Carbohydrate targeting ligands for metabolic stabilization of oligonucleotide conjugates

Abstract

The present disclosure relates to a delivery platform for payload-specific and efficient directing of stable RNAi agents to hepatocytes in a subject. The delivery platform disclosed herein includes a metabolically stable N-acetylgalactosamine (NAG or GalNAc) targeting ligand conjugated to one or more oligonucleotides by a metabolically stable bond that is more stable than a phosphodiester bond to facilitate delivery of oligonucleotide-based payloads to cells, including hepatocytes. Also described are pharmaceutical compositions comprising the metabolically stable RNAi agent conjugate delivery platform, as well as methods for treating various diseases and conditions in which a therapeutic payload is required to be delivered to hepatocytes.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 505,414, filed May 31, 2023, and U.S. Provisional Patent Application Serial No. 63 / 559,728, filed February 29, 2024, the contents of each of which are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application contains a sequence list, which has been submitted in XML format and is hereby incorporated in its entirety by reference. The XML copy is named 30725-WO_SeqListing_2024.05.30.XML, created on May 30, 2024, and is 250kb in size. Technical Field

[0005] This disclosure relates to a delivery platform for delivering oligonucleotides or oligonucleotide-based agents (e.g., antisense oligonucleotides (ASO), double-stranded RNAi agents, or small interfering RNA (siRNA)) into hepatocytes in vivo, and particularly to hepatocytes. Delivery of RNAi agents using the delivery platform disclosed herein provides inhibition of gene expression in hepatocytes. Background Technology

[0006] Delivering therapeutic payloads to specific target tissues and cells within a subject remains a major challenge in medicine. This is especially true for oligonucleotide-based therapeutics, such as antisense oligonucleotides (ASOs) and RNA interference (RNAi) agents (typically containing small (or short) interfering RNAs using chemically modified nucleotides), which have shown great promise and potential to revolutionize medicine and provide effective therapeutic options for previously incurable diseases; provided, of course, that the therapeutic oligonucleotides can reach the desired cells and tissues in the body. Indeed, achieving appropriate delivery of oligonucleotide-based therapeutics is, and remains, one of the most pressing challenges in the discovery and identification of viable therapeutics.

[0007] Despite decades of development leading to a better understanding of how to properly deliver oligonucleotides to hepatocytes by covalently linking oligonucleotide payloads to targeting ligands containing N-acetylgalactosamine (NAG or GalNAc), further improvements are needed and anticipated. Improved delivery could potentially allow for the administration of less drug to patients or subjects, providing the benefit of reduced toxicological side effects and potentially lowering the cost of therapeutic agents as less material would need to be manufactured.

[0008] One potential approach is to stabilize the NAG ligand to prevent its metabolic degradation. It has been shown that the NAG ligand degrades under physiological conditions before the oligonucleotide payload can be delivered to hepatocytes.

[0009] Therefore, a delivery mechanism or platform is still needed to specifically and efficiently deliver oligonucleotide-based therapeutics, especially RNAi agents, to hepatocytes.

[0010] Overview

[0011] This document discloses a delivery platform comprising a metabolically stable carbohydrate ligand linked to a therapeutic agent, such as an oligonucleotide-based therapeutic agent, including antisense oligonucleotides (ASO) or RNA interference (RNAi) agents (also referred to herein as RNAi agents, RNAi inducers, or inducers; e.g., double-stranded RNAi agents or small (or short) interfering RNA (siRNA)), for use in liver cells in vivo. Delivery of this therapeutic oligonucleotide facilitates the selective and efficient inhibition of gene expression present in the liver, particularly genes present in hepatocytes.

[0012] While metabolically stable NAG ligands have been previously proposed, reported data have shown no identifiable improvement or advantage compared to conventional standard NAG ligands. This is at least partly because others have failed to observe the benefits of metabolically stable NAG ligands by simultaneously stabilizing bonds with oligonucleotide molecules through bonds more stable than conventional phosphodiester bonds (e.g., by failing to introduce more stable thiophosphate or dithiophosphate bonds).

[0013] One aspect described herein is a compound or a pharmaceutically acceptable salt thereof, comprising: a. Oligonucleotides with a length between 12 and 49 nucleotides; and b. Metabolically stable carbohydrate ligands; The double-stranded RNAi agent and the metabolically stable carbohydrate ligand are covalently bound by thiophosphate bonds, dithiophosphate bonds, or bonds that are more metabolically stable than phosphodiester bonds.

[0014] In another respect, this article describes a compound or a pharmaceutically acceptable salt thereof, comprising: a. A double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand of the double-stranded RNAi agent comprises 15 to 23 nucleotides, and the antisense strand of the double-stranded RNAi agent comprises 18 to 23 nucleic acids; b. Metabolically stable carbohydrate ligands; The double-stranded RNAi agent and the metabolically stable carbohydrate ligand are covalently linked via thiophosphate bonds.

[0015] In some respects, this article provides a compound or a pharmaceutically acceptable salt thereof comprising: a. Oligonucleotides with a length between 12 and 49 nucleotides; and b. Metabolically stable carbohydrate ligands; The double-stranded RNAi agent and the metabolically stable carbohydrate ligand are covalently bound by phosphate thioester bonds, phosphate dithioester bonds, or bonds that are more metabolically stable than phosphate diester bonds. The metabolically stable carbohydrate ligands described therein have the following formula:

[0016] in: Each instance of the metabolically stable carbohydrate is independently a chemically modified carbohydrate portion; Each instance of the tether is independently defined as follows: or , where m 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); The branch point group is selected from the following structures:

[0017] The connector is selected from the following structures:

[0018] n is an integer from 1 to 4; and This indicates the attachment site of the oligonucleotide.

[0019] In some embodiments, the metabolically stable carbohydrate ligand includes an N-acetylgalactosamine (NAG or GalNAc) targeting ligand, which comprises the structure of the following formula: or Where X = CH2 or S, and RNAi agents.

[0020] As shown above, Formula I represents the β-anodic bonding of metabolically stable NAG ligands, while Formula II represents the α-anodic bonding of metabolically stable NAG ligands. (The symbols used herein are as follows.) "It refers to any one or more groups that can be connected to it according to the scope of the invention described herein."

[0021] Metabolically stable carbohydrate ligands and oligonucleotides can be covalently linked in any manner known in the art, provided that the linkers used for covalently linking the various components are metabolically stable bonds that are more stable in vivo than phosphodiester bonds. Exemplary embodiments of multimeric RNAi agent conjugates can be found in various examples herein, according to the scope of the invention disclosed herein.

[0022] Preferably, for metabolically stable compounds having the chemical structure of Formula I or Formula II, X is CH2, as shown in Formula Ia and Formula IIa below: or .

[0023] Preferably, the oligonucleotide is a double-stranded RNAi agent containing a sense strand and an antisense strand, and more preferably, a metabolically stable carbohydrate ligand is linked to the sense strand of the RNAi agent.

[0024] Although it has been previously reported that RNAi agents (especially siRNA) can be delivered to hepatocytes in vivo using metabolically stable NAG-RNAi agent conjugates, previously reported results have shown no difference in affinity for the asialic acid glycoprotein receptor (ASGPR) or gene silencing activity compared to previously known standard GalNAc ligands with metabolically unstable glycosidic bonds, as shown in the following structure:

[0025] (See Kandasmy et al., Metabolically stabilized Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways). As disclosed herein, this reported conclusion is inaccurate when metabolically stable compounds having the chemical structure of Formula I or II are covalently linked to components via metabolically stable linkers that are more stable than phosphodiester bonds, as supported by data shown in the examples herein (comparisons, for example, with the accompanying figure from the above abstract "Metabolic Unstable Glycoside Bonding" (showing unstable phosphate thioester bonds with RNAi agents and monomeric RNAi agent conjugates)).

[0026] In some embodiments, a metabolically stable compound having the structure of Formula I or Formula II is linked to an RNAi agent via a linker that is not a linker less stable than a phosphodiester bond, such as a thiophosphate bond. In some embodiments, a metabolically stable compound having the structure of Formula I or Formula II is linked to an RNAi agent via a linker that includes a stable thiophosphate bond or a dithiophosphate bond.

[0027] In another aspect disclosed herein, in some embodiments, the length of the RNAi agent used in the multimeric RNAi agent conjugate delivery platform described herein comprises a duplex having a sense strand of no more than 21 nucleotides in length and an antisense strand of no more than 20 nucleotides in length. In some embodiments, the multimeric RNAi agent conjugate delivery platform described herein comprises a duplex having a sense strand of no more than 19 nucleotides in length and an antisense strand of no more than 18 nucleotides in length. As illustrated in the embodiments described herein, data show that additional delivery advantages can be obtained when the length of one or both RNAi agents used in the multimeric RNAi agent conjugate delivery platform is limited, preferably wherein the RNAi agent comprises a sense strand and an antisense strand of no more than 21 nucleotides, no more than 20 nucleotides, or no more than 19 nucleotides in length.

[0028] The metabolically stable carbohydrate conjugates described herein can be used in methods of treating conditions and diseases mediated at least partially by reduced expression of target genes, including, for example, diseases mediated at least partially by reduced expression of one or more genes in hepatocytes. The compounds disclosed herein can selectively reduce the expression of target genes in subject cells, particularly hepatocytes in the liver. The methods disclosed herein include administering one or more multimeric RNAi agent conjugates or one or more ASO conjugates to a subject (such as a human or animal subject) using, for example, any suitable method known in the art, such as intravenous infusion, intravenous injection, or subcutaneous injection.

[0029] This document also describes pharmaceutical compositions comprising the oligonucleotide conjugates disclosed herein, which are capable of inhibiting the expression of one or more target genes, wherein the compositions further comprise at least one pharmaceutically acceptable excipient. The pharmaceutical compositions described herein comprise one or more of the disclosed RNAi agents or other oligonucleotide conjugates or oligonucleotide-based reagent conjugates, which are capable of selectively and effectively reducing or inhibiting the expression of target genes in vivo.

[0030] 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. While similar or equivalent methods and materials described herein may be used in the practice or testing of this invention, suitable methods and approaches are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0031] Other objects, features, aspects and advantages of the present invention will be set forth in the following detailed description, drawings and claims. Invention Details

[0033] definition

[0034] As used herein, the terms “oligonucleotide” and “polynucleotide” refer to polymers of linked nucleosides, wherein each nucleoside may be modified or unmodified independently.

[0035] As used herein, an "RNAi agent" (also known as an "RNAi inducer") refers to a composition containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules capable of degrading or inhibiting (e.g., under appropriate conditions) the translation of a messenger RNA (mRNA) transcript of a target mRNA in a sequence-specific manner. As used herein, RNAi agents may act via RNA interference mechanisms (i.e., by interacting with RNA interference pathways in mammalian cells, such as RNA-induced silencing complexes or RISC) or through any alternative mechanism or pathway. While it is believed that RNAi agents act primarily through RNA interference mechanisms as used herein, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action. The RNAi agents disclosed herein contain sense and antisense strands and include, but are not limited to, short (or small) interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), and dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the targeted mRNA. RNAi agents may include one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0036] As used herein, the terms “silence,” “reduction,” “inhibition,” “downregulation,” or “knockdown” when referring to the expression of a given gene mean that, when cells, cell populations, tissues, organs, or subjects are treated with the RNAi agents described herein, the expression of that gene is reduced compared to a second cell, cell population, tissue, organ, or subject that has not been so treated, as measured by the level of RNA transcribed from that gene or the level of polypeptides, proteins, or protein subunits translated from mRNA in the cells, cell populations, tissues, organs, or subjects in which gene transcription took place.

[0037] As used herein, the terms “sequence” and “nucleotide sequence” refer to a continuous sequence or order of nucleobases or nucleotides, described using standard nomenclature as a continuous sequence of letters.

[0038] As used herein, “base,” “nucleotide base,” or “nucleobase” refers to a heterocyclic pyrimidine or purine compound that is a component of a nucleotide, and includes major purine bases: adenine and guanine, and major pyrimidine bases: cytosine, thymine, and uracil. Nucleobases can be further modified to include, but are not limited to, universal bases, hydrophobic bases, hybrid bases, size-extended bases, and fluorinated bases. (See, for example, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed., Wiley VCH, 2008). The synthesis of such modified nucleobases (including phosphorous amide compounds containing modified nucleobases) is known in the art.

[0039] As used herein and unless otherwise stated, the term "complementary" in describing a first nucleobase or nucleotide sequence (e.g., a sense strand or targeted mRNA of an RNAi agent) relative to a second nucleobase or nucleotide sequence (e.g., an antisense strand of an RNAi agent or a single-stranded antisense oligonucleotide) refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide containing the second nucleotide sequence under specific standard conditions (forming base-pair hydrogen bonds under mammalian physiological conditions (or similar in vivo 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 native or modified nucleotides or nucleotide mimics to at least the extent required for hybridization described above. Sequence identity or complementarity is independent of modification. For example, for the purpose of determining identity or complementarity, a and Af, as defined herein, are complementary to U (or T) and identical to A.

[0040] As used herein, "perfect complementarity" or "complete complementarity" means that in a hybridization pair of nucleobase or nucleotide sequences, all (100%) bases in the sequential sequence of the first oligonucleotide will hybridize with the same number of bases in the sequential sequence of the second oligonucleotide. The sequential sequence may contain all or part of the first or second nucleotide sequence.

[0041] As used herein, "partial complementarity" means that in a hybridization pair of nucleobase or nucleotide sequences, at least 70% but not all of the bases in the sequential sequence of the first oligonucleotide will hybridize with the same number of bases in the sequential sequence of the second oligonucleotide. The sequential sequence may contain all or part of the first or second nucleotide sequence.

