the sense strand.

[0229] To monitor knockdown, serum samples were collected from treated mice on days 8, 15, 22, and 29. Knockdown was measured by quantifying circulating mouse F12 protein (mF12) levels in serum using an endogenously expressed mF12 alphaLISA® (Perkin Elmer). Expression on a particular bleed date was normalized to the mean of the saline control group on that same day.

[0230] Figure 12 shows the results of this study. At the lowest point (day 22), AD02803 showed a reduction of approximately 70% in circulating F12 levels, whereas AD02807 showed a reduction of more than 80%. The data also show differences in the duration of the knockdown effect, as AD02803-treated mice showed a faster return to baseline compared to AD2807-treated mice at day 29. These data confirm that linking the GalNAc ligand at the 5' end of the sense strand is superior to linking at the 3' sense strand.

[0231] Example 7. Further comparison of 3' and 5' sense strand attachment sites of GalNAc-targeting ligands using F12 expression-inhibiting oligomeric compounds in wild-type mice To further evaluate the attachment sites of GalNAc ligands at the 3' and 5' ends of the sense strand of double-stranded expression-inhibiting oligomeric compounds (double-stranded RNAi agents), compositions directed against F12 were prepared having the sequences shown in Table 3 below:

[0232] Table 3. F12 Expression Inhibitory Oligomeric Compounds (RNAi Agent Duplexes) of Example 7 [Table 3]

[0233] In Table 3 above, the following notation is used: (NAG20)= [ka]

[0234] (NAG20) has the chemical structure represented herein by Structure 4. Each strand of the F12 RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strands were mixed to form a duplex by combining equimolar RNA solutions (sense and antisense) in 0.2x PBS (phosphate-buffered saline, 1x, Corning, Cellgro) according to the method generally described in Example 4 herein.

[0235] The F12 RNAi agent linked to each GalNAc ligand (i.e., (NAG20)) was combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection.

[0236] The F12 RNAi agents linked to each GalNAc ligand were delivered by SC injection. On day 1, 200 μl of solution per 20 g mouse containing either saline or a 3 mg / kg (mpk) dose of one of the two F12 RNAi agents (AD02815 or AD02816) in buffered saline was injected SC into the loose skin on the back between the shoulders. Three (3) wild-type mice were present per treatment group. As previously shown in Table 3, AD2815 contains (NAG20) attached to the 5' end of the sense strand, whereas AD02816 contains (NAG20) attached to the 3' end of the sense strand.

[0237] To monitor knockdown, serum samples were collected from treated mice on days 8, 15, 22, and 29. Knockdown was measured by quantifying circulating mouse F12 protein (mF12) levels in serum using an endogenously expressed mF12 alphaLISA® (Perkin Elmer). Expression on a particular bleed date was normalized to the mean of the saline control group on that same day.

[0238] Figure 13 shows the results of this experiment. At the lowest point (day 22), AD02816 showed approximately a 60% reduction in circulating F12 protein levels, compared with a 79% reduction for AD02815. At day 29, AD02815-treated mice showed a 71% knockdown from saline levels, compared with a 40% knockdown for AD02816-treated mice. These data support the attachment of a GalNAc ligand at the 5' end of the sense strand.

[0239] Example 8. Inhibition of Lp(a) Expression in Lp(a) Transgenic (Tg) Mice. Oligomeric Compounds (Double-Stranded RNAi Agents) Linked to Targeting Ligands of Structure 101 Lp(a) expression inhibitory oligomeric compounds (double-stranded Lp(a) RNAi agents) were prepared having the sequences shown in Table 5 below:

[0240] Table 4. LP(a) Expression Inhibitory Oligomeric Compounds (Double-Stranded RNAi Agents) of Example 8 [Table 4]

[0241] In Table 4 above, the following notation is used: (NAG25)= [ka] (NAG29)= [ka]

[0242] (NAG25) has the chemical structure represented herein by structure 101. Each strand of the Lp(a) RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strands were mixed to form a duplex by combining equimolar RNA solutions (sense and antisense) in 0.2x PBS (phosphate-buffered saline, 1x, Corning, Cellgro) according to the method generally described in Example 4 herein.

[0243] Lp(a) transgenic (Tg) mice (Frazer KA et al. 1995, Nature Genetics 9:424-431) were used to evaluate the efficacy of double-stranded RNAi agents with conjugated N-acetylgalactosamine ligands in vivo. These mice express human apo(a) and human apoB-100 from a YAC containing the complete LPA gene (encoding the apo(a) protein) with additional 5' and 3' sequences, thereby producing humanized Lp(a) particles (hereafter referred to as "Lp(a) Tg mice") (Callow MJ et al. 1994, PNAS 91:2130-2134).

