Metabolically stable hydrocarbon target ligands for oligonucleotide conjugates

Abstract

This disclosure relates to a delivery platform for specifically and efficiently delivering a stable RNAi agent payload to target hepatocytes in vivo. The delivery platform disclosed herein comprises a metabolically stable N-acetylgalactosamine (NAG or GalNAc) target ligand conjugated to one or more oligonucleotides via a metabolically stable linkage that is more stable than phosphodiester linkage, thereby facilitating the delivery of oligonucleotide-based payloads to cells, including hepatocytes. Pharmaceutical compositions comprising the metabolically stable RNAi agent conjugate delivery platform, as well as methods of use for the treatment of various diseases and disorders in which delivery of a therapeutic payload to hepatocytes is desirable, are also described.

Description

[Technical Field]

[0001] References to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 63 / 505,414, filed on 31 May 2023, and U.S. Provisional Patent Application No. 63 / 559,728, filed on 29 February 2024, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted in XML format, which is incorporated in its entirety by reference. The XML copy is named 30725-WO_SeqListing_2024.05.30.xml, was created on May 30, 2024, and is 250kb in size.

[0003] This disclosure relates to a delivery platform for the in vivo delivery of oligonucleotides or oligonucleotide-based drugs (e.g., antisense oligonucleotides (ASOs), double-stranded RNAi agents, or small interfering RNAs (siRNAs)) to liver cells, particularly hepatocytes. The delivery of RNAi agents using the delivery platform disclosed herein is provided for the inhibition of genes expressed in hepatocytes. [Background technology]

[0004] Delivering therapeutic payloads to specific tissues and target cells in vivo remains a major challenge in the pharmaceutical field. This is especially true for oligonucleotide-based therapies, such as antisense oligonucleotides (ASOs) and RNA interference (RNAi) drugs (typically involving small (or short) interfering RNAs using chemically modified nucleotides), which have revolutionized the medical field and demonstrate great potential to provide powerful treatment options for diseases that were previously difficult to treat. However, this presupposes that therapeutic oligonucleotides can reach target cells and tissues in vivo. In reality, achieving proper delivery of oligonucleotide-based therapies remains one of the most pressing challenges to overcome in the discovery and identification of viable therapies.

[0005] While developments over the past few decades have led to a better understanding of how to properly deliver oligonucleotide payloads to hepatocytes by covalently linking them to target ligands, including N-acetylgalactosamine (NAG or GalNAc), further improvements are needed and desired. Improved delivery would allow for less drug to be administered to patients or subjects, offering the benefit of reducing the potential for toxicological side effects and potentially lowering the cost of therapeutics due to fewer materials required for manufacturing.

[0006] One potential solution is to stabilize the NAG ligand to prevent its metabolic degradation. NAG ligands have been shown to degrade under physiological conditions before they can deliver oligonucleotide payloads to hepatocytes.

[0007] Therefore, there remains a demand for mechanisms or platforms that can specifically and efficiently deliver oligonucleotide-based therapeutics, particularly RNAi agents, to hepatocytes. [Overview of the project]

[0008] Disclosed herein is an in vivo delivery platform to hepatocytes comprising a metabolically stable hydrocarbon ligand linked to an oligonucleotide-based therapeutic agent, such as an antisense oligonucleotide (ASO) or RNA interference (RNAi) agent (also referred herein as RNAi agent, RNAi trigger, or trigger; e.g., a double-stranded RNAi agent or a small (or short) interfering RNA (siRNA)). Delivery of such therapeutic oligonucleotides facilitates selective and efficient inhibition of the expression of genes present in the liver, particularly genes present in hepatocytes.

[0009] Metabolically stable NAG ligands have been proposed to date, but the reported data have not shown any identifiable improvements or advantages over conventional standard NAG ligands. As supported by the examples described herein, this is at least in part due to the failure of others to simultaneously stabilize linkage to oligonucleotide molecules through more stable linkages than conventional phosphodiester bonds (e.g., the lack of introduction of more stable phosphorothioate or phosphorodithioate linkages), and thus the benefits of metabolically stable NAG ligands can be found.

[0010] One embodiment described herein is as follows: a. Oligonucleotides with a nucleotide length of 12 to 49 nucleotides; and b. Metabolic stable hydrocarbon ligands; A compound containing or a pharmaceutically acceptable salt thereof, wherein the double-stranded RNAi agent and the metabolically stable hydrocarbon ligand are covalently linked by a linkage more metabolically stable than phosphorothioate linkage, phosphorodithioate linkage, or phosphodiester linkage.

[0011] Another aspect described herein is: a. A double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand of the double-stranded RNAi agent contains 15 to 23 nucleotides, and the antisense strand of the double-stranded RNAi agent contains 18 to 23 nucleotides; b. A metabolically stable hydrocarbon ligand; A compound or a pharmaceutically acceptable salt thereof, wherein the double-stranded RNAi agent and the metabolically stable hydrocarbon ligand are covalently bonded by phosphorothioate linkages.

[0012] Some embodiments provided herein are as follows: a. An oligonucleotide 12 to 49 nucleotides in length; and b. A metabolically stable hydrocarbon ligand; A compound or a pharmaceutically acceptable salt thereof, wherein the double-stranded RNAi agent and the metabolically stable hydrocarbon ligand are covalently bonded by a linkage that is more metabolically stable than phosphorothioate linkages, phosphorodithioate linkages, or phosphodiester linkages; wherein the metabolically stable hydrocarbon ligand has the following formula:

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[0013] In some embodiments, metabolically stable hydrocarbon ligands have the following structure: [ka] The formula includes an N-acetylgalactosamine (NAG or GalNAc) target ligand, where X is CH2 or S, and an RNAi agent. As shown above, Formula I represents the β-anomeric linkage of a metabolically stable NAG ligand, and Formula II represents the α-anomeric linkage of a metabolically stable NAG ligand. Symbols used herein: [ka] This means that any or more groups are linked thereto, in accordance with the scope of the invention described herein.

[0014] Metabolically stable hydrocarbon ligands and oligonucleotides may be covalently linked in any manner known in the art. However, it is assumed that all linkers used to covalently link the various components are metabolically stable linkages that are more stable in vivo than phosphodiester bonds. Exemplary embodiments of multimeric RNAi agent conjugates according to the inventions disclosed herein can be found in various examples herein.

[0015] Preferably, in a metabolically stable compound having the chemical structure of formula I or formula II, X is CH2, as shown in formulas Ia and IIa below. [ka]

[0016] Preferably, the oligonucleotide is a double-stranded RNAi agent comprising a sense strand and an antisense strand, and more preferably, a metabolically stable hydrocarbon ligand is ligated to the sense strand of the RNAi agent.