[0042] As used herein, "substantially complementary" means that in a hybridization pair of nucleobase or nucleotide sequences, at least 85% but not all of the bases in the sequential sequence of the first oligonucleotide will hybridize with the same number of bases in the sequential sequence of the second oligonucleotide. The sequential sequence may contain all or part of the first or second nucleotide sequence.

[0043] As used herein, the terms “complementary,” “fully complementary,” “partially complementary,” and “substantially complementary” are used in relation to nucleobase or nucleotide matching between the sense and antisense strands of an RNAi agent or between the antisense strand of an RNAi agent and the target mRNA sequence.

[0044] As used herein, "oligonucleotide-based reagents" refers to reagents containing approximately 10–50 oligonucleotides (e.g., 10–48, 10–46, 10–44, 10–42, 10–40, 10–38, 10–36, 10–34, 10–32, 10–30, 10–28, 10–26, 10–24, 10–22, 10–20, 10–18, 10–16, 10–14, 10–12, 12–50, 12–48, 12–46, 12–44, 12–42, 12–40, 12–38, 12–36, 12–34, 12–32, 12–30, 12–28, 12–26, 12–24, 12–22, 12–20, 12–18, 1). 2 to 16, 12 to 14, 14 to 50, 14 to 48, 14 to 46, 14 to 44, 14 to 42, 14 to 40, 14 to 38, 14 to 36, 14 to 34, 14 to 32, 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to 50 16 to 48, 16 to 46, 16 to 44, 16 to 42, 16 to 40, 16 to 38, 16 to 36, 16 to 34, 16 to 32, 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 50, 18 to 48, 18 to 46, 18 to 44, 18 to 42 18 to 40, 18 to 38, 18 to 36, 18 to 34, 18 to 32, 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, 18 to 20, 20 to 50, 20 to 48, 20 to 46, 20 to 44, 20 to 42, 20 to 40, 20 to 38, 20 to 36, 20 to 34, 20 to 3 2. 20 to 30, 20 to 28, 20 to 26, 20 to 24, 20 to 22, 22 to 50, 22 to 48, 22 to 46, 22 to 44, 22 to 42, 22 to 40, 22 to 38, 22 to 36, 22 to 34, 22 to 32, 22 to 30, 22 to 28, 22 to 26, 22 to 24, 24 to 50, 24 to 48, 24 to 46, 24 to 44, 24 to 42, 24 to 40, 24 to 38, 24 to 36, 24 to 34, 24 to 32, 24 to 30, 24 to 28, 24 to 26, 26 to 50, 26 to 48, 26 to 46, 26 to 44, 26 to 42, 26 to 40, 26 to 38, 26 to 36, 26 to 34, 26 To 32, 26 to 30, 26 to 28, 28 to 50, 28 to 48, 28 to 46, 28 to 44, 28 to 42, 28 to 40, 28 to 38, 28 to 36, 28 to 34, 28 to 32, to 28 to 30, 30 to 50, 30 to 48, 30 to 46, 30 to 44, 30 to 42, 30 to 40, 30 to 38,30 to 36, 30 to 34, 30 to 32, 32 to 50, 32 to 48, 32 to 46, 32 to 44, 32 to 42, 32 to 40, 32 to 38, 32 to 36, 32 to 34, 34 to 50, 34 to 48, 34 to 46, 34 to 44, 34 to 42, 34 to 40, 34 to 38, 34 to 36, 36 to 50, 36 to 48, 36 to 46, 36 to 44, 36 to 42, 36 to A nucleotide sequence of 40, 36 to 38, 38 to 50, 38 to 48, 38 to 46, 38 to 44, 38 to 42, 38 to 40, 40 to 50, 40 to 48, 40 to 46, 40 to 44, 40 to 42, 42 to 50, 42 to 48, 42 to 46, 42 to 44, 44 to 50, 44 to 48, 44 to 46, 46 to 50, 46 to 48, or 48 to 50 nucleotides or nucleotide base pairs. In some embodiments, the oligonucleotide-based reagent has a nucleobase sequence that is at least partially complementary to the coding sequence in the target nucleic acid or target gene expressed in the cell. In some embodiments, the oligonucleotide-based reagent, upon delivery to a cell expressing a gene, is capable of inhibiting the expression of the corresponding gene, and is referred to herein as an "oligonucleotide-based expression inhibitor." Gene expression can be inhibited in vitro or in vivo.

[0045] "Oligonucleotide-based reagents" include, but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides (ASO), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer substrates. In some embodiments, the oligonucleotide-based reagent is a single-stranded oligonucleotide, such as an antisense oligonucleotide. In some embodiments, the oligonucleotide-based reagent is a double-stranded oligonucleotide. In some embodiments, the oligonucleotide-based reagent is a double-stranded oligonucleotide used as an RNAi agent.

[0046] As used herein, the terms "substantially identical" or "substantially identical" when applied to nucleic acid sequences mean that a nucleotide sequence (or a portion thereof) has at least about 85% sequence identity or more compared to a reference sequence, for example, at least 90%, at least 95%, or at least 99% identity. The percentage of sequence identity is determined by comparing two best-aligned sequences within a comparison window. The percentage is calculated by determining the number of positions in both sequences where the same type of nucleic acid bases appears to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0047] As used herein, the terms “treat”, “treatment”, etc., refer to a method or procedure taken to provide relief or reduction in the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, “treat” and “treatment” may include preventative measures, management, preventative treatment, and / or suppression or reduction of the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0048] As used herein, the phrase “introduced into cells” in the context of RNAi agents refers to the functional delivery of RNAi agents into cells. The phrase “functional delivery” refers to the delivery of RNAi agents into cells in a manner that enables them to have the desired biological activity (e.g., sequence-specific inhibition of gene expression).

[0049] As used herein, the term "isomer" refers to compounds that have the same molecular formula but differ in the properties or bonding sequence of their atoms, or in the spatial arrangement of their atoms. Isomers that differ in the spatial arrangement of their atoms are called "stereoisomers." Stereoisomers that are not mirror images of each other are called "diastereomers," while stereoisomers that are non-overlapping mirror images of each other are called "enantiomers," or sometimes optical isomers. The carbon atom bonded to four different substituents is called the "chiral center."

[0050] As used herein, unless a specific conformation is explicitly specified in the structure, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure and racemic forms, for each structure having an asymmetry center and thus producing enantiomers, diastereomers, or other stereoisomers. For example, the structures disclosed herein are intended to cover mixtures of diastereomers as well as single stereoisomers.

[0051] As used in the claims herein, the phrase “consisting of…” excludes any element, step, or ingredient not specified in the claims. When used in the claims herein, the phrase “consisting substantially of…” limits the scope of the claims to the specified materials or steps, and those materials or steps that do not substantially affect the essential and novel features of the claimed invention.

[0052] It will be readily understood and appreciated by those skilled in the art that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound or composition is situated. Therefore, as used herein, the structures disclosed contemplate certain functional groups that can be protonated or deprotonated, such as, for example, OH, SH, or NH. As will be readily understood by those skilled in the art, the disclosure herein is intended to cover the disclosed compounds and compositions regardless of their protonation state based on environmental factors (e.g., pH).

[0053] As used herein, the terms “link” or “combination” referring to the connection between two compounds or molecules means that the two molecules are joined by a covalent bond or via a non-covalent bond (e.g., hydrogen bond or ionic bond). In some instances, where the term “link” or “combination” refers to the non-covalent bond between two molecules, the binding between two dissimilar molecules in a physiologically acceptable buffer solution (such as buffered saline) has a binding strength of less than 1 x 10⁻⁶. -4 M (such as less than 1 x 10) -5 M, less than 1 x 10 -6 M or less than 1 x 10 -7 M) of K D Unless otherwise stated, the terms “connection” and “combination” as used herein may refer to a connection between a first compound and a second compound, with or without any intermediate atoms or groups.

[0054] As used herein, a linking group is one or more atoms that connect one molecule or a portion of a molecule to another molecule, and then to a second molecule or a second portion of a molecule. Similarly, as used in the art, the term "scaffold" is sometimes used interchangeably with a linking group. A linking group may include any number of atoms or functional groups. In some embodiments, the linking group may not promote any biological or pharmaceutical response, but may simply be used to link two biologically active molecules.

[0055] As used herein, a “metabolicly stable carbohydrate ligand” is a carbohydrate ligand suitable for binding to desialyl glycoprotein receptors that are highly expressed on hepatocytes, wherein the carbohydrate ligand has been chemically modified to provide a more stable chemical composition in serum (e.g., human serum). Those skilled in the art can readily determine suitable tests to ascertain whether such compounds are metabolically more stable (e.g., more stable in human serum) and still retain the ability to deliver cargo molecules, such as RNAi agents, to hepatocytes compared to unmodified carbohydrate ligands.

[0056] Certain chemical modifications are provided for metabolically stable carbohydrate ligands, including but not limited to modifications at the atom adjacent to the anomeric carbon, or modifications for phosphodiester bonds (e.g., thiophosphate bonds or dithiophosphate bonds) linking the metabolically stable carbohydrate ligand to an oligonucleotide. In some embodiments, the metabolically stable carbohydrate ligand is chemically modified at the atom adjacent to the anomeric carbon. In some embodiments, the atom adjacent to the anomeric carbon is a second carbon atom, which may be a methylene (-CH2-) moiety. In other embodiments, the atom adjacent to the anomeric carbon is a sulfur (-S-) atom.

[0057] In some embodiments, the metabolically stable carbohydrate ligand comprises a sugar moiety. In some embodiments, the sugar moiety is selected from glucose, galactose, and N-acetylgalactosamine. Non-limiting examples of metabolically stable carbohydrate ligands are the metabolically stable N-acetylgalactosamine ligands of Formulas I and II disclosed herein.

[0058] In some embodiments, as measured by the half-life of the compound in in vitro serum, the stability of metabolically stable carbohydrate ligands in human serum is 1.5 times that of non-metabolicly stable carbohydrate ligands. In some embodiments, as measured by the half-life of the compound in in vitro serum, the stability of metabolically stable carbohydrate ligands in human serum is 2 times that of non-metabolicly stable carbohydrate ligands. In some embodiments, as measured by the half-life of the compound in in vitro serum, the stability of metabolically stable carbohydrate ligands in human serum is 5 times that of non-metabolicly stable carbohydrate ligands. In some embodiments, as measured by the half-life of the compound in in vitro serum, the stability of metabolically stable carbohydrate ligands in human serum is 10 times that of non-metabolicly stable carbohydrate ligands.

[0059] Unless otherwise stated, as used in this document. "It refers to any one or more groups that can be connected to it according to the scope of the invention described herein."

[0060] As used herein, the term "including" is used to mean the phrase "including but not limited to" and is used interchangeably with it. The term "or" is used to mean the term "and / or" and is used interchangeably with it unless the context clearly indicates otherwise.

[0061] The phrase “consisting of” as used in the claims herein excludes any element, step, or ingredient not specified in the claims. When used in the claims herein, the phrase “consisting substantially of” limits the scope of the claims to the specified materials or steps, and those materials or steps that do not substantially affect the essential and novel features of the claimed invention.

[0062] The term "pharmaceutically acceptable salt" refers to salts that, within reasonable medical judgment, are suitable for use in contact with human tissues without excessive toxicity, irritation, allergic reactions, etc., and in proportion to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., by reference incorporated herein by reference... J. Pharmaceutical Sciences Pharmaceutically acceptable salts are described in detail in , 1977, 66, 1-19.

[0063] Pharmaceutically acceptable salts of the compounds disclosed herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed by other methods known in the art (e.g., ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, disaccharide, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptahydrate, glyceryl phosphate, gluconate, hemisulfate, heptahydrate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and alkylammonium salts (i.e., C44-C ... 1-4 Alkyl)4N + Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts. Other pharmaceutically acceptable salts include (where applicable) those formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate ions to form non-toxic ammonium, quaternary ammonium, and amine cations.

[0064] Modified nucleotides

[0065] In some embodiments, the RNAi agent contains one or more modified nucleotides. As used herein, a “modified nucleotide” is a nucleotide other than a ribonucleotide (2’-hydroxynucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides may include, but are not limited to: deoxyribonucleotides, nucleotide mimics, abase-free nucleotides (represented herein as Ab), 2’-modified nucleotides, 3’ to 3’-bonded (reverse) nucleotides (represented herein as invdN, invN, invn), nucleotides containing modified nucleosides, bridging nucleotides, peptide nucleic acids (PNA), 2’,3’-open-ring nucleotide mimics (unlocked nucleobase analogs, referred to herein as N… UNA (or NUNA), locked nucleotides (referred to as N in this article) LNA (or NLNA), 3'-O-methoxy (2'-nucleotide linked) nucleotide (referred to as 3'-OMen in this article), 2'-F-arabinonucleotide (referred to as NfANA or Nf in this article) ANA The following are considered nucleotides: 5'-Me, 2'-fluoronucleotides (referred to herein as 5Me-Nf), morpholinonucleotides, vinylphosphonate deoxyribonucleotides (referred to herein as vpdN), vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides (cPrpN). 2'-Modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides (also known as 2'-methoxynucleotides, and referred to herein as lowercase 'n' in the nucleotide sequence), 2'-fluoronucleotides (also known herein as 2'-deoxy-2'-fluoronucleotides, and referred to herein as Nf), 2'-deoxynucleotides (referred to herein as dN), 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides (also known herein as 2'-MOE, and referred herein as NM), 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary to consistently modify all positions in a given compound. Conversely, more than one modification can be incorporated into a single RNAi agent or even a single nucleotide therein. The sense and antisense strands of RNAi agents can be synthesized and / or modified using methods known in the art. Modification of one nucleotide is independent of modification of another nucleotide.