[0244] The Lp(a) RNAi agent linked to the respective GalNAc ligand (i.e., (NAG25) or (NAG29)) was combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection.

[0245] Lp(a) RNAi agents linked to the respective GalNAc ligand (i.e., (NAG25) or (NAG29)) at the 5' end of the sense strand were delivered by SC injection. On day 1, SC injections of 200 μl solution / 20 g mouse containing either saline or a 1 mg / kg (mpk) dose of one of the respective Lp(a) RNAi agents (AD03547 or AD03549) in buffered saline were administered into the loose skin between the shoulders on the back. There were four (4) Lp(a) Tg mice per treatment group.

[0246] Serum samples from treated mice were collected on days -1 (pre-dose), 5, 11, 16, 22, 29, and 36. Knockdown was measured by calculating circulating Lp(a) particle levels in serum. Lp(a) particle levels were measured using a Cobas® Integra 400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, the Lp(a) level for each animal at a given time point was divided by that animal's pre-dose expression level (in this case, on day -1) to determine the "normalized to day -1" expression ratio. Expression at a particular time point was then normalized to the saline control group by dividing the "normalized to day -1" ratio for each individual animal by the average "normalized to day -1" ratio for all mice in the saline control group. This resulted in expression for each time point normalized to expression in the control group. Experimental error is shown as the standard deviation.

[0247] The results are shown in Figure 14. AD03549 (NAG25) showed 71% knockdown at its lowest point (day 16), and AD03547 (NAG29) showed 81% knockdown at its lowest point (day 11). Both triggers showed similar recovery curves after the nadir, with less than 26% knockdown at day 36. These data confirm that the indicated GalNAc ligands share similarities in both initial knockdown activity and durability of knockdown in Lp(a)Tg mice using a single 1 mg / kg dose.

[0248] Example 9. Lp(a) knockdown in Lp(a) transgenic (Tg) mice following administration of an Lp(a) expression-inhibiting oligomeric compound (double-stranded RNAi agent) linked to a targeting ligand of structure 101 Lp(a) expression inhibitory oligomeric compounds (double-stranded Lp(a) RNAi agents) were prepared having the sequences shown in Table 5 below:

[0249] Table 5. LP(a) Expression Inhibitory Oligomeric Compounds (Double-Stranded RNAi Agents) of Example 9 [Table 5]

[0250] In Table 5, (NAG25) is the same structure as shown above in Example 8, and has the chemical structure represented in Structure 101 herein. Each strand of the Lp(a) RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strands were mixed to form a duplex by combining equimolar RNA solutions (sense and antisense) in 0.2x PBS (phosphate-buffered saline, 1x, Corning, Cellgro) according to the method generally described in Example 4 herein.

[0251] Lp(a)Tg mice were used to evaluate the efficacy of double-stranded RNAi agents with conjugated N-acetyl-galactosamine ligands in vivo. The Lp(a) RNAi agent linked to the targeting ligand structure 101 was combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection. Lp(a) RNAi agents linked to a targeting ligand at the 5' end of the sense strand were delivered by SC injection. On day 1, SC injections were administered into the loose skin between the shoulders on the back of a 20g mouse containing a 1mg / kg (mpk) dose of either saline or the RNAi agent AD03272 in buffered saline. Four (4) Lp(a) Tg mice were administered per treatment group.

[0252] Serum samples from treated mice were collected on days -1 (pre-dose), 8, 15, 22, 29, 36, and 43. Knockdown was measured by calculating circulating Lp(a) particle levels in serum. Lp(a) particle levels were measured using a Cobas® Integra 400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, the Lp(a) level for each animal at a given time point was divided by that animal's pre-dose expression level (in this case, on day -1) to determine the "normalized to day -1" expression ratio. Expression at a particular time point was then normalized to the saline control group by dividing the "normalized to day -1" ratio for each individual animal by the average "normalized to day -1" ratio for all mice in the saline control group. This resulted in expression for each time point normalized to expression in the control group. Experimental error is shown as the standard deviation.

[0253] The results are shown in Figure 15. AD03272 showed 88% knockdown at its lowest (day 15) and remained at 75% knockdown at day 29. These data confirm that the targeting ligand of structure 1008 can target LPA-targeting RNAi agents to the liver and can obtain >85% knockdown in transgenic mice using a single 1 mg / kg dose.