[0017] Metabolically stable NAG-RNAi conjugates have been previously reported to be able to deliver RNAi agents (especially siRNAs) to hepatocytes in vivo, but the previously reported results were based on the following structure: [ka] It has been shown that there is no difference in asialoglycoprotein receptor (ASGPR) affinity or gene silencing activity compared to known standard GalNAc ligands with metabolically unstable glycosidic linkages as shown (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 a metabolically stable compound having the chemical structure of formula I or formula II is covalently linked to its components by a metabolically stable linker that is more stable than phosphodiester linkage, as supported by the data shown in the examples herein. (For comparison, see, for example, Abstract Figure Id in "Metabolically labileglycosidic linkage" (showing unstable phosphorothioate linkage to RNAi agents and monomeric RNAi agent conjugates)).

[0018] In some embodiments, a metabolically stable compound having the structure of formula I or formula II is linked to an RNAi agent by a linker that is less stable than a phosphodiester linkage, for example, a phosphorothioate linkage, or not a linker. In some embodiments, a metabolically stable compound having the structure of formula I or formula II is linked to an RNAi agent by a linker containing a stable phosphorothioate linkage or a phosphorodithioate linkage.

[0019] In further embodiments disclosed herein, in some embodiments, the length of the RNAi agent used in the multimeric RNAi agent conjugate delivery platform described herein includes a double strand having a sense strand of 21 nucleotides or less in length and an antisense strand of 21 nucleotides or less in length. In some embodiments, the multimeric RNAi agent conjugate delivery platform described herein includes a double strand having a sense strand of 19 nucleotides or less in length and an antisense strand of 19 nucleotides or less in length. As shown in the examples 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 the RNAi agent includes a sense strand and an antisense strand of 21 nucleotides or less, 20 nucleotides or less, or 19 nucleotides or less in length.

[0020] The metabolically stable hydrocarbon conjugates described herein can be used in methods for the treatment (preventive, interventional, and preventive therapies) of conditions and diseases that can be at least partially mediated by a reduction in the expression of a target gene, such as diseases that can be at least partially mediated by a reduction in the expression of one or more genes in hepatocytes. The compounds disclosed herein can selectively reduce the expression of a target gene in target cells, particularly hepatocytes of the liver. Methods disclosed herein include, for example, administering one or more multimeric RNAi agent conjugates or one or more ASO conjugates to a target, such as a human or animal, using any suitable method known in the art, such as intravenous infusion, intravenous injection, or subcutaneous injection.

[0021] Also described herein are pharmaceutical compositions comprising oligonucleotide conjugates disclosed herein that can inhibit the expression of one or more target genes, wherein the composition further comprises at least one pharmaceutically acceptable excipient. Pharmaceutical compositions described herein, comprising one or more disclosed RNAi agents or other oligonucleotide conjugates, or oligonucleotide-based drug conjugates, can selectively and efficiently reduce or inhibit the expression of target genes in vivo.

[0022] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Similar or equivalent methods and materials may be used in the practice or testing of the present invention, but appropriate methods and materials are described as such. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of any conflict, this specification, including definitions, shall prevail. Furthermore, the materials, methods, and examples are for illustrative purposes only and are not intended to limit the scope of this invention.

[0023] Other objects, features, embodiments, and advantages of the present invention will become apparent from the following detailed description, the accompanying drawings, and the claims. [Modes for carrying out the invention]

[0024] definition As used herein, the terms “oligonucleotide” and “polynucleotide” mean polymers of linked nucleosides, each of which may be independently modified or unmodified.

[0025] As used herein, “RNAi agent” (also referred to as “RNAi trigger”) means a composition comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of degrading or inhibiting (e.g., degradation or inhibition under appropriate conditions) the translation of a messenger RNA (mRNA) transcript of a target mRNA in a sequence-specific manner. As used herein, an RNAi agent may be operated through an RNA interference mechanism (i.e., through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) in mammalian cells) or by any alternative mechanism or pathway. As used herein, an RNAi agent is considered to be primarily operated through an RNA interference mechanism, but the disclosed RNAi agents are not constrained or limited to any particular pathway or mechanism of action. The RNAi agents disclosed herein include sense and antisense strands and, non-limitingly, include: short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), and Dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the target mRNA. The RNAi agent comprises one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0026] As used herein, the terms “silencing,” “reduction,” “inhibition,” “downregulate,” or “knockdown” mean, when referring to the expression of a particular gene, that gene expression, as measured at the RNA level transcribed from the gene or at the polypeptide, protein, or protein subunit level translated from mRNA, is reduced in cells, cell populations, tissues, organs, or subjects treated with the RNAi agents described herein compared to a second set of untreated cells, cell populations, tissues, organs, or subjects.

[0027] As used herein, the terms “sequence” and “nucleotide sequence” mean a sequence or order of nucleic acid bases or nucleosides, as written in string form using standard nomenclature.

[0028] As used herein, the terms “base,” “nucleotide base,” or “nucleic acid base” refer to heterocyclic pyrimidine or purine compounds that are components of nucleotides, including the major purine bases adenine and guanine, and the major pyrimidine bases cytosine, thymine, and uracil. Nucleic acid bases may be further modified, non-limitingly, to include universal bases, hydrophobic bases, polyvalent bases, size-extended bases, and fluorinated bases (see, for example, Modified nucleoside in Biochemistry, Biotechnology and Medicine. Herdewijn, P. ed. Wiley-VCH, 2008). The synthesis of such modified nucleic acid bases (including phosphoramidites containing modified nucleic acid bases) is known in the art.

[0029] Where used herein, unless otherwise specified, the term “complementary” means the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence under specific standard conditions (forming base-pair hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro)) to form a double-stranded or double-helical structure. This includes Watson-Crick base pairs or non-Watson-Crick base pairs, and includes natural or modified nucleotides or nucleotide mimetic derivatives, to the extent that the hybridization requirements described above are met. Sequence identity or complementarity is independent of modification. For example, as defined herein, a and Af are complementary to U (or T) and identical to A for the purposes of determining identity or complementarity.

[0030] As used herein, “perfectly complementary” or “fully complementary” means that in a hybridized pair of nucleic acid bases or nucleotide sequence molecules, all (100%) of the contiguous sequence of the first oligonucleotide hybridizes with the same number of contiguous sequences of the second oligonucleotide. The contiguous sequence may consist of all or part of the first or second nucleotide sequence.

[0031] As used herein, “partially complementary” means that in a hybridized pair of nucleic acid bases or nucleotide sequence molecules, at least 70%, but not all, of the contiguous sequence of the first oligonucleotide hybridizes with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first or second nucleotide sequence.