[0066] Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (such as 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, adenine, and guanine 6-alkyl (such as 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives, adenine and guanine 2-alkyl (such as 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives, 2-thiouracil, 2-thiouracil, and 2-thiouracil. Thymidine, 2-thiocytosine, 5-halogenated uracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymidine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-mercapto, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated (such as 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaaadenine, 7-deazoguanine, 7-deazoadenine, 3-deazoguanine and 3-deazoadenosine.

[0067] In some embodiments, all or substantially all of the nucleotides in the RNAi agent are modified nucleotides. As used herein, an RNAi agent in which substantially all of the present nucleotides are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides that are ribonucleotides (i.e., unmodified) in both the sense and antisense strands. As used herein, a sense strand in which substantially all of the present nucleotides are modified nucleotides is a sense strand in which two or fewer (i.e., 0, 1, or 2) nucleotides are unmodified ribonucleotides. As used herein, an antisense strand in which substantially all of the present nucleotides are modified nucleotides is an antisense strand in which two or fewer (i.e., 0, 1, or 2) nucleotides are unmodified ribonucleotides. In some embodiments, one or more nucleotides in the RNAi agent are unmodified ribonucleotides.

[0068] Modified nucleoside interbonding

[0069] In some implementations, one or more nucleotides of the RNAi agent are linked by non-standard bonding or a backbone (i.e., modified internucleotide bonding or a modified backbone). Modified internucleotide bonds or skeletons include, but are not limited to, phosphorothioate groups (represented herein by lowercase "s"), chiral phosphorothioates, thiophosphates, dithiophosphates, triphosphates, aminoalkyl phosphates, alkyl phosphonates (e.g., methylphosphonates or 3'-alkylenephosphonates), chiral phosphonates, hypophosphonates, phosphoramidates (e.g., 3'-aminophosphatate, aminoalkylphosphatate, or thionophosphaamidate), thioalkylphosphonates, thioalkyl phosphates, morpholino bonds, borophosphates with normal 3'-5' bonds, 2'-5' linked analogs of borophosphates, or borophosphates with reverse polarity (where adjacent nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'). In some embodiments, the modified internucleotide bonds or skeletons lack a phosphorus atom. Modified internucleotide bonds lacking phosphorus atoms include, but are not limited to, short-chain alkyl or cycloalkyl sugar bonds, mixed heteroatom and alkyl or cycloalkyl sugar bonds, or bonds between one or more short-chain heteroatoms or heterocyclic sugars. In some embodiments, the modified internucleotide skeleton includes, but is not limited to, siloxane skeletons, sulfide skeletons, sulfoxide skeletons, sulfone skeletons, formacetyl and thioformyl skeletons, methyleneformyl and thioformyl skeletons, olefin-containing skeletons, aminosulfonate skeletons, methyleneimino and methylenehydrazine skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and other skeletons having mixed N, O, S, and CH2 components.

[0070] In some embodiments, the sense strand of the RNAi agent may contain 1, 2, 3, 4, 5, or 6 phosphate thioester bonds, and the antisense strand of the RNAi agent may contain 1, 2, 3, 4, 5, or 6 phosphate thioester bonds, or both the sense and antisense strands may independently contain 1, 2, 3, 4, 5, or 6 phosphate thioester bonds. In some embodiments, the sense strand of the RNAi agent may contain 1, 2, 3, or 4 phosphate thioester bonds, and the antisense strand of the RNAi agent may contain 1, 2, 3, or 4 phosphate thioester bonds, or both the sense and antisense strands may independently contain 1, 2, 3, or 4 phosphate thioester bonds.

[0071] In some embodiments, the sense strand of the RNAi agent contains at least two phosphate-thioester nucleoside bonds. In some embodiments, the at least two phosphate-thioester nucleoside bonds are located between nucleotides at positions 1-3 starting from the 3' end of the sense strand. In some embodiments, one phosphate-thioester nucleoside bond is located at the 5' end of the sense strand, and the other phosphate-thioester nucleoside bond is located at the 3' end of the sense strand. In some embodiments, two phosphate-thioester nucleoside bonds are located at the 5' end of the sense strand, and the other phosphate-thioester nucleoside bond is located at the 3' end of the sense strand. In some embodiments, the sense strand does not include any phosphate-thioester nucleoside bonds between nucleotides, but contains one, two, or three phosphate-thioester bonds between the terminal nucleotides at both the 5' and 3' ends and optionally, a reverse debasement residue terminal cap. In some embodiments, the targeting ligand is linked to the sense strand via phosphate-thioester bonds.

[0072] In some embodiments, the antisense strand of the RNAi agent contains four phosphate-thioester nucleoside bonds. In some embodiments, the four phosphate-thioester nucleoside bonds are between nucleotides at positions 1-3 from the 5' end of the antisense strand, and between nucleotides at positions 17-19, 18-20, 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end. In some embodiments, three phosphate-thioester nucleoside bonds are located between positions 1-4 from the 5' end of the antisense strand, and the fourth phosphate-thioester nucleoside bond is located between positions 20-21 from the 5' end of the antisense strand. In some embodiments, the RNAi agent contains at least three or four phosphate-thioester nucleoside bonds in the antisense strand.

[0073] In some embodiments, the RNAi agent contains one or more modified nucleotides and one or more modified nucleoside bonds. In some embodiments, a 2'-modified nucleoside is combined with a modified nucleoside bond.

[0074] Targeting ligands and targeting groups

[0075] As disclosed herein, multimeric RNAi agent conjugate delivery platforms comprise one or more targeting groups. The targeting group or targeting portion enhances the pharmacokinetic or biodistributional properties of the conjugate or RNAi agent to which it is attached, thereby improving the cell-specific (in some cases, organ-specific) distribution and cell-specific (or organ-specific) uptake of the conjugate or RNAi agent. The targeting group may be monovalent, divalent, trivalent, tetravalent, or have a higher valence relative to its target. Representative targeting groups include, but are not limited to, compounds with affinity for cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity for cell surface molecules. In some embodiments, a linker, such as a PEG linker or one, two, or three debased and / or ribitol (debased ribose) residues (which may act as linkers in some cases), is used to attach the targeting group to the RNAi agent.

[0076] In some embodiments, the targeting group is covalently attached to the 3' and / or 5' ends of the sense and / or antisense strands of an RNAi agent. In some embodiments, the targeting ligand is attached to the 3' and / or 5' ends of the sense strand of one of the RNAi agents. In some embodiments, the targeting group is attached to the 5' end of the sense strand of an RNAi agent. In some embodiments, the targeting group is internally attached to one or more nucleotides of the sense strand of an RNAi agent. In some embodiments, the targeting ligand is located between two RNAi agents in a multimeric RNAi agent conjugate. The targeting group can be linked to the RNAi agent directly or indirectly via a linker / linker group. In some embodiments, the targeting group is linked to the RNAi agent via a metabolically stable bond or linkage.

[0077] In some embodiments, the targeting group includes a desialyl glycoprotein receptor ligand. As used herein, a desialyl glycoprotein receptor ligand is a ligand containing a portion having affinity for the desialyl glycoprotein receptor. As described herein, the desialyl glycoprotein receptor is highly expressed on hepatocytes. In some embodiments, the desialyl glycoprotein receptor ligand comprises or is composed of one or more galactose derivatives. As used herein, the term "galactose derivative" includes galactose and galactose derivatives having an affinity for the desialyl glycoprotein receptor equal to or greater than that of galactose. Galactose derivatives include, but are not limited to: galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butylgalactosamine, and N-isobutyrylgalactosamine (see, for example: ST. Iobst and K. Drickamer, JBC, 19962716686), and metabolically stable glycoside-linked N-acetylgalactosamine. Galactose derivatives and galactose derivative clusters that can be used to target oligonucleotides and other molecules in vivo to the liver are known in the art (see, for example, Baenziger and Fiete, 1980, Cell, 22611-620; Connolly et al., 1982, J. Biol. Chem., 257939-945).

[0078] Galactose derivatives have been used to target molecules to hepatocytes in vivo by binding to desialyl glycoprotein receptors expressed on the surface of hepatocytes. The binding of desialyl glycoprotein receptor ligands to the desialyl glycoprotein receptors facilitates cell-specific targeting of hepatocytes and endocytosis of molecules into hepatocytes. Desialyl glycoprotein receptor ligands can be monomers (e.g., having a single galactose derivative, also known as monovalent or monodentate) or polymers (e.g., having multiple galactose derivatives). Galactose derivatives or clusters of galactose derivatives can be attached to the 3' or 5' end of the sense or antisense strand of an RNAi agent using methods known in the art. Alternatively, galactose derivatives or clusters of galactose derivatives can be internally attached to one or more nucleotides of the sense or antisense strand of an RNAi agent using methods known in the art.

[0079] In some embodiments, the targeting ligand comprises one or more metabolically stable N-acetylgalactosamine (NAG or GalNAc) targeting ligands, which include structures of the following formula: or , where X = CH2 or S.

[0080] In some embodiments, the metabolically stable NAG targeting ligand is a trimer (also known as a trianthal or trivalent), wherein three portions of Formula I or Formula II are attached via a central branch point. (See, for example, the chemical structure referred to herein as NAG52). In some embodiments, the targeting ligand is a cluster of four metabolically stable NAG portions, thus forming a tetrameric (also known as a tetraanthal or tetravalent) targeting ligand. In some embodiments, the metabolically stable NAG targeting ligand is a bianthal or bivalent), wherein two portions of Formula I or Formula II are attached via a central branch point.

[0081] As used herein, metabolically stable NAG-targeting ligands contain one or more portions of Formula I or Formula II, each portion being attached to a central branch point. In some embodiments, the targeting ligand is attached to the branch point via a linker or spacer. In some embodiments, the linker or spacer is a flexible hydrophilic spacer, such as a PEG group (see, for example, U.S. Patent No. 5,885,968; Biessen et al., Med. Chem. 1995 Vol. 39 p. 1538-1546). The branch point can be any small molecule that allows attachment of three galactose derivatives and further allows attachment of the branch point to an RNAi agent. Examples of branch point groups are dilysine or diglutamic acid. Attachment of the branch point to the RNAi agent can occur via a linker or spacer. In some embodiments, the linker or spacer comprises a flexible hydrophilic spacer, such as, but not limited to, a PEG spacer. In some embodiments, the linker comprises a rigid linker, such as a cyclic group.

[0082] In some embodiments, the delivery platforms or compounds disclosed herein comprise one or more targeting ligands, said targeting ligands comprising compounds of Formula I or II or pharmaceutically acceptable salts thereof: or , Where X = CH2 or S.

[0083] In some embodiments, the delivery platform or compound disclosed herein comprises one or more targeting ligands, said targeting ligands comprising compounds of formula 1a or 1b: or .

[0084] The method for preparing compounds of formula Ia is described in the examples below.

[0085] In some embodiments, compounds that can be conjugated with RNAi agents to synthesize RNAi agent delivery platforms are shown in Table 1 below.

[0086] In some embodiments, the compounds described herein comprise: a. Oligonucleotides with a length between 12 and 49 nucleotides; and b. Metabolically stable carbohydrate ligands; The double-stranded RNAi agent and the metabolically stable carbohydrate ligand are covalently bound by thiophosphate ester bond, dithiophosphate ester bond, or a bond that is more metabolically stable than phosphodiester bond. Metabolically stable carbohydrate ligands have the following formula:

[0087] in: Each instance of the metabolically stable carbohydrate is independently a chemically modified carbohydrate portion; Each instance of the tethering is independently represented by the following formula: or , where m 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); The branch point group is selected from the following structures: and ; The connector is selected from the following structures:

[0088] n is an integer from 1 to 4; and This indicates the attachment site to the oligonucleotide.

[0089] In some implementations, the tether has the following formula: , where m 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). In some embodiments, the tether has the following formula: , where m 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). In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11. In some embodiments, m is 12. In some embodiments, m is 13. In some embodiments, m is 14. In some embodiments, m is 15. In some embodiments, m is 16. In some embodiments, m is 17. In some embodiments, m is 18. In some embodiments, m is 19. In some embodiments, m is 20.

[0090] In some implementations, the tether has the following formula: In some implementations, the tether has the following formula: .

[0091] In some implementations, the branch point group has the following formula: In some embodiments, the branch point group has the following formula: In some embodiments, the branch point group has the following formula: In some embodiments, the branch point group has the following formula: In some embodiments, the branch point group has the following formula: In some embodiments, the branch point group has the following formula: In some embodiments, the branch point group has the following formula: In some embodiments, the branch point group has the following formula: In some embodiments, the branch point group has the following formula: In some embodiments, the branch point group has the following formula: In some embodiments, the branch point group has the following formula: In some embodiments, the branch point group has the following formula: In some embodiments, the branch point group has the following formula: .

[0092] In some implementations, the joint structure has the following formula: In some implementations, the joint structure has the following formula: In some implementations, the joint structure has the following formula: In some implementations, the joint structure has the following formula: In some implementations, the joint structure has the following formula: In some implementations, the joint structure has the following formula: In some implementations, the joint structure has the following formula: .

[0093] In some implementations, n is 1. In some implementations, n is 2. In some implementations, n is 3. In some implementations, n is 4.

[0094] In some embodiments, the compounds described herein include any of the metabolically stable N-acetylgalactosamine targeting ligands shown in Table 1 below.