[0254] Example 10. Apolipoprotein(a) (apo(a)) knockdown in apo(a) transgenic (Tg) mice following administration of Lp(a) expression-inhibiting oligomeric compounds (double-stranded RNAi agents) linked to targeting ligands of structures 101, 102, and 103. Lp(a) expression inhibitory oligomeric compounds (double-stranded Lp(a) RNAi agents) were prepared having the sequences shown in Table 4 below:

[0255] Table 6. LP(a) Expression Inhibitory Oligomeric Compounds (Double-Stranded RNAi Agents) of Example 10 [Table 6]

[0256] In Table 6 above, the following notation is used: (NAG26)= [ka] ;(NAG27)= [ka]

[0257] Additionally, (NAG25) is the same structure as previously shown in Example 8 and has the chemical structure represented herein as Structure 101. (NAG26) has the chemical structure represented herein as Structure 102. (NAG27) has the chemical structure represented herein as Structure 103. As previously shown in Table 8, the compositions are identical except for the different targeting ligands selected.

[0258] Each strand of the Lp(a) RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strands were mixed to form a duplex by combining equimolar RNA solutions (sense and antisense) in 0.2x PBS (phosphate-buffered saline, 1x, Corning, Cellgro) according to the method generally described in Example 10 herein.

[0259] Apo(a) transgenic (Tg) mice were used to evaluate the efficacy of double-stranded RNAi agents with conjugated N-acetyl-galactosamine ligands in vivo. Apo(a) Tg mice (Frazer KA et al. 1995, Nature Genetics 9:424-431) generate human apo(a) (hereafter referred to as "apo(a) Tg mice") from a YAC containing the complete LPA gene (encoding the apo(a) protein) with both 5' and 3' additional sequences.

[0260] The Lp(a) RNAi agent linked to each GalNAc ligand (i.e., (NAG25), (NAG26), or (NAG27)) was combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection. Lp(a) RNAi agents linked to the respective GalNAc ligand (i.e., (NAG25), (NAG26), or (NAG27)) at the 5' end of the sense strand were delivered by SC injection. On day 1, SC injections were administered into the loose skin on the back between the shoulders of a 200 μl solution / 20 g mouse containing either saline or a 1 mg / kg (mpk) dose of the respective RNAi agent (AD03275, AD03341, or AD03421) in buffered saline. There were three (3) apo(a) Tg mice per treatment group.

[0261] Serum samples from treated mice were collected on days -1 (pre-dose), 8, 15, 22, 29, 36, and 43. Knockdown was measured by monitoring circulating apo(a) protein levels in serum using an apo(a) ELISA (Abcam). Lp(a) particle levels were measured using a Cobas® Integra 400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, the Lp(a) level for each animal at a given time point was divided by that animal's pre-dose expression level (in this case, on day -1) to determine the "normalized to day -1" expression ratio. Expression at a particular time point was then normalized to the saline control group by dividing the "normalized to day -1" ratio for each individual animal by the average "normalized to day -1" ratio for all mice in the saline control group. This resulted in expression for each time point normalized to expression in the control group. Experimental error is shown as the standard error of the mean.

[0262] The results are shown in Figure 16. Lp(a) RNAi agent AD03275, containing targeting ligand structure 101 (NAG25), showed 82% knockdown at its lowest (day 22) and remained at 72% knockdown at day 29. Lp(a) RNAi agent AD03341, containing targeting ligand structure 102 (NAG26), showed 87% knockdown at its lowest (day 15), however, knockdown at day 29 was only 45%, indicating an increase back to pre-administration apo(a) levels. Lp(a) RNAi agent AD03421, containing targeting ligand structure 103 (NAG27), showed 70% knockdown at its lowest (day 15) and 50% knockdown at day 29. These data confirm that Structure 101 (NAG25), Structure 102 (NAG26), and Structure 103 (NAG27) all exhibit similar knockdown activity. However, these data also demonstrate that AD03275 (Structure 101 (NAG25)) has superior duration and maintains better knockdown (72% knockdown at day 29) than Structures 102 (NAG26) and 103 (NAG27).