[0032] As used herein, “substantially complementary” means that in a hybridized pair of nucleic acid bases or nucleotide sequence molecules, at least 85%, but not all, of the contiguous sequence of the first oligonucleotide hybridizes with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first or second nucleotide sequence.

[0033] As used herein, the terms “complementary,” “fully complementary,” “partially complementary,” and “substantially complementary” are used in reference to the matching of nucleic acid bases or nucleotides between the sense strand and antisense strand of an RNAi agent, or between the antisense strand of an RNAi agent and a target mRNA sequence.

[0034] As used herein, "oligonucleotide-based drug" means approximately 10 to 50 oligonucleotides (for example, 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, 12-2 4, 12-22, 12-20, 12-18, 12-16, 12-14, 14-50, 14-48, 14-46, 14-44, 14-42, 14-40, 14-38, 14-36, 14-34, 14-32, 14-30, 14-28, 14-26, 14-24, 14-22, 14-20, 14-18, 14-16, 16-50, 16-48, 16-46, 16-44, 16-42, 16-40, 16-38, 16-36, 16-34, 16-32, 16-30, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18 , 18~50, 18~48, 18~46, 18~44, 18~42, 18~40, 18~38, 18~36, 18~34, 18~32, 18~30, 18~28, 18~26, 18~24, 18~22, 18~20, 20~50, 20~48, 20~46, 20~44, 20~42, 20~40, 20~38, 20~36, 20~34, 20~32, 20~30, 20~28, 20~26, 20~24, 20~22, 22~50, 22~48, 22~46, 22~44, 22~42, 22~40, 22~38, 22~36, 22~34 , 22~32, 22~30, 22~28, 22~26, 22~24, 24~50, 24~48, 24~46, 24~44, 24~42, 24~40, 24~38, 24~36, 24~34, 24~32, 24~30, 24~28, 24~26, 26~50, 26~48, 26~46, 26~44, 26~42, 26~40, 26~38, 26~36, 26~34, 26~32, 26~30, 26~28, 28~50, 28~48, 28~46, 28~44, 28~42, 28~40, 28~38, 28~36, 28~34, 28~32,~28~30, 30~50, 30~48, 30~46, 30~44, 30~42, 30~40, 30~38, 30~36, 30~34, 30~32, 32~50, 32~48, 32~46, 32~44, 32~42, 32~40, 32~38, 32~36, 32~34, 34~50, 34~48, 34~46, 34~44, 34~42, 34~40, 34~38, 34~36, 36~50, 36~48, 36~46, The oligonucleotide is a nucleotide or nucleotide base pair sequence comprising nucleotides or nucleotide base pairs (36-44, 36-42, 36-40, 36-38, 38-50, 38-48, 38-46, 38-44, 38-42, 38-40, 40-50, 40-48, 40-46, 40-44, 40-42, 42-50, 42-48, 42-46, 42-44, 44-50, 44-48, 44-46, 46-50, 46-48, or 48-50). In some embodiments, the oligonucleotide-based agent has a nucleic acid base sequence that is at least partially complementary to the coding sequence of the target nucleic acid or target gene expressed in the cell. In some embodiments, the oligonucleotide-based agent, when delivered to a cell expressing a gene, can inhibit the expression of the underlying gene and is referred to herein as an “expression-inhibiting oligonucleotide-based agent”. Gene expression can be inhibited in vitro or in vivo.

[0035] "Oligononucleotide-based drugs" include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, 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 drug is a single-stranded oligonucleotide, e.g., an antisense oligonucleotide. In some embodiments, the oligonucleotide-based drug is a double-stranded oligonucleotide. In some embodiments, the oligonucleotide-based drug is a double-stranded oligonucleotide that is an RNAi agent.

[0036] As used herein, the terms “substantially identical” or “substantially identical” mean, when applied to nucleic acid sequences, that a nucleotide sequence (or a portion of a nucleotide sequence) has at least approximately 85% sequence identity, e.g., at least 90%, at least 95%, or at least 99% identity, compared to a reference sequence. The percentage of sequence identity is determined by comparing two sequences that are optimally aligned within a comparison window. The percentage is calculated by determining the number of positions where homogeneous nucleic acid bases occur in both sequences 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.

[0037] As used herein, terms such as “treat” and “treatment” mean methods or processes taken to reduce or alleviate the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, “treat” and “treatment” may include preventive treatment, control, prophylactic treatment, and / or inhibition or reduction of the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0038] As used herein, the phrase “intracellular delivery” means functionally delivering an RNAi agent into a cell, when referring to an RNAi agent. The phrase “functional delivery” means delivering an RNAi agent into a cell in a manner that enables it to possess the expected biological activity, such as sequence-specific inhibition of gene expression.

[0039] As used herein, the term "isomer" refers to compounds that have the same molecular formula but differ in the nature or arrangement of the bonds between their atoms, or in the arrangement of those atoms in space. Isomers that differ in the arrangement of their atoms in space are called "stereoisomers." Stereoisomers that are not mirror images of each other are called "diastereomers," and stereoisomers that are non-superimal mirror images are called "enantiomers," or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is called a "chiral center."

[0040] Where used herein, unless expressly identified as having a specific stereoconfiguration in the structure, each structure disclosed herein is intended to represent all possible isomers, including their optically pure and racemic forms, for each structure in which a chiral center is present, thereby resulting in an enantiomer, diastereomer, or other stereoisomer configuration. For example, the structures disclosed herein are intended to encompass mixtures of diastereomers as well as single stereoisomers.

[0041] As used in the claims of this specification, the phrase "consists of" excludes any element, process, or component not expressed in the claim. As used in the claims of this specification, the phrase "essentially consists of" limits the scope of the claim to certain materials or processes that do not substantially affect the basic and novel characteristics of the invention described in the claim.

[0042] Those skilled in the art will readily understand and recognize 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 located. Therefore, when used herein, the structures disclosed herein assume that certain functional groups, such as OH, SH, or NH, may be protonated or deprotonated. This disclosure is intended to encompass the disclosed compounds and compositions regardless of their protonation state based on the environment (e.g., pH), as will be readily understood by those skilled in the art.

[0043] As used herein, the terms “linked” or “conjugated” refer to a bond between two compounds or molecules, meaning that the two molecules are covalently bonded or related via non-covalent bonds (e.g., hydrogen bonds or ionic bonds). In some examples, when the terms “linked” or “conjugated” refer to a bond between two molecules via non-covalent bonds, the bond between the two different molecules is 1 x 10⁻¹⁰ in a physiologically acceptable buffer (e.g., saline). -4 Less than M (for example, 1 x 10) -5 Less than M, 1x10 -6 Less than M, or 1x10 -7 K (less than M) D It has. Unless otherwise specified, the terms “linked” or “conjugated” as used herein may refer to a bond between the first compound and the second compound, with or without intervening atoms or groups of atoms.