[0095] Table 1: Examples of metabolically stable N-acetylgalactosamine targeting ligands

[0096]

[0097] Linking group

[0098] In some embodiments, the RNAi agent contains or is conjugated to one or more nonnucleotide groups, including but not limited to linker groups, delivery polymers, or delivery carriers. Nonnucleotide groups can enhance the targeting, delivery, or attachment of the RNAi agent. Examples of linker groups are provided in Table 2. Nonnucleotide groups can be covalently linked to the 3' and / or 5' ends of the sense and / or antisense strands. In some embodiments, the RNAi agent contains a nonnucleotide group linked to the 3' and / or 5' end of the sense strand. In some embodiments, the nonnucleotide group is linked to the 5' end of the sense strand of the RNAi agent. The nonnucleotide group can be directly or indirectly linked to the RNAi agent via a linker / connector group. In some embodiments, the nonnucleotide group is linked to the RNAi agent via an unstable, cleavable, or reversible bond or linker.

[0099] In some embodiments, the nonnucleotide group enhances the pharmacokinetic or biodistribution properties of the RNAi agent or conjugate to which it is attached, thereby improving the cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, the nonnucleotide group enhances the endocytosis of the RNAi agent.

[0100] The RNAi agents described herein can be synthesized having reactive groups, such as amino groups (also referred to herein as amines), at the 5'-terminus and / or 3'-terminus. These reactive groups can then be used to attach the target moiety using methods typical of the art.

[0101] A linker or connecting group is a connection between two atoms that links a chemical group (such as an RNAi agent) or fragment of interest to another chemical group (such as a targeting ligand, targeting group, PK / PD modulator, or delivery polymer) or fragment of interest via one or more covalent bonds. Unstable bonds contain unstable bonds. Bonding may optionally include spacers that increase the distance between the two bonding atoms. Spacers may further increase the flexibility and / or length of the bond. Spacers include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, aralkyl, areneyl, and arynyl; each may contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and sugars. Spacer groups are well known in the art, and the foregoing list is not intended to limit the scope of this specification.

[0102] In some embodiments, the target group is attached to the RNAi agent without the use of an additional adapter. In some embodiments, the target group is designed to have readily available adapters to facilitate bonding with the RNAi agent. In some embodiments, when the composition contains two or more RNAi agents, the same adapter can be used to attach the two or more RNAi agents to their respective target groups. In some embodiments, when the composition contains two or more RNAi agents, different adapters can be used to attach the two or more RNAi agents to their respective target groups.

[0103] In some embodiments, the linker group may be synthetically conjugated to the 5' or 3' end of the sense strand of the RNAi agent described herein. In some embodiments, the linker group is synthetically conjugated to the 5' end of the sense strand of the RNAi agent. In some embodiments, the linker group conjugated to the RNAi agent may be a triyne linker group.

[0104] Table 2 provides examples of some modified nucleotides and linker groups.

[0105] Table 2: Structures of various modified nucleotides and linker groups

[0106]

[0107] Alternatively, other linking groups known in the art can be used.

[0108] As a complement or alternative to RNAi agents and one or more targeting ligands, targeting groups, and / or PK / PD modulators, in some embodiments, a delivery vehicle may be used to deliver the RNAi agent to cells or tissues. The delivery vehicle is a compound that can improve the delivery of RNAi agents to cells or tissues and may include, but is not limited to: polymers, such as amphiphilic polymers, membrane-active polymers, peptides, melittin, melittin-like peptides (MLPs), lipids, reversibly modified polymers or peptides, or reversibly modified membrane-active polyamines, or a combination thereof.

[0109] In some embodiments, RNAi agents may be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs, or other delivery systems available in the art. RNAi agents may also be chemically conjugated to targeting groups, lipids (including but not limited to cholesterol and cholesterol derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example, WO 2000 / 053722, WO 2008 / 022309, WO 2011 / 104169 and WO 2012 / 083185, WO 2013 / 032829, WO 2013 / 158141, each of which is incorporated herein by reference) or other delivery systems available in the art.

[0110] Pharmaceutical Composition

[0111] In some embodiments, this disclosure provides pharmaceutical compositions comprising, consisting of, or substantially consisting of one or more delivery platforms disclosed herein.

[0112] As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of an active pharmaceutical ingredient (API) and optionally one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance, in addition to the active pharmaceutical ingredient (API, therapeutic product), intentionally included in a drug delivery system. The excipient does not exert or is not intended to exert a therapeutic effect at the intended dose. The excipient may function to: a) assist in the processing of the drug delivery system during manufacturing; b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API; c) assist in product identification; and / or d) enhance the overall safety, effectiveness, or any other property of delivery of the API during storage or use. Pharmaceutically acceptable excipients may or may not be inert substances.

[0113] Excipients include, but are not limited to: absorption promoters, anti-adhesion agents, defoamers, antioxidants, adhesives, buffers, carriers, coating agents, colorants, delivery promoters, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, flow aids, humectants, lubricants, oils, polymers, preservatives, brine, salt, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, tensioning agents, mediators, waterproofing agents, and wetting agents.

[0114] The pharmaceutical compositions described herein may contain other additional components commonly found in pharmaceutical compositions. In some embodiments, the additional components are pharmaceutically active materials. Pharmaceutically active materials include, but are not limited to: antipruritics, astringents, local anesthetics or anti-inflammatory agents (such as antihistamines, diphenhydramine, etc.), small molecule drugs, antibodies, antibody fragments, aptamers and / or vaccines.

[0115] Pharmaceutical compositions may also contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for altering osmotic pressure, buffers, coating agents, or antioxidants. They may also contain other agents with known therapeutic benefits.

[0116] The pharmaceutical composition can be administered in a variety of ways, depending on whether local or systemic treatment is required and the area to be treated. Administration can be performed in any manner known in the art, such as, but not limited to, local (e.g., via a transdermal patch), pulmonary (e.g., by inhalation or blowing of powder or aerosol, including via nebulizer, intratracheal, or intranasal), epidermal, transdermal, oral, or parenteral administration. Parenteral administration includes, but is not limited to, intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous (e.g., via an implanted device), intracranial, intraparenchymal, intrathecal, and intraventricular administration. In some embodiments, the pharmaceutical composition described herein is administered via subcutaneous injection. The pharmaceutical composition can be administered orally, for example, in the form of tablets, coated tablets, dragées, hard or soft gelatin capsules, solutions, emulsions, or suspensions. It can also be administered rectally, for example, using suppositories; locally or transdermally, for example, using ointments, creams, gels, or solutions; or parenterally, for example, using injectable solutions.

[0117] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor® EL (BASF, Parsippany, NJ), or phosphate-buffered saline. They should be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings (e.g., lecithin), in the case of dispersions by maintaining the desired particle size, and by using surfactants. In many cases, isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), and sodium chloride, are preferably included in the composition. Prolonged absorption of the injectable composition can be caused by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0118] Sterile injection solutions can be prepared by combining a desired amount of the active compound in a suitable solvent with one or a combination of the ingredients listed above (as needed), followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and any other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injection solutions, preparation methods include vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any other desired ingredients from a pre-sterile filtered solution.

[0119] Formulations suitable for intra-articular application may be in the form of a sterile aqueous formulation of any ligand described herein (which may be in microcrystalline form), such as an aqueous microcrystalline suspension. Liposome formulations or biodegradable polymer systems may also be used to deliver any ligand described herein for intra-articular and ophthalmic application.

[0120] The active compound can be prepared together with a carrier that protects the compound from rapid elimination from the body, such as controlled-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 be used. Methods for preparing such formulations are readily apparent to those skilled in the art. Liposome 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.

[0121] Pharmaceutical compositions may contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). As used herein, “pharmacologically effective amount,” “therapeuticly effective amount,” or simply “effective amount,” refers to the amount of a pharmaceutically active agent that produces a pharmacological, therapeutic, or preventative effect.

[0122] Drugs containing RNAi agents are also an object of this invention, as are methods for preparing such drugs, which include preparing one or more compounds containing RNAi agents, and (if necessary) one or more other substances having known therapeutic benefits, into a pharmaceutically acceptable form.

[0123] The RNAi agents and pharmaceutical compositions containing RNAi agents disclosed herein can be packaged or contained in kits, containers, pouches, or dispensers. The RNAi agents and pharmaceutical compositions containing RNAi agents can be packaged in pre-filled syringes or vials.

[0124] Treatment methods and expression inhibition

[0125] The delivery platform disclosed herein can be used to treat subjects (e.g., humans or other mammals) suffering from a disease or condition that will benefit from the administration of an RNAi agent. In some embodiments, the RNAi agent delivery platform disclosed herein can be used to treat subjects (e.g., humans) who will benefit from reduced and / or inhibited mRNA expression and / or target protein levels.

[0126] In some embodiments, a therapeutically effective amount of any one or more RNAi agents is administered to the subject. Treatment of the subject may include therapeutic and / or prophylactic treatment. A therapeutically effective amount of any one or more RNAi agents described herein is administered to the subject. The subject may be a person, a patient, or a human patient. The subject may be an adult, adolescent, child, or infant. The pharmaceutical compositions described herein may be administered to humans or animals.

[0127] The RNAi agents described herein can be used to treat at least one symptom in a subject suffering from a disease or condition associated with a target gene, or a disease or condition at least partially mediated by the expression of the target gene. In some embodiments, the RNAi agent is used to treat or manage the clinical presentation of a subject suffering from a disease or condition that would benefit from a reduction in the target mRNA or is at least partially mediated by a reduction in the target mRNA. A therapeutically effective amount of one or more of the RNAi agents described herein, or a composition containing an RNAi agent, is administered to the subject. In some embodiments, the methods disclosed herein include administering a composition containing an RNAi agent described herein to a subject to be treated. In some embodiments, a preventatively effective amount of any one or more of the RNAi agents is administered to the subject, thereby treating the subject by preventing or suppressing at least one symptom.

[0128] In some embodiments, this disclosure provides methods for treating a disease, symptom, condition, or pathological state in patients in need that is at least partially mediated by the expression of a target gene, wherein said methods include administering any of the RNAi agents described herein to the patient.

[0129] In some implementations, compared to subjects who had not received RNAi or before, subjects who received RNAi showed a reduction in the gene expression level and / or mRNA level of target genes by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or greater than 99%. The gene expression level and / or mRNA level in the subjects may be reduced in the subjects' cells, cell populations, and / or tissues.

[0130] In some implementations, compared to subjects who had not received RNAi, subjects who received RNAi showed a reduction in protein levels of at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99%. The reduction in protein levels can occur in the subject's cells, cell groups, tissues, blood, and / or other fluids.

[0131] Decreases in mRNA and protein levels can be assessed using any method known in the art. As used herein, a decrease or reduction in mRNA and / or protein levels is collectively referred to herein as a decrease or reduction in target gene expression, or inhibition or reduction of target gene expression. The examples shown herein illustrate known methods for assessing gene expression inhibition.

[0132] In some embodiments, RNAi agents can be used to prepare pharmaceutical compositions for treating diseases, conditions, or symptoms that are at least partially mediated by the expression of a target gene.

[0133] In some embodiments, the method of treating the subject depends on the subject's weight. In some embodiments, the RNAi agent may be administered at a dose of about 0.05 mg / kg to about 40.0 mg / kg of the subject's body weight. In other embodiments, the RNAi agent may be administered at a dose of about 5 mg / kg to about 20 mg / kg of the subject's body weight.

[0134] In some implementations, the RNAi agent can be administered in multiple doses, meaning that the subject is given two doses over a short period of time (e.g., less than 24 hours). In some implementations, approximately half of the required daily dose is administered in the initial administration, and the remaining approximately half of the required daily dose is administered approximately four hours after the initial administration.

[0135] In some implementations, the RNAi agent can be administered weekly. In other implementations, the RNAi agent can be administered every two weeks (once every other week).

[0136] In some implementations, RNAi agents or compositions containing RNAi agents can be used to treat diseases, conditions, or symptoms that are at least partially mediated by the expression of target genes.

[0137] Cells, tissues and non-human objects

[0138] Cells, tissues, and non-human objects are considered, including at least one delivery platform containing the RNAi agent described herein. The cells, tissues, or non-human objects are prepared by delivering the RNAi agent to the cells, tissues, or non-human objects using any method available in the art. In some embodiments, the cells are mammalian cells, including but not limited to human cells.

[0139] The implementation schemes and projects provided above will now be illustrated by the following non-limiting examples.

[0140] Explanatory Implementation Plan

[0141] Illustrative embodiments of the disclosed technology are provided herein. These embodiments are merely illustrative and do not limit the scope of this disclosure or the appended claims.

[0142] Implementation Scheme 1: A compound or a pharmaceutically acceptable salt thereof for inhibiting the expression of one or more genes, comprising: a. Oligonucleotides, which consist of chains of 12-49 nucleotides in length; and b. Metabolically stable carbohydrate ligands; The oligonucleotide and the metabolically stable carbohydrate ligand therein are covalently linked by a thiophosphate bond, a dithiophosphate bond, or another bond that is more metabolically stable than a phosphodiester bond.

[0143] Implementation Scheme 2: The compound of Implementation Scheme 1 or a pharmaceutically acceptable salt thereof, wherein the metabolically stable carbohydrate ligand has the following formula:

[0144] in: Each instance of the metabolically stable carbohydrate is independently a chemically modified carbohydrate portion; Each instance of the tethering is independently represented by the following formula: or , where m is an integer selected from 1 to 20; The branch point group is selected from the following structures:

[0145] The connector is selected from the following structures:

[0146] n is an integer from 1 to 4, which is allowed for the valence; and Indicates the attachment point to the rest of the compound.

[0147] Implementation Scheme 3: The compound of Implementation Scheme 2, wherein the tether has the following formula: .

[0148] Implementation Scheme 4: The compound of Implementation Scheme 2, wherein the tether has the following formula: .

[0149] Implementation Scheme 5: The compound of any one of Implementation Schemes 2-4, wherein the branch point group has the following formula: .

[0150] Implementation Scheme 6: The compound of any one of Implementation Schemes 2-5, wherein the branch point group has the following formula: .