[0263] Example 11. LP(a) Expression Inhibition Oligomeric Compounds (Double-Stranded RNAi Agents) Linked to Targeting Ligand Structure 101 in Cynomolgus Monkeys Five different LPA RNAi agents linked to a targeting ligand represented by structure 101 were prepared to evaluate their performance in cynomolgus macaque (Macaca fascicularis) primates: AD03460, AD03536, AD03851, AD03853, and AD04110.

[0264] Each strand of the Lp(a) RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strands were mixed to form a duplex by combining equimolar RNA solutions (sense and antisense) in 0.2x PBS (phosphate-buffered saline, 1x, Corning, Cellgro) according to the method generally described in Example 4 herein.

[0265] The targeting ligands for all five (5) Lp(a) RNAi agents were added to the 5' end of the sense strand using non-nucleoside phosphoramidite synthesis generally as described herein and known in the art. The targeting ligand for each of the Lp(a) RNAi agents was linked to the 5' end of the respective RNAi agent using the following phosphoramidite compounds: [ka] (Structure 101d).

[0266] Targeting ligands (NAG25), including AD03460 and AD03536, were attached to the 5' end of the sense strand of each RNAi agent. (NAG25) has the same structure as shown in Example 8 above.

[0267] Targeting ligands (NAG25)s, including AD03851, AD03853, and AD04110, were attached to the 5' end of the sense strand of the respective RNAi agents. (NAG25)s= [ka] .

[0268] Blood samples were profiled and analyzed for lipoprotein(a) levels on days 8 and 15. Lp(a) levels were normalized to the mean of the three pre-dose values. Normalized Lp(a) levels are reported in the table below: [Table 7]

[0269] These data show that significant knockdown was achieved in cynomolgus monkeys at a dose of 2 mg / kg (mpk) of multiple different Lp(a) RNAi agents bound to the same targeting ligand structure of structure 101 herein.

[0270] Example 12: F12 expression inhibition oligomeric compound linked to a targeting ligand of structure 101 (double-stranded RNAi agent) in cynomolgus monkeys F12 expression-inhibiting oligomeric compounds (double-stranded F12 RNAi agents) were prepared having the sequences shown in Table 8 below:

[0271] Table 8. F12 expression-inhibiting oligomeric compounds (double-stranded RNAi agents) of Example 12 [Table 8]

[0272] In Table 8 above, (NAG25) represents the same structure as shown in Example 8, and is represented by structure 101 herein. Each strand of the Lp(a) RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strands were mixed to form a duplex by combining equimolar RNA solutions (sense and antisense) in 0.2x PBS (phosphate-buffered saline, 1x, Corning, Cellgro) according to the method generally described in Example 4 herein.

[0273] An F12 RNAi agent linked to a targeting ligand at the 5' end of the sense strand was generated and combined in a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection. Cynomolgus macaque (Macaca fascicularis) primates were injected subcutaneously with 3 mg / kg of AD03635 on Day 1. Three (3) monkeys were dosed per treatment group.

[0274] To observe knockdown, serum samples from treated cynomolgus monkeys were collected on days -7 and 1 (pre-dose), and on days 8, 15, and 22. Knockdown was measured by quantifying circulating cyno F12 protein (cF12) levels in serum using a human F12 ELISA kit (Molecular Innovations). The cF12 level for each animal at each time point was divided by the pre-treatment level of expression in that animal (average of days -7 and 1) to determine the "pre-dose normalized" expression rate. Experimental error is shown as the standard deviation.

[0275] The results are shown in Figure 17. The F12 RNAi agent linked to (NAG25) (structure 101 herein) showed knockdown in cynomolgus monkeys.

[0276] Example 13: Alpha-1 Antitrypsin Expression Inhibition Oligomeric Compounds Linked to Targeting Ligands of Structure 101 (Double-Stranded RNAi Agents) in PiZ Transgenic Mice To evaluate RNAi agents directed against the alpha-1 antitrypsin (AAT) gene in vivo, we used the transgenic PiZ mouse model (PiZ mice), which harbors the human PiZ AAT mutant allele and models human AATD (Carlson et al., Journal of Clinical Investigation 1989).

[0277] AAT expression-inhibiting oligomeric compounds (double-stranded RNAi agents) were prepared having the sequences shown in Table 9 below: Table 9. AAT Expression Inhibitory Oligomeric Compounds (RNAi Agent Duplexes) of Example 13 [Table 9]

[0278] In Table 9, (NAG25)s has the chemical structure shown in Example 11 above. AAT RNAi agents were prepared in a pharmaceutically acceptable saline buffer and administered to PiZ mice via subcutaneous (SC) injection of 200 μl of solution per 20 g mouse into the loose skin between the shoulders on the back to assess knockdown of AAT gene expression. Each mouse received a single SC dose of 5 mg / kg (mpk) of AD04454. Three mice were administered AAT RNAi agents (n=3).