[0044] As used herein, a linking group is one or more atoms that bond one molecule or part of a molecule to another second molecule or part of a molecule. Similarly, as used in the art, the term skeleton is sometimes used interchangeably with linking group. A linking group may contain any number of atoms or functional groups. In some embodiments, the linking group may not facilitate any biological or pharmaceutical response and may simply serve to link two biologically active molecules.

[0045] As used herein, “metabolically stable hydrocarbon ligand” is a hydrocarbon ligand suitable for binding to asialoclycoprotein receptors, which are abundantly expressed on hepatocytes, and where the hydrocarbon ligand is chemically modified to provide a more stable chemical composition in serum (e.g., human serum). Appropriate tests to determine whether such a compound is more metabolically stable (e.g., more stable in human serum) than an unmodified hydrocarbon ligand and retains the ability to deliver cargo molecules, e.g., RNAi agents, to hepatocytes can be readily determined by those skilled in the art.

[0046] Certain chemical modifications that provide a metabolically stable hydrocarbon ligand include, but are not limited to, modifications at an atom adjacent to the anomeric carbon, or modifications to phosphodiester linkages (e.g., phosphorothioate linkages or phosphorodithioate linkages) that attach the metabolically stable hydrocarbon ligand to an oligonucleotide. In some embodiments, the metabolically stable hydrocarbon ligand is chemically modified at an 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.

[0047] In some embodiments, the metabolically stable hydrocarbon ligand includes a sugar moiety. In some embodiments, the sugar moiety is selected from the group consisting of glucose, galactose, and N-acetylgalactosamine. Non-limiting examples of metabolically stable hydrocarbon ligands are the metabolically stable N-acetylgalactosamine ligands of formulas I and II disclosed herein.

[0048] In some embodiments, metabolically stable hydrocarbon ligands are 1.5 times more stable in human serum than non-metabolic, metabolically stable hydrocarbon ligands, as measured by the compound half-life in in vitro serum. In some embodiments, metabolically stable hydrocarbon ligands are 2 times more stable in human serum than non-metabolic, metabolically stable hydrocarbon ligands, as measured by the compound half-life in in vitro serum. In some embodiments, metabolically stable hydrocarbon ligands are 5 times more stable in human serum than non-metabolic, metabolically stable hydrocarbon ligands, as measured by the compound half-life in in vitro serum. In some embodiments, metabolically stable hydrocarbon ligands are 10 times more stable in human serum than non-metabolic, metabolically stable hydrocarbon ligands, as measured by the compound half-life in in vitro serum.

[0049] Unless otherwise specified, the symbols used in this specification are: [ka] This means that any or more groups in accordance with the scope of the invention described herein are linked thereto.

[0050] Where used herein, the term “includes” is used herein to mean and interchangeably with the phrase “non-restrictively include.” Unless otherwise clearly indicated in context, “or” is used herein to mean and interchangeably with “and / or.”

[0051] As used in the claims of this specification, the phrase "consists of" excludes any element, process, or component not expressed in the claim. As used in the claims of this specification, the phrase "essentially consists of" limits the scope of the claim to certain materials or processes that do not substantially affect the basic and novel characteristics of the invention described in the claim.

[0052] The term "pharmaceutically acceptable salt" refers to a salt that, within the bounds of proper medical judgment, is suitable for use in contact with human tissue without causing excessive toxicity, irritation, or allergic reactions, and that has a reasonable benefit / risk ratio. pharmaceutically acceptable salts are well known in the field. For example, Berge et al., in J. Pharmaceutical Sciences, 1977, 66, 1-19, describe pharmaceutically acceptable salts in detail, which are incorporated herein by reference.

[0053] pharmaceutically acceptable salts of the compounds of this disclosure include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable and non-toxic acid addition salts are those formed with inorganic salts, e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids, e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by other methods known in the art, e.g., ion exchange. Other pharmaceutically acceptable salts include adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hydroiodide, 2-hydroxyethanesulfonate, and rhynchophosphate. This includes ctobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, p-toluenesulfons, undecanoates, valersates, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and alkylammonium salts (i.e., (C 1-4 (alkyl) 4N + ) are included. Typical alkali or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, etc. Furthermore, pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed with counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkali sulfons, and aryl sulfons.

[0054] Modified nucleotides In some embodiments, the RNAi agent comprises one or more modified nucleotides. As used herein, a "modified nucleotide" is a nucleotide other than a nucleotide (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 include, without limitation, deoxyribonucleotides, nucleotide mimics, abasic nucleotides (represented herein as Ab), 2'-modified nucleotides, 3'-3' linked (inverted) nucleotides (represented herein as invdN, invN, invn), nucleotides containing modified nucleobases, bridged nucleotides, peptide nucleic acids (PNAs), 2',3'-seco nucleotide mimics (open-ring nucleobase analogs, represented herein as N UNA or NUNA), cyclic nucleotides (represented herein as N LNA or NLNA), 3'-O-methoxy (2'-nucleoside internucleotide linkage) nucleotides (represented herein as 3'-OMen), 2'-F-arabinonucleotides (represented herein as NfANA or Nf ANAThis includes 5'-Me, 2'-fluoronucleotides (represented herein as 5Me-Nf), morpholinonucleotides, vinyl phosphonate deoxyribonucleotides (represented herein as vpdN), vinyl phosphonate-containing nucleotides, and cyclopropylphosphonic acid-containing nucleotides (cPrpN). 2'-Modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of a five-membered sugar ring) include, non-limitingly, 2'-O-methylnucleotides (also referred to as 2'-methoxynucleotides and represented herein as a lowercase 'n' in the nucleotide sequence), 2'-fluoronucleotides (also referred to herein as 2'-deoxy-2'-fluoronucleotides and represented herein as Nf), 2'-deoxynucleotides (represented herein as dN), 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides (also referred to herein as 2'-MOE and represented herein as NM), 2'-aminonucleotides, and 2'-alkylnucleotides. In a given compound, it is not necessary for all positions to be uniformly modified. Conversely, multiple modifications can be incorporated into a single RNAi agent or a single nucleotide. The sense and antisense strands of an RNAi agent can be synthesized and / or modified by methods known in the art. Modifications at one nucleotide are independent of modifications at another nucleotide.