[0151] Implementation Scheme 7: The compound of any one of Implementation Schemes 2-4, wherein the branch point group has the following formula: .

[0152] Implementation Scheme 8: The compound of any one of Implementation Schemes 2-4 or 7, wherein the branch point group has the following formula: .

[0153] Implementation Scheme 9: The compound of any one of Implementation Schemes 1-8, wherein the metabolically stable carbohydrate ligand comprises metabolically stable N-acetylgalactosamine.

[0154] Implementation Scheme 10: The compound of any one of Implementation Schemes 1-9, wherein the metabolically stable carbohydrate has the following formula: , where X is CH2 or S, and represents the connection point with the rest of the compound.

[0155] Implementation Scheme 11: A compound of any one of Implementation Schemes 1-9, wherein the metabolically stable carbohydrate has the following formula: , where X is CH2 or S, and represents the connection point with the rest of the compound.

[0156] Implementation Scheme 12: The compound described in Implementation Scheme 10 or 11, wherein X is CH2.

[0157] Implementation Scheme 13: The compound described in Implementation Scheme 10 or 11, wherein X is S.

[0158] Implementation Scheme 14: The compound of any one of Implementation Schemes 1-6 or 9-13, wherein the metabolically stable carbohydrate ligand comprises three metabolically stable N-acetylgalactosamine moieties.

[0159] Implementation Scheme 15: The compound of any one of Implementation Schemes 2-6 or 9-13, wherein n is 3.

[0160] Implementation Scheme 16: The compound of any one of Implementation Schemes 2-15, wherein the connector has the following formula: .

[0161] Implementation Scheme 17: The compound of any one of Implementation Schemes 1-16, wherein the metabolically stable carbohydrate ligand comprises a structure selected from the following:

[0162] in Indicates the connection point with the rest of the compound.

[0163] Implementation Scheme 18: The compound of any one of Implementation Schemes 1-17, wherein the oligonucleotide is a double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein: (i) The sense strand of the double-stranded RNAi agent contains 19-23 nucleotides; and (ii) The antisense strand of the double-stranded RNAi agent contains 19-23 nucleotides.

[0164] Implementation Scheme 19: The compound of any one of Implementation Schemes 1-18, wherein the sense strand comprises 19-21 nucleotides and the antisense strand comprises 19-21 nucleotides.

[0165] Implementation Scheme 20: The compound of any one of Implementation Schemes 1-19, wherein the antisense strand of the double-stranded RNAi agent consists of 19 nucleotides.

[0166] Implementation Scheme 21: The compound of any one of Implementation Schemes 1-20, wherein the antisense strand is at least partially complementary to the mRNA sequence encoded by a gene expressed in human hepatocytes.

[0167] Implementation Scheme 22: The compound of any one of Implementation Schemes 1-21, wherein the antisense strand is completely complementary to the mRNA sequence encoded by a gene expressed in human hepatocytes.

[0168] Implementation Scheme 23: The compound of any one of Implementation Schemes 1-22, wherein the metabolically stable carbohydrate ligand is conjugated to the 3' end of the sense chain.

[0169] Implementation Scheme 24: The compound of any one of Implementation Schemes 1-23, wherein the metabolically stable carbohydrate ligand is conjugated to the 5' end of the sense chain.

[0170] Implementation Scheme 25: The compound of any one of Implementation Schemes 1-24, wherein the terminal cap is located at the 3' end of the first sense chain, the 3' end of the second sense chain, or the 3' end of both the first sense chain and the second sense chain.

[0171] Implementation Scheme 26: The compound described in Implementation Scheme 25, wherein the end cap is a reverse non-base moiety or NH2-C6.

[0172] Implementation Scheme 27: A compound comprising a structure selected from the following:

[0173] Or its pharmaceutically acceptable salt, wherein Indicates the connection point with the rest of the compound.

[0174] Implementation Scheme 28: The compound of Implementation Scheme 27, wherein the compound has the following formula: , Or its pharmaceutically acceptable salt, wherein Indicates the connection point with the rest of the compound.

[0175] Implementation Scheme 29: The compound of Implementation Scheme 27, wherein the compound has the following formula: , Or its pharmaceutically acceptable salt, wherein Indicates the connection point with the rest of the compound.

[0176] Implementation Scheme 30: The compound of Implementation Scheme 27, wherein the compound has the following formula: , Or its pharmaceutically acceptable salt, wherein Indicates the connection point with the rest of the compound.

[0177] Implementation Scheme 31: The compound of Implementation Scheme 27, wherein the compound has the following formula: , Or its pharmaceutically acceptable salt, wherein Indicates the connection point with the rest of the compound.

[0178] Implementation Scheme 32: The compound of any one of Implementation Schemes 27-31, wherein the remainder of the compound comprises an oligonucleotide chain of 12-49 nucleotides in length.

[0179] Implementation Scheme 33: The compound of Implementation Scheme 32, wherein the oligonucleotide is a double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein: (i) The sense strand of the double-stranded RNAi agent contains 19-23 nucleotides; and (ii) The antisense strand of the double-stranded RNAi agent contains 19-23 nucleotides.

[0180] Implementation Scheme 34: The compound of Implementation Scheme 33, wherein the sense strand comprises 19-21 nucleotides and the antisense strand comprises 19-21 nucleotides.

[0181] Implementation Scheme 35: The compound described in Implementation Scheme 33 or 34, wherein the antisense strand of the double-stranded RNAi agent consists of 19 nucleotides.

[0182] Implementation Scheme 36: The compound of any one of Implementation Schemes 33-35, wherein the antisense strand is at least partially complementary to the mRNA encoded by a gene expressed in human hepatocytes.

[0183] Implementation Scheme 37: The compound of any one of Implementation Schemes 33-36, wherein the antisense strand is completely complementary to the mRNA encoded by a gene expressed in human hepatocytes.

[0184] Implementation Scheme 38: The compound of any one of Implementation Schemes 33-37, wherein the end cap is located at the 3' end of the sense chain.

[0185] Implementation Scheme 39: The compound of any one of Implementation Schemes 33-38, wherein the end cap is located at the 5' end of the sense chain.

[0186] Implementation Scheme 40: A pharmaceutical composition comprising the compound of any one of Implementation Schemes 1-39 and a pharmaceutically acceptable excipient.

[0187] Implementation Scheme 41: A method for inhibiting gene expression, comprising administering to a subject in need of the compound described in any one of Implementation Schemes 1-39.

[0188] Example

[0189] The following examples are not limiting and are intended to illustrate certain implementations disclosed herein.

[0190] Example 1. Synthesis of RNAi agents and multimeric RNAi agent conjugates.

[0191] The following describes the general procedure for synthesizing certain RNAi agents and their conjugates (including the RNAi conjugates shown in the non-limiting examples described herein).

[0192] Synthesis of RNAi agents.RNAi agents can be synthesized using methods generally known in the art. For the synthesis of the RNAi agents shown in the examples described herein, the sense and antisense strands of the RNAi agent were synthesized using a solid-phase phosphorus amide technique used in oligonucleotide synthesis. Depending on scale, MerMade96E® (Bioautomation), MerMade12® (Bioautomation), or Oligopilot 100 (GE Healthcare) were used. Synthesis was performed on a solid-phase support made of controlled-aperture glass (CPG, 500 Å or 600 Å, available from Prime Synthesis, Aston, PA, USA) or polystyrene (available from Kinovate, Oceanside, CA, USA). All RNA and 2'-modified RNA phosphorus amides were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA), ChemGenes (Wilmington, MA, USA), or Hongene Biotech (Morrisville, NC, USA). Specifically, the 2'-O-methylphosphoramide used includes the following: (5'-O-dimethoxytriphenylmethyl-N 6 -(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide, 5'-O-dimethoxytriphenylmethyl-N 4 -(acetyl)-2'-O-methylcytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide, (5'-O-dimethoxytriphenylmethyl-N 2 -(isobutyryl)-2'-O-methylguanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide and 5'-O-dimethoxytriphenylmethyl-2'-O-methyluridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide. 2'-deoxy-2'-fluorophosphamide and 2'-O-propargylphosphamide have the same protecting group as 2'-O-methylphosphamide. 5'-Dimethoxytriphenylmethyl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide was purchased from Glen Research (Virginia). The reverse debasing (3'-O-dimethoxytriphenylmethyl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide was purchased from ChemGenes. The UNA phosphoramide used included the following: 5'-(4,4'-dimethoxytriphenylmethyl)-N... 6-(benzoyl)-2',3'-open-ring adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 5'-(4,4'-dimethoxytriphenylmethyl)-N-acetyl-2',3'-open-ring cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 5'-(4 4'-Dimethoxytriphenylmethyl)-N-isobutyryl-2',3'-open-ring guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, and 5'-(4,4'-dimethoxytriphenylmethyl)-2',3'-open-ring uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide. To introduce thiophosphate bonding, an anhydrous acetonitrile solution of 100 mM 3-phenyl-1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) or a pyridine solution of 200 mM hydroflavin (TCI America, Portland, OR, USA) was used.

[0193] TFA amino-linked phosphorus amides are also commercially available (ThermoFisher) to introduce (NH2-C6) reactive linkers. TFA amino-linked phosphorus amides were dissolved in anhydrous acetonitrile (50 mM) and a molecular sieve (3 Å) was added. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) were used as activating solutions. Coupling times were 10 min (RNA), 90 s (2'O-Me), and 60 s (2'F). Triyne-containing phosphorus amides were synthesized to introduce the corresponding (TriAlk#) linkers. When used in conjunction with the RNAi agents presented in some examples herein, the triyne-containing phosphorus amides were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidates were dissolved in anhydrous acetonitrile (50 mM) and a molecular sieve (3 Å) was added. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) were used as activating solutions. Coupling times were 10 min (RNA), 90 s (2'O-Me), and 60 s (2'F).

[0194] For certain RNAi agents, adapters, such as C6-SS-C6 or 6-SS-6 groups, and C6-SS(Me)-C5, are introduced at the 3' end of the sense strand. Pre-loaded resins with the corresponding adapters are obtained commercially. Alternatively, for certain sense strands, dT resin is used, and the corresponding adapters are then added via standard phosphorous amide synthesis.

[0195] Cleavage and deprotection of support-bound oligomers. After solid-phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt% methylamine / water and 28% to 31% ammonium hydroxide (Aldrich) at 30 °C for 1.5 h. The solution was evaporated, and the solid residue was reconstituted in water (see below).

[0196] Purification. Crude oligomers were purified by anion-exchange HPLC using a TSKgel SuperQ-5PW 13µm column and a Shimadzu LC-8 system. Buffer A consisted of 20 mM Tris, 5 mM EDTA, pH 9.0, and 20% acetonitrile. Buffer B was identical to buffer A, but with the addition of 1.5 M sodium chloride. UV traces were recorded at 260 nm. Appropriate fractions were combined, and size exclusion HPLC was run using a run buffer containing a GE Healthcare XK 16 / 40 column packed with Sephadex G25 fine particles and 100 mM ammonium bicarbonate, pH 6.7, and 20% acetonitrile or filtered water.

[0197] Annealing. Complementary strands were mixed in equimolar amounts of RNA solution (sense and antisense) in 1X PBS (phosphate-buffered saline, 1X, Corning, Cellgro) to form an RNAi agent. Some of the RNAi agent was lyophilized and stored at -15 to -25°C. The duplex concentration was determined by measuring the absorbance of the solution in 1X PBS using a UV-Vis spectrometer. The duplex concentration was then determined by multiplying the absorbance at 260 nm by a conversion factor and a dilution factor. A conversion factor of 0.037 mg / (mL) was used. (cm) or calculated based on the extinction coefficient determined experimentally.

[0198] Example 2. Synthesis of targeting ligands

[0199] Synthesis of NAG52 (Compound 9) .