[0279] Plasma samples were plotted and analyzed for AAT (Z-AAT) protein levels on days -1, 1 (pre-dose), 8, and 15. AAT levels were normalized to AAT plasma levels on day 1 (pre-dose). Protein levels were measured by quantifying circulating human Z-AAT levels in plasma with an ELISA kit.

[0280] The average normalized AAT (Z-AAT) levels are shown in Figure 18. An AAT RNAi agent linked to a targeting ligand of structure 101 herein showed knockdown in PiZ transgenic mice.

[0281] Example 14: F12 knockdown in wild-type mice following administration of an F12 expression-inhibiting oligomeric compound (double-stranded RNAi agent) linked to a targeting ligand of structure 101 F12 expression-inhibiting oligomeric compounds (double-stranded F12 RNAi agents) were prepared having the sequences shown in Table 10 below:

[0282] Table 10. F12 expression-inhibiting oligomeric compounds (double-stranded RNAi agents) of Example 14 [Table 10]

[0283] In Table 10, (NAG25) has the chemical structure shown in Example 8 and is represented by structure 101 disclosed herein. Each strand of the F12 RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strands were mixed to form a duplex by combining equimolar RNA solutions (sense and antisense) in 0.2x PBS (phosphate-buffered saline, 1x, Corning, Cellgro) according to the method generally described in Example 10 herein.

[0284] The F12 RNAi agent conjugated to each GalNAc targeting ligand (i.e., (NAG25)) was combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection.

[0285] The composition was delivered by SC injection. On day 1, a 200 μl solution / 20 g mouse containing either saline or a 3 mg / kg (mpk) dose of AD03632 in buffered saline was administered SC into the loose skin on the back between the shoulders. There were three (3) wild-type mice per treatment group. As previously noted, AD03632 contains a structure (NAG25) linked to the 5' end of the sense strand.

[0286] To monitor knockdown, serum samples from treated mice were collected on days -1 (pre-dose), 8, 15, 22, 29, and 36. Knockdown was measured by quantifying circulating mouse F12 protein (mF12) levels in serum using an endogenously expressed mF12 alphaLISA® (Perkin Elmer). Each animal's mF12 level at each time point was divided by the animal's pretreatment expression level to determine a "normalized to pre-dose" expression ratio. Expression at a particular time point was then normalized to the saline control group by dividing the "normalized to pre-dose" ratio for each individual animal by the average "normalized to pre-dose" ratio for all mice in the saline control group. This resulted in expression at each time point normalized to control expression. Experimental error is shown as the standard deviation.

[0287] Results from this study are shown in Figure 19. AD03632, comprising the targeting ligand structure 101 disclosed herein, shows significant knockdown across all time points.

[0288] Other embodiments While the present invention has been described in conjunction with the detailed description thereof, it should be understood that the foregoing description is intended to be illustrative and not limiting of 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. In the presence of 4,5-dicyanoimidazole, 【Chemical 1】 of, 【Chemistry 2】 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite React with 【Chemistry 3】 A method for preparing compound 10, comprising:

2. In the presence of triethylamine, 【Chemistry 4】 of, 【Chemistry 5】 10. The method of claim 1, further comprising reacting TFP with tetrafluorophenyl to form compound 9.

3. In the presence of N,N-diisopropylethylamine, 【Chemistry 6】 of, 【Chemistry 7】 10. The method of claim 1, further comprising reacting TFP with tetrafluorophenyl to form compound 9.

4. In the presence of Pd / C and p-toluenesulfonic acid, H ２ in 【Chemistry 8】 4. The method of claim 2 or 3, further comprising hydrogenating to form the tosylate salt 7.

5. In the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) and N,N-diisopropylethylamine (DIPEA), 【Chemistry 9】 of, 【Chemistry 10】 5. The method of claim 4, further comprising reacting with to form compound 6.

6. 【Catalog 11】 with formic acid to produce compound 4.

7. In the presence of isobutyl chloroformate and N-methylmorpholine (NMM), 【Chemistry 12】 of, 【Chemistry 13】 7. The method of claim 6, further comprising reacting with to form compound 3.