[0055] Modified nucleic acid bases include synthetic and natural nucleic acid bases, e.g., 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and guanine 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) adenine and guanine and other adenine and guanine alkyl derivatives. This includes 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azocymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0056] In some embodiments, all or substantially all nucleotides of the RNAi agent are modified nucleotides. As used herein, an RNAi agent in which substantially all present nucleotides are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotide ribonucleotides (i.e., unmodified) in both the sense and antisense strands. As used herein, a sense strand in which substantially all present nucleotides are modified nucleotides is a sense strand having two or more (i.e., 0, 1, or 2) nucleotide unmodified ribonucleotides in the sense strand. As used herein, an antisense sense strand in which substantially all present nucleotides are modified nucleotides is an antisense strand having two or more (i.e., 0, 1, or 2) nucleotide unmodified ribonucleotides in the sense strand. In some embodiments, one or more nucleotides of the RNAi agent are unmodified ribonucleotides.

[0057] Modified internucleoside linkages In some embodiments, one or more nucleotides of the RNAi agent are linked by non-standard ligatures or a backbone (i.e., modified nucleoside ligatures or a modified backbone). Modified internucleoside linkages or skeletons include, but are not limited to, phosphorothioate groups (represented herein by lowercase "s"), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotryesters, aminoalkyl-phosphotryesters, alkylphosphonic acids (e.g., methyl phosphonate or 3'-alkylene phosphonate), chiral phosphonic acids, phosphinic acids, phosphoramidates (e.g., 3'-aminophosphoramidates, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkylphosphonic acids, thionoalkylphosphotryesters, morpholino linkages, boranophosphates typically having 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates having inverted polarity in which pairs of adjacent nucleoside units are linked at 3'-5'~5'-3' or 2'-5'~5'-2'. In some embodiments, the modified internucleoside linkages or skeletons lack a phosphorus atom. The modified internucleoside linkages lacking a phosphorus atom include, non-limitingly, short-chain alkyl or cycloalkyl sugar linkages, mixed heteroatoms and alkyl or cycloalkyl sugar linkages, or one or more short-chain heteroatoms or heterocyclic sugar linkages. In some embodiments, the modified internucleoside skeletons include, non-limitingly, siloxane skeletons, sulfide skeletons, sulfoxide skeletons, sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamic acid skeletons, methyleneimino and methylenehydrazino skeletons, sulfonic acid and sulfonamide skeletons, amide skeletons, and other skeletons having mixed N, O, S, and CH2 components.

[0058] In some embodiments, the sense strand of the RNAi agent may contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, the antisense strand of the RNAi agent may contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand may independently contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, the sense strand of the RNAi agent may contain 1, 2, 3, or 4 phosphorothioate linkages, the antisense strand of the RNAi agent may contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand may independently contain 1, 2, 3, or 4 phosphorothioate linkages.

[0059] In some embodiments, the sense strand of the RNAi agent contains at least two phosphorothioate nucleoside linkages. In some embodiments, at least two phosphorothioate nucleoside linkages are located between nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate nucleoside linkage is located at the 5' end of the sense strand, and another phosphorothioate linkage is located at the 3' end of the sense strand. In some embodiments, two phosphorothioate nucleoside linkages are located at the 5' end of the sense strand, and another phosphorothioate linkage is located at the 3' end of the sense strand. In some embodiments, the sense strand does not contain any phosphorothioate nucleoside linkages between nucleotides, but contains one, two, or three phosphorothioate linkages between both the 5' and 3' terminal nucleotides and an optionally present inverted base-deleted end cap. In some embodiments, the target ligand is linked to the sense strand via phosphorothioate linkages.

[0060] In some embodiments, the antisense strand of the RNAi agent contains four phosphorothioate nucleoside linkages. In some embodiments, the four phosphorothioate nucleoside linkages are located between nucleotides 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 phosphorothioate nucleoside linkages are located between positions 1-4 from the 5' end of the antisense strand, and a fourth phosphorothioate nucleoside linkage 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 phosphorothioate nucleoside linkages in the antisense strand.

[0061] In some embodiments, the RNAi agent comprises one or more modified nucleotides and one or more modified nucleoside linkages. In some embodiments, the 2'-modified nucleoside is combined with the modified nucleoside linkage.

[0062] Target ligand and target group As disclosed herein, a multimeric RNAi agent conjugate delivery platform comprises one or more target groups. The target groups or target moieties enhance the pharmacokinetic or biodistribution properties of the conjugate or RNAi agent, and their binding improves cell-specific (and in some cases, organ-specific) distribution and cell-specific (or organ-specific) uptake of the conjugate or RNAi agent. The target groups may be monovalent, divalent, trivalent, tetravalent, or higher valencies relative to the target in question. Typical target groups include, but are not limited to, compounds with affinity to cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimetic compounds with affinity to cell surface molecules. In some embodiments, the target groups are linked to the RNAi agent using linkers, e.g., PEG linkers, or one, two, or three base-deleted and / or ribitol (base-deleted ribose) residues, which in some cases may function as linkers.

[0063] In some embodiments, the target group is covalently linked to the 3' and / or 5' ends of either the sense strand and / or antisense strand of the RNAi agent. In some embodiments, the target group is linked to the 3' and / or 5' ends of the sense strand of the RNAi agent. In some embodiments, the target group is linked to the 5' end of the RNAi agent sense strand of one RNAi agent. In some embodiments, the target group is internally linked to one or more nucleotides on the sense strand of the RNAi agent. In some embodiments, the target ligand is located between the two RNAi agents in a multimeric RNAi agent conjugate. The target group may be linked directly or indirectly to the RNAi agent via a linker / linking group. In some embodiments, the target group is linked to the RNAi agent via a metabolically stable bond or linkage.

[0064] In some embodiments, the target group comprises an asialoglycoprotein receptor ligand. As used herein, an asialoglycoprotein receptor ligand is a ligand comprising a moiety having affinity for the asialoglycoprotein receptor. As described herein, the asialoglycoprotein receptor is highly expressed in hepatocytes. In some embodiments, the asialoglycoprotein receptor ligand comprises or consists of one or more galactose derivatives. As used herein, the term galactose derivative includes galactose and galactose derivatives having an affinity for the asialoglycoprotein receptor equivalent to or higher than that of galactose. Galactose derivatives include, non-limitingly, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, Nn-butanoylgalactosamine, and N-isobutanoylgalactosamine (see, e.g., STIobst and K. Drickamer, JBC, 996, 271, 6686), as well as metabolically stable glycoside-linked N-acetylgalactosamine. Galactose derivatives and clusters of galactose derivatives useful for in vivo targeting of oligonucleotides and other molecules to the liver are known in the art (see, e.g., Baenziger and Fiete, 198, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945).