[0200] Compounds 1, 2, and 3 were each synthesized according to previously published procedures (see, for example, U.S. Patent Application Publication No. 2002 / 0107224 A1), and Cbz-NH-Glu-Glu-OH: According to the synthetic procedure described in Arrowhead Pharmaceuticals, Inc. International Patent Application Publication No.: WO2017 / 156012, the contents of these references are incorporated herein by reference as if fully set forth herein. More specifically, 1 is synthesized via the following synthetic route: . Synthesis of 5: 3 (4.61 g, 12.3 mmol) and Boc-N-amino-PEG2-NHS ester (CAS 2183440-73-3, 4.61 g, 12.3 mmol) were dissolved in anhydrous DCM (100 mL), followed by the addition of triethylamine (3.4 mL, 24.6 mmol). The reaction mixture was stirred at room temperature (rt) for 2 hours, and the solution was concentrated to 30 mL under reduced pressure and diluted with chloroform (300 mL). The resulting solution was first washed with brine / citric acid (1:1, 30 mL), and then with brine / saturated bicarbonate solution (1:1, 30 mL). The organic layer was dried over Na2SO4, concentrated under reduced pressure, and purified on a silica gel column (100% DCM to 20% MeOH in DCM). The fractions containing desired product 4 were combined, the solvent was removed under reduced pressure, and the resulting foamed residue was redissolved in 4M HCl / 1,4-dioxane (100 mL). The reaction mixture was stirred at room temperature for 1 hour, and the solvent was removed under reduced pressure. The resulting residue was suspended in toluene and dried under reduced pressure to give desired product 5 as an HCl salt (5.30 g, 9.30 mmol, 76% yield). MW 533.26 Measured ESIMS + m / z = 534.23 [M+H + ]. Synthesis of 7 5 (5.30 g, 9.94 mmol) was dissolved in anhydrous DMF, followed by the addition of DIPEA (4.4 mmol, 45.97 mmol). Z-BisGlu (1.12 g, 2.73 mmol) and TBTU (3.03 g, 7.99 mmol) were added under vigorous stirring. The solution turned pale brown, which deepened over time. When unreacted starting material could no longer be detected by LC-MS, the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed by co-evaporation with toluene, and the residue was redissolved in chloroform (400 mL). The resulting solution was first washed with brine / water (1:1, 60 mL), and then with brine / bicarbonate solution (1:1, 60 mL). The organic layer was dried over Na2SO4, concentrated under reduced pressure, and purified on a silica gel column (100% DCM to 20% MeOH in DCM). The fractions containing the desired product 6 were combined, the solvent was removed under reduced pressure, and the resulting foaming residue was redissolved in methanol (200 mL). Pd / C (0.70 g) was added to the solution, and the suspension was hydrogenated overnight at 1 atm. The reaction mixture was stirred overnight at room temperature under hydrogen. The solution was filtered through a diatomaceous earth mat and concentrated under reduced pressure to give the desired product 7, which was used unchanged in the next step (3.80 g, 2.09 mmol, 77% yield). MW was calculated as 1821.84. Measured ESIMS + m / z = 912.13 [M+2H + ]. Synthesis of 8 7 (3.80 g, 2.09 mmol) was dissolved in anhydrous DMF (30 mL) and slowly added to a solution of anhydrous DMF (30 mL) containing 4-hydroxycyclohexanecarboxylic acid (0.35 g, 2.43 mmol), TBTU (0.82 g, 2.16 mmol), and DIPEA (1.12 mL, 6.44 mmol). The reaction mixture was stirred at room temperature for 2 hours. The solvent was removed by co-evaporation with toluene, and the residue was redissolved in chloroform (300 mL). The solution was washed with brine / 5% citric acid (1:1, 30 mL), dried over Na2SO4, concentrated under reduced pressure, and purified on a silica gel column (100% DCM to 30% MeOH in DCM) to give the desired product 8 (2.40 g, 1.23 mmol, 59% yield). MW 1947.90 Measured ESIMS + m / z = 975.46 [M+2H + ]. Synthesis of 9 Compound 8 (2.40 g, 1.23 mmol) was thoroughly dried by co-evaporation of DCM with toluene and then under vacuum for 30 min. A round-bottom flask was loaded with a stir bar and pretreated molecular sieves and purged with nitrogen. DCM (100 mL) was added to the flask, and the molecular sieves were gently stirred for 10 min. Diisopropyltetrazolammonium (1.40 g, 8.19 mmol) was added to the solution, and the reaction mixture was stirred for another 30 min. 2-Cyanoethyl N,N,N',N'-tetraisopropyldiphosphonamide (0.55 mL, 1.73 mmol) was added, and the reaction mixture was stirred at room temperature for 1 h until unreacted starting material could not be detected by LC-MS. The solution was filtered through a diatomaceous earth mat to remove the molecular sieves and diluted with a saturated bicarbonate solution (100 mL) with stirring. After 15 min, the organic fraction was separated, and the aqueous layer was extracted with chloroform (2 x 200 mL). The organic fractions were combined, dried over Na₂SO₄, concentrated under reduced pressure, and purified on a silica gel column (100% DCM (+0.1% triethylamine) to 10% MeOH (+0.1% triethylamine) in DCM). The fractions containing the desired product 9 were combined, concentrated under reduced pressure, and the product was co-evaporated twice with toluene to remove any residual triethylamine, to give the desired product as a grayish-white solid (2.4 g, 1.16 mmol, 94% yield). Characterization of compound 9: 1 H NMR (DMSO-d6): 1.14 d (12H), 1.44 m (7H), 1.62-1.90 m (11H), 1.80s (9H), 1.94 s (9H), 2.00 s (9H), 2.07 s (9H), 2.03-2.16 m (4H), 2.20-2.31 m(6H), 2.76 t (2H), 2.88-2.98 m (3H), 3.12- 3.23 m (10H), 3.34-3.42 m (6H), 3.46 s (12 H), 3.57 t (8H), 3.62- 3.76 m (2H), 3.98-4.20 m (15H), 4.20-4.30 m(3H), 4.96 dd(3H), 5.28, d(3H), 7.56- 8.00 m(8H), 8.12 d(3H). 31 P NMR (DMSO-d6): 145.84, 146.01. Synthesis of NAG42 (Compound 9B) The synthesis of NAG42 follows the same route as NAG52 described above, with the only difference being the use of a β-anodic stable bond instead of an α-anodic bond. More specifically, compound 1B with a β-anodic bond can be synthesized as follows: . The remaining synthesis follows the description of the NAG52 synthesis above, replacing 1 with 1B to obtain compound 9B: . Characterization of compound 9B: 1 H NMR (DMSO-d6): 1.14 d (12H), 1.36-1.54 m (7H), 1.60-1.86 m (11H), 1.79 s (9H), 1.89 s (9H), 1.99 s (9H), 2.10 s (9H), 2-02-2.16 m (4H), 2.24-2.30 m (6H), 2.76 t (2H), 2.98-3.08 m (3H), 3.12-3.24 m (10H), 3.30-3.42 m(8H), 3.47 s (12H), 3.58 t (8H), 3.62-3.76 m (2H), 3.80- 4.06 m (14H), 4.10-4.20 m (2H), 4.88 dd (3H), 5.26 d (3H), 7.55-8.00 m (11H). 31 P NMR (DMSO-d6): 145.84, 145.89. Synthesis of NAG1008 phosphoramidites Synthesis of 3 Under anhydrous conditions, α-C-Nag-Phe-2-amine hydrochloride 1 (3.864 g, 6.79 mmol) was obtained. DMF (250 mL), glutamate derivative 2 (734 mg, 2.61 mmol), DIEA (4 mL, 23 mmol), and TBTU (1.860 g, 5.8 mmol) were added. The pH was checked to confirm alkalinity. The reaction was stirred for 1.5 h, and all volatiles were removed under vacuum at 40 °C. Toluene was evaporated twice to remove residual DMF. The product was dissolved in CHCl3, washed twice with 10% NaCl aqueous solution and saturated NaHCO3 aqueous solution, and dried (Na2SO4). Combiflash purification was performed using an 80 g column with eluent A = DCM; B = 20% MeOH in DCM, 0-60% within 60 min. Yield: 2.915 g, 81%. MW: 1311.59. ESIMS: 1311.59. + m / z = 1312.42 [M+H + ]. Synthesis of 4 Product 3 (2.910 g, 2.222 mmol) was hydrogenated for 16 hours using a hydrogen balloon in 10% Pd / C (330 mg) / methanol (60 mL). The product was filtered through diatomaceous earth, concentrated, and vacuum dried. The product was further dried by evaporation of toluene and used directly in the next step. MW was calculated as 1177.55. Measured ESIMS: 1177.55. + m / z = 1179.02 [M+H + ]. Synthesis of 5 Cis-4-hydroxycyclohexanecarboxylic acid (368 mg, 2.56 mmol) was treated with TBTU (855 mg, 2.66 mmol) and DIEA (1.16 mL, 6.67 mmol) in anhydrous DMF (20 mL) for 3 min. Compound 4 (2.222 mmol) from the previous step was dissolved in anhydrous DMF (40 mL) and added to a solution containing the activating acid. After stirring for 1.5 hours, the DMF was removed under vacuum at 40 °C, and toluene was evaporated twice to remove residual DMF. The residue was placed in chloroform (150 mL), washed twice with 10% NaCl aqueous solution and NaHCO3 aqueous solution, and dried (Na2SO4). Yield: 1.89 g (65%). MS: MW calculated as 1303.62. Measured values: ESIMS + m / z = 1304.42 [M+H + The crude product is used directly in the next step. Synthesis of NAG1008 phosphorous amide. The crude precursor 5 (1.449 mmol, 1.89 g) was dried by double toluene evaporation and redissolved in anhydrous DCM (60 mL). N,N,-diisopropyltetrazolium ammonium (348 mg, 2 mmol) and molecular sieve (100 mg) were added, and the mixture was stirred for 45 min. 2-Cyanoethyl N,N,N',N'-tetraisopropyl diphosphonamide (611 mg, 2 mmol) was added, and the mixture was stirred for 4 h. The mixture was filtered and stirred for 15 min with cold NaHCO3 (vv 10%) in DCM. The aqueous layer was separated, extracted with CHCl3 (x2), dried (Na2SO4), filtered, and concentrated. The crude product (2.02 g) was purified on a CombiFlash column. Column: 24 g, liquid-loaded. Eluent: A = DCM; B = MeOH 20% in DCM, 0-25%, 30 min. Yield: 1.357 g (62%). MS: Calculated MW 1503.72 measured value ESIMS - m / z = 1502.42 [MH + ]. NMR P-31, (DMSO, d6): 144.913, 144.944. Synthesis of compound 2: Cool the solution of compound 1 (1 equivalent) in MeOH (12 volumes) to 0°C. Add TFA (0.8 volumes) and water (0.8 volumes) to the stirred solution. Allow the solution to warm to room temperature and stir for 2 hours. Concentrate the reaction mixture and use the crude product of compound 2 for the next step. Synthesis of compound 3: Under a nitrogen atmosphere, crude compound 2 (1 equivalent) was dissolved in acetonitrile (8 volumes) and cooled in an acetone / dry ice bath. DBU (1.5 equivalent) and Br-PEG3-NHBoc (1.05 equivalent) were added, and the reaction was heated to room temperature and stirred overnight. The solvent was evaporated, and the crude product was purified by column chromatography to obtain compound 3. Synthesis of compound 4: Compound 3 (1 equivalent) was added to a flask, along with 3.7 times its volume of HCl solution (4 M in 1,4-dioxane), and the mixture was stirred at room temperature for 2.5 hours. The solution was concentrated, and the crude product of compound 4 was used in the next step. Synthesis of compound 6: Crude compounds 4 (3.7 equivalents) and 5 (1 equivalent) were dissolved in DMF (50 volumes). DIEA (15 equivalents) and TBTU (3.5 equivalents) were added to this solution, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated, and the crude product was then dissolved in chloroform (3.5 volumes) and washed with water / saline (1:1) and water / saturated NaHCO3 (1:1) solutions. The organic layer was dried over Na2SO4 and concentrated. The crude product was purified by column chromatography to give compound 6. Synthesis of compound 7: A solution of compound 6 (1 equivalent) and TFA (10 equivalent) was stirred in MeOH for 2 hours at room temperature in the presence of Pd / C (50 wt%) under a H2 atmosphere and at room temperature. The solution was filtered and concentrated to give compound 7. Synthesis of compound 9: Compound 7 was dissolved in DCM (32 volumes) under a nitrogen atmosphere and cooled to 0°C. In another flask, compound 8 (1.1 equivalents), TBTU (1.1 equivalents), and DIEA (3.5 equivalents) were stirred in DCM (20 volumes) for 15 minutes, then added to the compound 7 solution and stirred at room temperature. After 3 hours, compound 8 (1.1 equivalents), TBTU (1.1 equivalents), and DIEA (3.5 equivalents) were again stirred in DCM for 10 minutes in another flask and added to the main reaction flask. After stirring for another 15 minutes, saturated NH4Cl was added and extracted with DCM (3 times). The combined organic layers were washed with saturated NaHCO3 solution and brine. The mixture was then dried over Na2SO4 and concentrated. The crude product was purified by column chromatography to give compound 9. Synthesis of NAG55 phosphorous amide: Compound 9 was dissolved in DCM (15 times its volume) under a nitrogen atmosphere. 2-Cyanoethyl N,N,N',N'-tetraisopropyl diphosphonamide (2.7 equivalents) and diisopropyltetraazole ammonium (0.7 equivalents) were added, and the mixture was stirred for 2 hours. Then, more 2-cyanoethyl N,N,N',N'-tetraisopropyl diphosphonamide (0.5 equivalents) and diisopropyltetraazole ammonium (0.3 equivalents) were added, and the mixture was stirred for another 1.5 hours. The reaction mixture was cooled to 0°C and washed with a saturated NaHCO3 solution. The organic layer was dried over Na2SO4 and concentrated. The crude product was purified by column chromatography to give NAG55 phosphoramide. Example 3. Conjugation of linkers and targeting ligands to RNAi agents A. Concatenation of activated ester joints One potential method for linker conjugation is through coupling with an activated ester. In some embodiments, the following procedure can be used to conjugate a linker group having a terminal propargyl group to an RNAi agent having an amine-functionalized sense chain, such as C6-NH2, NH2-C6, or (NH2-C6)s, as shown in Table 2 above. The lyophilized annealed RNAi agent is dissolved at 25 mg / mL in DMSO and 10% water (v / v%). Then, 50-100 equivalents of TEA and 3 equivalents of the activated ester linker are added to the solution. The solution is allowed to react for 1-2 hours, monitored by RP-HPLC-MS (mobile phase A: 100 mM HFIP, 14 mM TEA; mobile phase B: acetonitrile, on an XBridge C18 column, Waters Corp.). The product can then be precipitated by adding 12 mL of acetonitrile and 0.4 mL of PBS and centrifuging the solid. The precipitate is then redissolved in 0.4 mL of 1XPBS and 12 mL of acetonitrile. The resulting granules are dried under high vacuum for one hour. B. Conjugation of the targeted ligand to the propargyl linker Similarly, another acceptable method for conjugating the targeted ligands of the compounds disclosed herein is through their conjugation with propargyl linkers. In some embodiments, the 5' or 3' tridentate alkyne functionalized sense chain can be conjugated with the NAG ligand before or after annealing. A possible method for conjugating the metabolically stable NAG for α / β-anodes with the annealed duplex is described below: A stock solution of 0.5 M tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 0.5 M copper(II) sulfate pentahydrate (Cu(II)SO4·5H2O), and 2 M sodium ascorbate solution is prepared in deionized water. A 75 mg / mL solution of the NAG ligand azide is prepared in DMSO. 25 µL of 1 M Hepes pH 8.5 buffer is added to a 1.5 mL centrifuge tube containing the trikyne functionalized duplex (3 mg, 75 µL, 40 mg / mL in deionized water, approximately 15000 g / mol). After vortexing, add 35 µL of DMSO and vortex the solution. Then, the ligand can be added to the reaction (e.g., 6 equivalents / dichain, 2 equivalents / acetylene, approximately 15 µL), and the solution vortexed. Check the pH using pH paper and confirm that the pH is approximately 8. In a separate 1.5 mL centrifuge tube, mix 50 µL of 0.5 M THPTA with 10 µL of 0.5 M Cu(II)SO4·5 H2O, vortex, and incubate at room temperature for 5 minutes. After 5 minutes, add the THPTA / Cu solution (7.2 µL, 6 equivalents 5:1 THPTA:Cu) to the reaction flask and vortex. Immediately afterwards, add 2 M ascorbic acid (5 µL, 50 equivalents / dichain, 16.7 equivalents / acetylene) to the reaction flask and vortex. Once the reaction is complete (usually within 0.5–1 h), the reaction mixture is immediately purified by non-denaturing anion exchange chromatography. C. Conjugation of targeted ligands to amine-functionalized sense chains In some embodiments, the following procedure can be used to conjugate an activated ester-functionalized targeting ligand (such as a metabolically stable carbohydrate ligand) with an amine-functionalized RNAi agent containing amines (such as C6-NH2, NH2-C6, or (NH2-C6)s, as shown in Table 2): The annealed lyophilized RNAi agent is dissolved at 25 mg / mL in DMSO and 10% water (v / v%). Then, 50-100 equivalents of TEA and three equivalents of the activated ester-functionalized ligand are added to the mixture. The reaction mixture is stirred for 1-2 hours while being monitored by RP-HPLC-MS (mobile phase A: 100 mM HFIP, 14 mM TEA; mobile phase B: acetonitrile; column: XBridge C18). After the reaction mixture is complete, 12 mL of acetonitrile is added, followed by 0.4 mL of PBS, and the mixture is then centrifuged. The solid particles are collected and dissolved in 0.4 mL of 1xPBS, followed by 12 mL of acetonitrile. The resulting particles are collected and dried under vacuum for 1 hour. D. By synthesizing phosphorusamide or by adding a targeted ligand to the resin. Other acceptable methods for coupling targeting ligands include preparing the desired ligand as a phosphoramidite compound, which can be added to the 5' end of the chain using standard solid-phase synthesis, or preparing the targeting ligand on a resin using standard solid-phase oligonucleotide synthesis, which can be placed at the 3' end of the chain after cleavage. Example 4. In vivo administration of metabolically stabilized RNAi conjugates in cynomolgus monkeys Crab-eating macaques ( Macaca fascicularis Gene silencing activity of multimeric (dimeric) RNAi agent conjugates, including metabolically stable NAG-targeting ligands, was evaluated in primates (referred to herein as "cynos"). Each multimeric RNAi agent conjugate evaluated consisted of one RNAi agent with sufficient complementarity to the mouse angiopoietin-like 3 (ANGPTL3) gene transcript and a second RNAi agent with sufficient complementarity to the mouse factor 12 (FXII) gene transcript. The expression levels of both the FXII and ANGPTL3 genes were evaluated, as discussed in more detail below. According to Table 3 below, on day 1, male cynomolgus monkeys were given a single subcutaneous administration of 0.3 mL / kg of animal body weight (20 mg / mL concentration), which contained a multimeric RNAi conjugate prepared in isotonic saline at 6.0 mg / kg (mpk), or two separate RNAi conjugates at 3.0 mg / kg. Table 3: Dosage groups for Example 4. Each multimeric RNAi conjugate and single monomeric RNAi conjugate in Group 1 contains a modified nucleotide. The RNAi conjugates were synthesized on a solid phase using a phosphoramidite