8. In the presence of isobutyl chloroformate and N-methylmorpholine (NMM), 【Chemistry 14】 of, 【Chemistry 15】 React with 【Chemistry 16】 generating Compound 3 is reacted with formic acid to give 【Chemistry 17】 generating Compound 4 was synthesized in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) and N,N-diisopropylethylamine (DIPEA). 【Chemistry 18】 React with 【Chemistry 19】 generating In the presence of Pd / C and p-toluenesulfonic acid, H ２ Compound 6 is hydrogenated with 【Chemistry 20】 generating Tosylate salt 7 in the presence of triethylamine 【Chemical 21】 where TFP is tetrafluorophenyl, 【Chemical 22】 generating Compound 9 in the presence of 4,5-dicyanoimidazole 【Chemical 23】 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite React with 【Chemistry 24】 A method for preparing compound 10, comprising:

9. In the presence of isobutyl chloroformate and N-methylmorpholine (NMM), 【Chemistry 25】 of, ​ React with 【Chemical 27】 generating Compound 3 is reacted with formic acid to give 【Chemical 28】 generating Compound 4 was synthesized in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) and N,N-diisopropylethylamine (DIPEA). 【Chemical 29】 React with 【Chemistry 30】 generating In the presence of Pd / C and p-toluenesulfonic acid, H ２ Compound 6 is hydrogenated with 【Chemical 31】 generating Tosylate salt 7 in the presence of N,N-diisopropylethylamine 【Chemical 32】 where TFP is tetrafluorophenyl, 【Chemical 33】 generating Compound 9 in the presence of 4,5-dicyanoimidazole 【Chemical 34】 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite React with 【Chemical 35】 A method for preparing compound 10, comprising:

10. A targeting ligand comprising the following structure: or a pharmaceutically acceptable salt thereof: 【Chemical 36】 10. A pharmaceutical composition for inhibiting expression of a target nucleic acid in a subject, comprising an expression-inhibiting oligomeric compound linked to:

11. A targeting ligand comprising the following structure, or a pharmaceutically acceptable salt thereof, linked to an expression-inhibiting oligomeric compound: 【Chemical 37】 1. A pharmaceutical composition for introducing an expression-inhibiting oligomeric compound into a mammalian cell, comprising: wherein the targeting ligand or a pharmaceutically acceptable salt thereof is in contact with the mammalian cell, and the mammalian cell is present in a human subject.

12. The pharmaceutical composition of claim 11 , wherein the expression-inhibiting oligomeric compound is an RNAi agent.

13. 1. A pharmaceutical composition for treating a disease or disorder that would benefit from the administration of an expression-inhibiting oligomeric compound, comprising: A targeting ligand comprising the following structure, or a pharmaceutically acceptable salt thereof, linked to an expression-inhibiting oligomeric compound: 【Chemical 38】 10. A pharmaceutical composition comprising a therapeutic amount of

14. The pharmaceutical composition of claim 13 , wherein the expression-inhibiting oligomeric compound is an RNAi agent.

15. A targeting ligand comprising the following structure: or a pharmaceutically acceptable salt thereof: 【Chemical 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 Or, 【Chemistry 43】

16. 16. The targeting ligand of claim 15, wherein the targeting ligand is further linked to an expression-inhibiting oligomeric compound.

17. A compound selected from the group consisting of the following structures: or a pharmaceutically acceptable salt thereof: 【Chemical 44】 wherein Z consists of or comprises an expression-inhibiting oligomeric compound, A is O or S, and A' is O － , S － , or NH － ); 【Chemistry 45】 wherein Z consists of or comprises an expression-inhibiting oligomeric compound, A is O or S, and A' is O － , S － , or NH － ); 【Chemistry 46】 wherein Z consists of or comprises an expression-inhibiting oligomeric compound, A is O or S, and A' is O － , S － , or NH － ); 【Chemistry 47】 wherein Z consists of or comprises an expression-inhibiting oligomeric compound, A is O or S, and A' is O － , S － , or NH － ) and 【Chemistry 48】 wherein Z consists of or comprises an expression-inhibiting oligomeric compound, A is O or S, and A' is O － , S － , or NH － (It is).

18. A compound selected from the group consisting of the following structures: or a pharmaceutically acceptable salt thereof: 【Chemistry 49】 【Chemistry 50】

19. A targeting ligand comprising a structure selected from the group consisting of the following structures, or a pharmaceutically acceptable salt thereof: 【Chemistry 51】 【Chemistry 52】 and 【Chemistry 53】

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