[0065] Galactose derivatives are used in vivo for molecular targeting of hepatocytes through their binding to asialoglycoprotein receptors expressed on the surface of hepatocytes. Binding of asialoglycoprotein receptor ligands to asialoglycoprotein receptors promotes cell-specific targeting of hepatocytes and endocytosis of molecules into hepatocytes. Asialoglycoprotein receptor ligands can be monomeric (e.g., having a single galactose derivative, also referred to as monovalent or monodentate) or polymeric (e.g., having multiple galactose derivatives). Galactose derivatives or galactose derivative clusters can be bound to the 3' or 5' end of the sense or antisense strand of an RNAi agent using methods well known in the art. Galactose derivatives or galactose derivative clusters can also be internally bound to one or more nucleotides of the sense or antisense strand of an RNAi agent using methods well known in the art.

[0066] In some embodiments, the target ligand has the following structure: [ka] The formula comprises one or more metabolically stable N-acetylgalactosamine (NAG or GalNAc) target ligands, where X is CH2 or S.

[0067] In some embodiments, the metabolically stable NAG target ligand is a trimer (also referred to as tripolar or trivalent), where the three parts of formula I or formula II are bound through a central branching point. (See, for example, the chemical structure shown for NAG52 herein). In some embodiments, the target ligand is a cluster of four metabolically stable NAG moieties, thereby forming a tetrameric (also referred to as tetrapolar or tetravalent) target ligand. In some embodiments, the metabolically stable NAG target ligand is bipolar or bivalent, where the two parts of formula I or formula II are bound through a central branching point.

[0068] As used herein, a metabolically stable NAG target ligand comprises one or more parts of Formula I or Formula II, each linked to a central branching point. In some embodiments, the target ligand is linked to the branching point via a linker or spacer. In some embodiments, the linker or spacer is a flexible hydrophilic spacer, such as a PEG group (e.g., U.S. Patent No. 5,885,968; see Biessen et al. J. Med. Chem. 1995 Vol. 39 pp. 1538-1546). The branching point can be any small molecule that allows the binding of three galactose derivatives and further allows the branching point to bind to an RNAi agent. Examples of branching point groups are dyridine or diglutamic acid. Binding of the branching point to the RNAi agent may occur via the linker or spacer. In some embodiments, the linker or spacer includes a flexible hydrophilic spacer, such as a PEG spacer, for example. In some embodiments, the linker includes a rigid linker, such as a cyclic group.

[0069] In some embodiments, the delivery platform or compound disclosed herein is a compound of formula I or formula II: [ka] comprising one or more target ligands containing, or a pharmaceutically acceptable salt thereof, In the formula, X is either CH2 or S.

[0070] In some embodiments, the delivery platform or compound disclosed herein is a compound of formula 1a or formula 1b: [ka] It contains one or more target ligands, including the following:

[0071] The method for preparing the compound of formula Ia is described in the following examples.

[0072] In some embodiments, compounds conjugated to RNAi agents for synthesizing RNAi agent delivery platforms are shown in Table 1 below.

[0073] In some embodiments, the compounds described herein are as follows: a. Oligonucleotides with a nucleotide length of 12 to 49 nucleotides; and b. Metabolic stable hydrocarbon ligands; This includes, where the double-stranded RNAi agent and the metabolically stable hydrocarbon ligand are covalently linked by a linkage more metabolically stable than phosphorothioate linkage, phosphorodithioate linkage, or phosphodiester linkage; Metabolically stable hydrocarbon ligands are given by the following formula: [ka] In the formula, each example of a metabolically stable hydrocarbon is independently a chemically modified hydrocarbon moiety; Each example of a tether is independently expressed by the following formula: [ka] In the formula, m is an integer selected from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); The branching points are as follows: [ka] It is a structure selected from the group consisting of; The linker is as follows: [ka] It is a structure selected from the group consisting of; n is an integer from 1 to 4; and [ka] This indicates the binding site with the oligonucleotide.

[0074] In certain embodiments, the tether is as follows: [ka] The formula is as follows, 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 a particular embodiment, the tether is as follows: [ka] In the formula, m is an integer selected from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In a particular embodiment, m is 1. In a particular embodiment, m is 2. In a particular embodiment, m is 3. In a particular embodiment, m is 4. In a particular embodiment, m is 5. In a particular embodiment, m is 6. In a particular embodiment, m is 7. In a particular embodiment, m is 8. In a particular embodiment, m is 9. In a particular embodiment, m is 10. In a particular embodiment, m is 11. In a particular embodiment, m is 12. In a particular embodiment, m is 13. In a particular embodiment, m is 14. In a particular embodiment, m is 15. In a particular embodiment, m is 16. In a particular embodiment, m is 17. In a particular embodiment, m is 18. In certain embodiments, m is 19. In certain embodiments, m is 20.

[0075] In a particular embodiment, the tether is expressed by the following formula: [ka] In a particular embodiment, the tether is given by the following formula: [ka] That is the case.

[0076] In a particular embodiment, the branching point group is given by the following equation: [ka] In a particular embodiment, the branching point group is given by the following equation: [ka] In a particular embodiment, the branching point group is given by the following equation: [ka] In a particular embodiment, the branching point group is given by the following equation: [ka] In a particular embodiment, the branching point group is given by the following equation: [ka] In a particular embodiment, the branching point group is given by the following equation: [ka] In a particular embodiment, the branching point group is given by the following equation: [ka] In a particular embodiment, the branching point group is given by the following equation: [ka] In a particular embodiment, the branching point group is given by the following equation: [ka] In a particular embodiment, the branching point group is given by the following equation: [ka] In a particular embodiment, the branching point group is given by the following equation: [ka] In a particular embodiment, the branching point group is given by the following equation: [ka] In a particular embodiment, the branching point group is given by the following equation: [ka] That is the case.

[0077] In a particular embodiment, the linker is given by the following formula: [ka] In a particular embodiment, the linker is given by the following formula: [ka] In a particular embodiment, the linker is given by the following formula: [ka] In a particular embodiment, the linker is given by the following formula: [ka] In a particular embodiment, the linker is given by the following formula: [ka] In a particular embodiment, the linker is given by the following formula: [ka] In a particular embodiment, the linker is given by the following formula: [ka] That is the case.

[0078] In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, n is 3. In certain embodiments, n is 4.

[0079] In some embodiments, the compounds described herein include one of the metabolically stable N-acetylgalactosamine target ligands shown in Table 1.