technique, following general procedures known in the art and commonly used in oligonucleotide synthesis, as described in Example 1 of this document. For all RNAi conjugates, the targeting ligand was positioned at the 5' end of the sense strand, as shown in Table 4 below: Table 4 Abbreviations: a, c, g, and u represent 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methylguanosine, and 2'-O-methyluridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoroadenosine, 2'-fluorocytidine, 2'-fluoroguanosine, and 2'-fluorouridine, respectively; s represents thiophosphate ester bonding; invAb represents reverse debased deoxyribose residues (see Table 2); sp18 represents the spacer 18-polyethylene glycol (PEG) linker shown in Table 2; C6-NH2 represents the amino linker or end cap shown in Table 2; NAG37s represents an N-acetylgalactosamine trimer composed of the following structures: NAG42s represents a metabolically stable NAG trimer with the structure shown in Table 2; and NAG52s represents a metabolically stable NAG trimer with the structure shown in Table 2. The NAG37s structure was added to the sense chain as a phosphoramidite compound and was typically synthesized according to International Patent Application Publication No. WO 2018 / 044350 of Arrowhead Pharmaceuticals, Inc., which is incorporated herein by reference in its entirety as if fully set forth herein. The NAG42s and NAG52s structures were also added to the sense chain according to Examples 1, 2, and 3 herein. In Table 4 above, AS refers to the antisense strand and SS refers to the sense strand. Individual nucleotides in the strands, though separated by commas in the table for convenience, are linked together by phosphodiester bonds, unless an "s" is present, in which case thiophosphate bonds replace phosphodiester bonds, linking the nucleotide or non-nucleotide components of each corresponding strand. The antisense strand is then annealed to the corresponding sense strand. As used throughout this document for the disclosed multimeric RNAi agent conjugates, the "first" antisense strand or As(1) in Table 4 above refers to the antisense strand located at the 3' end of the sense strand-multimeric RNAi conjugate complex. Each additional RNAi agent added to the multimeric conjugate (e.g., AS(2), AS(3), etc.) is located closer to the 5' end of the sense strand. As described above and discussed herein, the group 3 multimeric RNAi agent conjugate (AD14217) comprises a metabolically stable NAG of formula II (and more specifically, formula IIa), and the group 4 multimeric RNAi agent conjugate (AD14228) comprises a metabolically stable NAG of formula I (and more specifically, formula Ia). The group 2 multimeric RNAi agent conjugate (AD14216) and the group 1 monomeric RNAi agent (AD14219+AD14220) each comprise an unmetabolic N-acetylgalactosamine targeting ligand and have the structure of NAG37 described above. Each targeting ligand is linked to its respective RNAi agent via a phosphate thioester bond. Each group was administered to three (3) cynomolgus monkeys (n=3). Serum samples were collected on day -14, day -7, and day 1 (before administration). The monkeys were then administered to the corresponding groups as shown in Table 5. Serum samples were then collected on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 98, 106, 120, 133, 148, and 162. Serum ANGPTL3 protein levels were measured via ELISA assay (R&D Systems) as recommended by the manufacturer. Serum FXII protein levels were also measured via ELISA assay (R&D Systems) as recommended by the manufacturer. ANGPTL3 and FXII protein levels for each animal were normalized. To normalize, the ANGPTL3 or FXII protein level for each animal at a given time point was divided by the geometric mean of the animal's pre-treatment expression levels (in this case, at day -14, day -7, and day 1 (before administration)) to determine the "normalized / pre-treatment" expression ratio. Expression at a specific time point was then normalized relative to the saline control group by dividing the individual animal's "normalized / pre-treatment" ratio by the mean "normalized / pre-treatment" ratio of all mice in the saline control group. This resulted in normalized expression at each time point relative to the control group. The research data in this embodiment are shown in Tables 5 and 6 below: This embodiment illustrates the practicality of the delivery platform of the present invention. Regarding ANGPTL3 protein levels in cynomolgus monkeys, for example, on day 22 (i.e., 3 weeks after administration), the co-administered monomeric conjugate in group 1 achieved approximately 76% cANGPTL3 knockdown (0.244), group 2 (using a dimer RNAi agent conjugate with a NAG-targeting moiety) achieved approximately 66% cANGPTL3 knockdown (0.339), while the metabolically stable multimeric RNAi conjugates in group 3 (with a metabolically stable NAG with a β-anodic bond) and group 4 (with a metabolically stable NAG with an α-anodic bond) showed approximately 81% (0.194) and 86% (0.137) cANGPTL3 knockdown, respectively. On day 106 (more than 3 months post-dose), the co-administered monomeric conjugate in group 1 and the dimeric RNAi conjugate with the NAG-targeting moiety in group 2 essentially returned to baseline, showing no substantial knockdown of cANGPTL3. Conversely, on day 106, the dimeric RNAi conjugates with metabolically stable NAG-targeting ligands in groups 3 (knockdown of approximately 33% (0.777)) and 4 (knockdown of approximately 45% (0.565)) still showed gene silencing activity. Regarding FXII protein levels in cynomolgus monkeys, for example, on day 22 (3 weeks post-dose), the co-administered monomeric conjugate in group 1 achieved approximately 71% cFXII knockdown (0.295); group 2 (using a dimeric RNAi conjugate with a NAG-targeting moiety) and group 3 (a dimeric RNAi conjugate with a metabolically stable NAG anomeric linker linked to an α-anomeric linker) both achieved approximately 78% cFXII knockdown, and group 4 (a dimeric RNAi conjugate with a metabolically stable NAG anomeric linker linked to a β-anomeric linker) showed approximately 85% knockdown (0.156). On day 98 (more than 3 months post-dose), the co-administered monomeric conjugate in group 1 regressed to showing only 25% cFXII knockdown, and similarly, the dimeric RNAi conjugate with a NAG-targeting moiety in group 2 showed only approximately 41% knockdown (0.592). Meanwhile, the dimer RNAi conjugates with metabolically stable NAG-targeting ligands in Group 3 (approximately 69% knockdown (0.316)) and Group 4 (approximately 86% knockdown (0.139)) provided significantly higher gene knockdown on day 98, indicating a longer duration of silencing activity in this study. In fact, even on day 162 (more than 5 months post-dose), the dimer RNAi conjugate with metabolically stable NAG-targeting ligand (AD14218) in Group 4 still showed more than 70% (0.285) cFXII inhibition. While this particular embodiment includes RNAi agents for inhibiting ANGPTL3 and FXII, the same multimeric RNAi agent delivery platform can be used to inhibit gene expression of other genes present in the liver (including hepatocytes). Example 5 : In vivo administration of APOC3-PCSK9 RNAi agents in cynomolgus monkeys. The inhibitory effects of the APOC3-PCSK9 RNAi agent on APOC3 and PCSK9 were tested in cynomolgus monkeys. On day 1, using a syringe and needle, three (n=3) male cynomolgus monkeys in each test group were administered APOC3-PCSK9 RNAi agent (6.0 mg / kg) via subcutaneous (SQ) injection into the mid-scapular region at a dose volume of 20.0 mL / kg. Crab-eating macaques were acclimatized to their environment for at least one (1) day. These animals were 2 to 7 years old. Animals were not mixed for at least 24 hours after administration of the test substance (RNAi agent) to monitor for any effects associated with the test substance. Certified Primate Diet Feed the animals with 5048 (PMI, Inc.) and provide Greenfield municipal water for free drinking. Keep the animals in a temperature of 20 to 26 degrees Celsius, relative humidity of 50 + / - 20%, and a 12-hour light / 12-hour dark cycle. The dosing regimen is shown in Table 7 below. Table 7: Dosing regimen for cynomolgus monkeys in Example 5. Following general procedures known in the art and commonly used in oligonucleotide synthesis, each multimeric RNAi conjugate and single-monomer RNAi conjugate were synthesized on a solid phase using phosphoramide technology. For all RNAi conjugates, the targeting ligand was positioned at the 5' end of the sense strand, as described in Table 8 below: Table 8 Abbreviations: a, c, g, and u represent 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methylguanosine, and 2'-O-methyluridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoroadenosine, 2'-fluorocytidine, 2'-fluoroguanosine, and 2'-fluorouridine, respectively; s represents thiophosphate bond; invAb represents reverse debased deoxyribose residue (see Table 2); sp18 represents the spacer 18-polyethylene glycol (PEG) linker shown in Table 2; C6-NH2 represents the amino linker or end cap shown in Table 2, and NAG52s represents a metabolically stable NAG trimer with the structure shown in Table 2. Before each SQ injection, the test animals were first sedated. Sedation was achieved using ketamine hydrochloride (10 mg / kg) or teprazole (5-8 mg / kg) administered as an intramuscular (IM) injection, with ketamine supplemented as needed (5 mg / kg). Administer the APOC3-PCSK9 RNAi agent to test animals via subcutaneous injection (SQ) using a syringe and needle in the scapular region (upper left, upper right, lower left, or lower right scapular region). Shave the hair at the administration site at least one day prior to each dose administration. Calculate the individual dose of the APOC3-PCSK9 RNAi agent based on the body weight recorded on each administration day. On each administration day, allow the APOC3-PCSK9 RNAi agent to warm to ambient temperature for at least 30 minutes at approximately room temperature before administration. Fast the animals overnight before administration. Serum blood (approximately 5.0 mL) was collected on days -6, 8, 15, 22, 29, 36, 43, 50, 57, and 63 prior to liver biopsy sample collection or dosage administration (if applicable), and collected from any animal in a near-death state or euthanized at an unplanned time. The femoral vein was the collection site, with the great saphenous vein used as an alternative collection site. Liver biopsies and serum collected from test animals were used to analyze APOC3 and PCSK9 expression, as well as other biological parameters. Liver biopsies were collected on days -6, 15, 36, 50, and 64 (autopsy). Liver biopsy collection was performed under sedation. Animals were fasted overnight (at least 12 hours but less than 18 hours) prior to each liver biopsy collection. The liver biopsy sample collected from each animal was approximately 100 mg (80 to 120 mg). The expression of APOC3 and PCSK9, as well as other biological parameters, in collected liver biopsies were analyzed. Using cARL1 as an endogenous control gene, the expression levels of APOC3 and PCSK9 mRNA in the liver were quantified by qPCR and normalized relative to day 6 (before administration). The qPCR APOC3 and PCSK9 expression data are shown in Tables 9 and 10 below. Table 9: APOC3 expression in the liver of cynomolgus monkeys in Example 5. The APOC3-PCSK9 RNAi agent achieved APOC3 transcript knockdown for at least 64 days via subcutaneous SQ injection at 6.0 mg / kg on day 1. APOC3 knockdown was achieved in both groups 1 and 2. More specifically, at 6.0 mg / kg, AC003791 achieved approximately 66% inhibition (0.336) on day 50. At a single 6.0 mg / kg dose, AC003791 achieved approximately 50% inhibition (0.500) on day 64. Table 10: PCSK9 expression in the liver of cynomolgus monkeys in Example 5. The APOC3-PCSK9 RNAi agent achieved PCSK9 transcript knockdown for at least 64 days via a single subcutaneous SQ injection of 6.0 mg / kg on day 1. PCSK9 knockdown was achieved in both groups 1 and 2. More specifically, at a single 6.0 mg / kg dose, AC003791 achieved approximately 64% inhibition (0.351) on day 64. Serum PCSK9 was quantified via ELISA (R&D Systems, catalog number DPC900) according to the manufacturer's instructions. Relative PCSK9 levels were normalized relative to day -6 before administration. Data are shown in Table 11 below. Table 11: Serum PCSK9 expression in cynomolgus monkeys of Example 5. The APOC3-PCSK9 RNAi agent achieved serum PCSK9 knockdown for at least 64 days via a single subcutaneous SQ injection of 6.0 mg / kg on day 1. PCSK9 knockdown was achieved in both groups 1 and 2. More specifically, at a single 6.0 mg / kg dose, AC003791 achieved approximately 67% inhibition (0.323) on day 36 (the lowest point). At a single 6.0 mg / kg dose, AC003791 achieved approximately 46% inhibition (0.538) on day 64. Serum APOC3 was quantified according to the manufacturer's instructions using the Roche Cobas® assay for APOC3. The data are shown in Table 12 below. Table 12: Serum APOC3 expression in cynomolgus monkeys in Example 5. The APOC3-PCSK9 RNAi agent achieved serum APOC3 knockdown for at least 64 days via a single subcutaneous SQ injection of 6.0 mg / kg on day 1. APOC3 knockdown was achieved in both groups 1 and 2. More specifically, at a single 6.0 mg / kg dose, AC003791 achieved approximately 51% inhibition on day 36 (the lowest point) (2.96 mg / dL APOC3 on day 36, compared to 6.15 mg / dL APOC3 on day -6). Furthermore, at a single 6.0 mg / kg dose, AC005898 achieved approximately 40% inhibition on day 22 (the lowest point) (3.50 mg / dL APOC3 on day 22, compared to 5.84 mg / dL on day -6). At a single dose of 6.0 mg / kg, AC003791 achieved approximately 24% inhibition on day 64 (4.65 mg / dL, compared to 6.15 mg / dL on day -6). At a single dose of 6.0 mg / kg, AC005898 achieved approximately 23% inhibition on day 64 (4.47 mg / dL, compared to 5.84 mg / dL on day -6). Example 6: In vivo administration of multimeric FXII gene X RNAi agent in cynomolgus monkeys. The inhibitory effects of FXII and single gene target (referred to as "gene X") multimeric RNAi agents produced in human hepatocytes on factor XII (FXII) and gene X were tested in cynomolgus monkeys. The AS(1) of each dimer used in this embodiment is the same as the AS(1) of AD14217 and AD14218 shown in Example 4 above. The AS(2) of each dimer used in this embodiment is the same in groups 1, 2 and 3 and is complementary to the 19 nucleotide sequence of the mRNA encoded by gene X. The dimers used in this embodiment include the same sense strand sequence as AD14217 and AD14218 shown in Example 4 above, except for the metabolically stable carbohydrate ligands as shown in Table 13, and in this case, the sense strand portion complementary to ANGPTL3 AS(2) in AD14217 and AD14218 is a modified nucleotide complementary to gene X AS(2). On day 1, using a syringe and needle, three (n=3) male cynomolgus monkeys in each test group were given a saline-prepared multimeric FXII gene XRNAi agent (6.0 mg / kg) via subcutaneous (SQ) injection into the mid-scapular region at a dose volume of 0.3 mL / kg. The test animals were male cynomolgus monkeys (non-naïve). The RNAi agent was administered subcutaneously (SQ) into the mid-scapular region using a syringe and needle. The dosing regimen is shown in Table 13 below. Table 13: Dosing regimen for cynomolgus monkeys in Example 6. Each multimeric RNAi conjugate was synthesized on a solid phase using phosphoramide technology, following general procedures known in the art and commonly used in oligonucleotide synthesis. According to embodiments 1, 2 and 3 of this paper, the (NAG52)s, (NAG55)s and (NAG1008)s structures are also added to the meaningful chain. As described above and discussed herein, the group 1 of multimeric RNAi agent conjugates includes metabolically stable targeting ligands (NAG52)s (see Table 1), the group 2 of multimeric RNAi agent conjugates includes metabolically stable targeting ligands (NAG55)s (see Table 1), and the group 3 of multimeric RNAi agent conjugates includes metabolically stable targeting ligands (NAG1008)s (see Table 1). Prior to each SQ injection, the test animals were sedated. Sedation was achieved using ketamine hydrochloride (10 mg / kg) administered intramuscularly (IM). The individual dose of the multimeric FXII-gene X RNAi agent was calculated based on the body weight recorded on each dosing day. Animals were fasted overnight for at least 12 hours but less than 24 hours before administration. Serum blood (approximately 5.0 mL) was collected on days -14, -7, day 1 (before administration), day 8, day 15, day 22, day 29, day 36, day 43, day 50, day 57, day 64, day 71, day 78, day 85, day 92, and day 99, and was collected from any animal in a near-death state or euthanized at an unplanned time. The femoral vein was the collection site, with the great saphenous vein used as an alternative site. The expression of FXII and X genes, as well as other biological parameters, in collected serum samples were analyzed. Serum FXII and gene X protein levels were quantified by ELISA according to the manufacturer's instructions, and relative expression was normalized for each experimental group prior to drug administration. The quantified FXII and gene X protein levels are shown in Tables 14 and 15 below. Table 14: Serum FXII levels in cynomolgus monkeys of Example 6. A single subcutaneous SQ injection of 6.0 mg / kg on day 1 achieved FXII inhibition with the FXII-gene X-polymer RNAi agent until at least day 99. At the lowest point, a single 6.0 mg / kg dose of the NAG55S conjugate dimer achieved ~76% FXII inhibition on day 36 (0.238). On day 99, a single 6.0 mg / kg dose of the NAG52S conjugate dimer achieved ~63% FXII inhibition (0.363). Table 15: Serum gene X levels in cynomolgus monkeys of Example 6. Gene X inhibition was achieved by a single subcutaneous SQ injection of 6.0 mg / kg on day 1, with the FXII-Gene X multimer RNAi agent lasting until at least day 99. At its lowest point, a single 6.0 mg / kg dose of the NAG52s conjugate dimer achieved ~87% inhibition of Gene X (0.129) on day 22. On day 99, a single 6.0 mg / kg dose of the NAG52s conjugate dimer achieved ~71% inhibition of Gene X (0.284). Other implementation plans It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and improvements are within the scope of the following claims.