[0080] [Table 1-1] [Table 1-2]

[0081] linking group In some embodiments, the RNAi agent comprises, or is conjugated to, one or more non-nucleotide groups, including, non-limiting linking groups, delivery polymers, or delivery vehicles. The non-nucleotide groups enhance the targeting, delivery, or binding of the RNAi agent. Examples of linking groups are provided in Table 2. The non-nucleotide groups may be covalently linked to the 3' and / or 5' ends of either the sense strand and / or antisense strand. In some embodiments, the RNAi agent comprises a non-nucleotide group linked to the 3' and / or 5' ends of the sense strand. In some embodiments, the non-nucleotide group is linked to the 5' end of the sense strand of the RNAi agent. The non-nucleotide group may be linked directly or indirectly to the RNAi agent via a linker / linking group. In some embodiments, the non-nucleotide group is linked to the RNAi agent via an unstable, cleavable, or reversible bond or linker.

[0082] In some embodiments, non-nucleotide groups enhance the pharmacokinetic or biodistribution characteristics of the RNAi agent or conjugate to which they bind, improving cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, non-nucleotide groups enhance the endocytosis of the RNAi agent.

[0083] The RNAi agents described herein can be synthesized to have a reactive group, such as an amino group (also referred to herein as an amine), at its 5' and / or 3' ends. The reactive group can then be used to bind to a target site using methods typical in the art.

[0084] A linker or linking group is a connection between two atoms that links one chemical group (e.g., an RNAi agent) or target segment to another chemical group (e.g., a target ligand, target group, PK / PD modulator, or delivery polymer) or target segment via one or more covalent bonds. Unstable linkages include unstable bonds. Linkages may optionally include spacers that increase the distance between the two bonded atoms. Spacers may further add flexibility and / or length to the linkage. Spacers include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkylyl groups; each of which may include one or more heteroatoms, heterocycles, amino acids, nucleotides, and sugars. Spacer groups are well known in the art, and the preceding list is not intended to limit the scope of this description.

[0085] In some embodiments, the target group is linked to the RNAi agent without the use of an additional linker. In some embodiments, the target group is designed to have a linker to facilitate linkage to the RNAi agent. In some embodiments, when two or more RNAi agents are included in the composition, the two or more RNAi agents can be linked to their respective target groups using the same linker. In some embodiments, when two or more RNAi agents are included in the composition, the two or more RNAi agents can be linked to their respective target groups using different linkers.

[0086] In some embodiments, the linking group may be synthetically conjugated to the 5' or 3' end of the RNAi agent described herein. In some embodiments, the linking group is synthetically conjugated to the 5' end of the sense strand of the RNAi agent. In some embodiments, the linking group conjugated to the RNAi agent may be a trialkin linking group.

[0087] Examples of specific modified nucleotides and linking groups are provided in Table 2.

[0088] [Table 2-1] [Table 2-2] [Table 2-3]

[0089] Alternatively, other linking groups well known in the field may be used.

[0090] In addition to or instead of linking the RNAi agent to one or more target ligands, target 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 the RNAi agent to cells or tissues and may comprise, but is not limited to: polymers, e.g., amphiphilic polymers, membrane-active polymers, peptides, melittin peptides, melittin-like peptides (MLPs), lipids, reversibly modified polymers or peptides, or reversibly modified membrane-active polyamines.

[0091] In some embodiments, RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs, or other delivery systems available in the art. RNAi agents can also be chemically conjugated with target groups, lipids (including, but not limited to, cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, e.g., WO 2000 / 053722, WO 2008 / 022309, WO 2011 / 104169, and WO 2012 / 083185, WO 2013 / 032829, WO 2013 / 158141, each incorporated herein by reference), or other delivery systems available in the art.

[0092] Pharmaceutical composition In some embodiments, the present disclosure provides pharmaceutical compositions comprising, or essentially comprising, one or more delivery platforms disclosed herein.

[0093] As used herein, “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 other than the active ingredient (API, therapeutic product) that is intentionally included in a drug delivery system. Excipients do not, or are not intended to, exert a therapeutic effect at the intended dose. Excipients may act 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 any of the attributes of overall safety, efficacy, or efficacy in API delivery during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0094] Excipients include, but are not limited to: absorption enhancers, anti-adhesion agents, defoamers, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, fragrances, lubricants, humectants, lubricants, oils, polymers, preservatives, saline solutions, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, isotonic agents, vehicles, water repellents, and wetting agents.

[0095] The pharmaceutical compositions described herein may include 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, topical anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.), small molecule drugs, antibodies, antibody fragments, aptamers, and / or vaccines.

[0096] The pharmaceutical composition may also contain preservatives, solubilizers, stabilizers, humectants, emulsifiers, sweeteners, colorants, flavorings, osmotic salts, buffers, coatings, or antioxidants. They may also contain other agents with known therapeutic effects.

[0097] Pharmaceutical compositions can be administered in a number of ways, depending on whether topical or systemic treatment is preferred and on the area of ​​treatment. Administration may be carried out by any method commonly known in the art, for example, not limited to topical (e.g., by transdermal patch), pulmonary (e.g., intratracheal, intranasal, by inhalation or spraying of powder or aerosol, including by nebulizer), epidermal, transdermal, oral or parenteral. Parenteral administration may include, not limited to, intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous (e.g., by implantable device), intracranial, intracerebrospinal, intrathecal, and intraventricular administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection. Pharmaceutical compositions may be administered orally, for example, in the form of tablets, coated tablets, dragées, hard or soft gelatin capsules, solutions, emulsions or suspensions. Administration may be carried out rectally, for example, using suppositories; topically or percutaneously, for example, using ointments, creams, gels, or solutions; or parenterally, for example, using injections.

[0098] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if soluble) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremofor® EL (BASF, Parsippani, NJ), or phosphate-buffered saline. They should be stable under manufacturing and storage conditions and protected from microbial contamination, such as bacteria and fungi. The carrier may be a solvent or dispersion medium, for example, containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coating agents, such as lectins; in the case of dispersions, by maintaining particle size; and by the use of surfactants. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride, in the composition. Long-term absorption of injectable compositions can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0099] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in a suitable solvent having one or a combination of the components listed above, and then sterilizing by filtration. Generally, dispersions can be prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders in the preparation of sterile injectable solutions, the preparation method includes vacuum drying and lyophilization, which yield powders of the active component and any additional desired components derived from those solutions that have been previously sterilized by filtration.

[0100] Formulations suitable for intra-articular administration may be in the form of a sterile aqueous preparation of any of the ligands described herein, which may be in the form of a microcrystalline form, for example, an aqueous microcrystalline suspension. Liposome formulations or biodegradable polymer systems may be used with any of the ligands described herein for intra-articular and ophthalmic administration.