Claims

1. A compound or a pharmaceutically acceptable salt thereof for inhibiting the expression of one or more genes, comprising: a. Oligonucleotides, which consist of chains of 12-49 nucleotides in length; and b. Metabolically stable carbohydrate ligands; The oligonucleotide and the metabolically stable carbohydrate ligand therein are covalently linked by a thiophosphate bond, a dithiophosphate bond, or another bond that is more metabolically stable than a phosphodiester bond.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the metabolically stable carbohydrate ligand has the following formula: ， in: Each instance of the metabolically stable carbohydrate is independently a chemically modified carbohydrate portion; Each instance of the tethering is independently represented by the following formula: or , where m is an integer selected from 1 to 20; The branch point group is selected from the following structures: and ； The connector is selected from the following structures: and ; n is an integer from 1 to 4, which is allowed for the valence. and Indicates the attachment point of the rest of the compound.

3. The compound of claim 2, wherein the tether has the following formula: .

4. The compound of claim 2, wherein the tether has the following formula: .

5. The compound of any one of claims 2-4, wherein the branching group has the following formula: .

6. The compound of any one of claims 2-5, wherein the branching group has the following formula: .

7. The compound of any one of claims 2-4, wherein the branching group has the following formula: .

8. The compound of any one of claims 2-4 or 7, wherein the branch point group has the following formula: .

9. The compound of any one of claims 1-8, wherein the metabolically stable carbohydrate ligand comprises metabolically stable N-acetylgalactosamine.

10. The compound of any one of claims 1-9, wherein the metabolically stable carbohydrate has the following formula: Where X is CH2 or S, and Indicates the connection point with the rest of the compound.

11. The compound of any one of claims 1-9, wherein the metabolically stable carbohydrate has the following formula: Where X is CH2 or S, and Indicates the connection point with the rest of the compound.

12. The compound of claim 10 or 11, wherein X is CH2.

13. The compound of claim 10 or 11, wherein X is S.

14. The compound of any one of claims 1-6 or 9-13, wherein the metabolically stable carbohydrate ligand comprises three metabolically stable N-acetylgalactosamine moieties.

15. The compound of any one of claims 2-6 or 9-13, wherein n is 3.

16. The compound of any one of claims 2-15, wherein the connector has the following formula: .

17. The compound of any one of claims 1-16, wherein the metabolically stable carbohydrate ligand comprises a structure selected from: in Indicates the connection point with the rest of the compound.

18. The compound of any one of claims 1-17, wherein the oligonucleotide is a double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein: (i) The sense strand of the double-stranded RNAi agent contains 19-23 nucleotides; and (ii) The antisense strand of the double-stranded RNAi agent contains 19-23 nucleotides.

19. The compound of any one of claims 1-18, wherein the sense strand comprises 19-21 nucleotides and the antisense strand comprises 19-21 nucleotides.

20. The compound of any one of claims 1-19, wherein the antisense strand of the double-stranded RNAi agent consists of 19 nucleotides.

21. The compound of any one of claims 1-20, wherein the antisense strand is at least partially complementary to the mRNA sequence encoded by a gene expressed in human hepatocytes.

22. The compound of any one of claims 1-21, wherein the antisense strand is completely complementary to the mRNA sequence encoded by a gene expressed in human hepatocytes.

23. The compound of any one of claims 1-22, wherein the metabolically stable carbohydrate ligand is conjugated to the 3' end of the sense chain.

24. The compound of any one of claims 1-23, wherein the metabolically stable carbohydrate ligand is conjugated to the 5' end of the sense chain.

25. The compound of any one of claims 1-24, wherein the end cap is located at the 3' end of the first sense chain, the 3' end of the second sense chain, or the 3' end of both the first sense chain and the second sense chain.

26. The compound of claim 25, wherein the end cap is a reverse non-base moiety or NH2-C6.

27. A compound comprising a structure selected from the following: Or its pharmaceutically acceptable salt, wherein Indicates the connection point with the rest of the compound.

28. The compound of claim 27, wherein the compound has the following formula: ， Or its pharmaceutically acceptable salt, wherein Indicates the connection point with the rest of the compound.

29. The compound of claim 27, wherein the compound has the following formula: ， Or its pharmaceutically acceptable salt, wherein Indicates the connection point with the rest of the compound.

30. The compound of claim 27, wherein the compound has the following formula: ， Or its pharmaceutically acceptable salt, wherein Indicates the connection point with the rest of the compound.

31. The compound of claim 27, wherein the compound has the following formula: ， Or its pharmaceutically acceptable salt, wherein Indicates the connection point with the rest of the compound.

32. The compound of any one of claims 27-31, wherein the remainder of the compound comprises an oligonucleotide chain of 12-49 nucleotides in length.

33. The compound of claim 32, wherein the oligonucleotide is a double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein: (i) The sense strand of the double-stranded RNAi agent contains 19-23 nucleotides; and (ii) The antisense strand of the double-stranded RNAi agent contains 19-23 nucleotides.

34. The compound of claim 33, wherein the sense strand comprises 19-21 nucleotides and the antisense strand comprises 19-21 nucleotides.

35. The compound of claim 33 or 34, wherein the antisense strand of the double-stranded RNAi agent consists of 19 nucleotides.

36. The compound of any one of claims 33-35, wherein the antisense strand is at least partially complementary to the mRNA encoded by a gene expressed in human hepatocytes.

37. The compound of any one of claims 33-36, wherein the antisense strand is completely complementary to the mRNA encoded by a gene expressed in human hepatocytes.

38. The compound of any one of claims 33-37, wherein the end cap is located at the 3' end of the sense chain.

39. The compound of any one of claims 33-38, wherein the end cap is located at the 5' end of the sense chain.

40. A pharmaceutical composition comprising the compound of any one of claims 1-39 and a pharmaceutically acceptable excipient.

41. A method of inhibiting gene expression, comprising administering to a subject in need of such a method a compound of any one of claims 1-39.

42. A method of inhibiting gene expression, comprising administering the pharmaceutical composition of claim 40 to a subject in need of such inhibition.

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