[0101] The active compound can be prepared with a carrier that protects the compound from rapid elimination from the body, for example, from sustained-release formulations including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations are apparent to those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods well known to those skilled in the art, for example, the method described in U.S. Patent No. 4,522,811.

[0102] A pharmaceutical composition may include other additional components commonly found in pharmaceutical compositions. Such additional components may include, but are not limited to: antipruritic, astringent, topical anesthetic, or anti-inflammatory agents (e.g., antihistamines, diphenylhydramine, etc.). As used herein, “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” means the amount of a pharmaceutically active agent necessary to produce a pharmacological, therapeutic, or prophylactic result.

[0103] Pharmaceuticals containing RNAi agents are also subject to the present invention, along with a manufacturing process for such pharmaceuticals, which includes making one or more compounds containing RNAi agents and, optionally, one or more substances having known therapeutic effects, into a pharmaceutically acceptable form.

[0104] The RNAi agents and pharmaceutical compositions described herein, including the RNAi agents disclosed herein, may be packaged or contained in kits, containers, packs, or dispensers. The RNAi agents and pharmaceutical compositions, including the RNAi agents, may be packaged in pre-filled syringes or vials.

[0105] Treatment methods and inhibition of expression The delivery platforms disclosed herein can be used to treat subjects (e.g., humans or other mammals) with diseases or disorders for which the administration of RNAi agents is expected to be beneficial. In some embodiments, the RNAi agent delivery platforms disclosed herein can be used to treat subjects (e.g., humans) for which the reduction and / or inhibition of mRNA expression and / or target protein levels is expected to be beneficial.

[0106] In some embodiments, a subject is administered a therapeutically effective dose of any one or more RNAi agents. Treatment of a subject may include therapeutic and / or prophylactic treatment. A subject is administered a therapeutically effective dose of any one or more RNAi agents described herein. A subject may be a human, a patient, or a human patient. A subject may be an adult, adolescent, child, or infant. Administration of the pharmaceutical compositions described herein may be to humans or animals.

[0107] The RNAi agents described herein can be used to treat at least one symptom in subjects having a disease or disorder associated with a target gene, or a disease or disorder at least partially mediated by the expression of a target gene. In some embodiments, the RNAi agents are used to treat or manage clinical symptoms in subjects having a disease or disorder from which a reduction in the target mRNA is expected to be beneficial or at least partially mediated. The subject is administered a therapeutically effective dose of one or more RNAi agents or RNAi agent-containing compositions described herein. In some embodiments, the methods disclosed herein include administering a composition comprising an RNAi agent described herein to the subject to be treated. In some embodiments, the subject is treated by being administered a prophylactically effective dose of any one or more described RNAi agents, thereby preventing or inhibiting at least one symptom.

[0108] In certain embodiments, the present disclosure provides a method for treating patients with diseases, disorders, conditions, or pathological conditions that are at least partially mediated by target gene expression, wherein the method comprises administering one of the RNAi agents described herein to the patient.

[0109] In some embodiments, the gene expression level and / or mRNA level of the target gene in subjects administered with the RNAi agent is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or more than 99% compared to subjects before or without the RNAi agent. The gene expression level and / or mRNA level in the subjects may be reduced in the cells, cell populations, and / or tissues of the subjects.

[0110] In some embodiments, protein levels in subjects administered with the RNAi agent are reduced by at least approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and more than 99% compared to subjects before or without the RNAi agent. Protein levels in subjects may be reduced in the subjects' cells, cell populations, tissues, blood, and / or other bodily fluids.

[0111] Decreases in mRNA and protein levels can be assessed by any method known in the art. As used herein, decreases or reductions in mRNA and protein levels collectively refer to decreases or reductions in the target gene, or inhibition or reductions in the expression of the target gene. The examples described herein illustrate known methods for assessing inhibition of gene expression.

[0112] In some embodiments, RNAi agents may be used to prepare pharmaceutical compositions for use in treating diseases, disorders, or symptoms that are at least partially mediated by the expression of a target gene.

[0113] In some embodiments, the method of treating the subject depends on the subject's body weight. In some embodiments, the RNAi agent may be administered in doses ranging from 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 in doses ranging from about 5 mg / kg to about 20 mg / kg of the subject's body weight.

[0114] In some embodiments, the RNAi agent may be administered in divided doses, meaning that the subject is given two doses within a short period of time (e.g., less than 24 hours). In some embodiments, half of the desired daily dose is given in the first dose, and the remaining half of the desired daily dose is given about 4 hours after the first dose.

[0115] In some embodiments, the RNAi agent may be administered once a week (i.e., weekly). In other embodiments, the RNAi agent may be administered every other week (once every two weeks).

[0116] In some embodiments, RNAi agents or RNAi agent-containing compositions may be used to treat diseases, disorders, or symptoms that are at least partially mediated by the expression of a target gene.

[0117] Cells, tissues, and non-human organisms Cells, tissues, and non-human organisms are envisioned that include at least one delivery platform containing the RNAi agent described herein. These cells, tissues, and non-human organisms are constructed by delivering the RNAi agent to the cells, tissues, and non-human organisms by any means available in the art. Cells are, in no particular way, mammalian cells, including human cells.

[0118] The embodiments and items provided above are illustrated in the following non-limiting embodiments.

[0119] Exemplary Embodiments Provided herein are exemplary embodiments of the disclosed technology. These embodiments are for illustrative purposes only and do not limit the scope of the disclosure or the appended claims.

[0120] Embodiment 1. A compound that inhibits the expression of one or more genes, the following: a. Oligonucleotides containing chains of 12 to 49 nucleotides in length; and b. Metabolic stable hydrocarbon ligands; A compound or a pharmaceutically acceptable salt thereof, comprising, where the oligonucleotide and metabolically stable hydrocarbon ligand are covalently bonded by a linkage more metabolically stable than phosphorothioate linkage, phosphorodithioate linkage, or phosphodiester linkage.

[0121] Embodiment 2. A metabolically stable hydrocarbon ligand is given by the following formula: [ka] And here: Each example of a metabolically stable hydrocarbon is, independently, a chemically modified hydrocarbon moiety; Each example of a tether is independently expressed by the following formula: [ka] And in the formula, m is an integer selected from 1 to 20; The branching point group is as follows: [ka] It is a structure selected from the group consisting of; The linker is as follows: [ka] It is a structure selected from the group consisting of; n is an integer between 1 and 4, as long as its valence allows; and [ka] The compound described in Embodiment 1 or a pharmaceutically acceptable salt thereof, where is a bonding site with the rest of the compound.

[0122] Embodiment 3. The tether is given by the following formula: [ka] The compound described in Embodiment 2.

[0123] Embodiment 4. The tether is given by the following formula: [ka] The compound described in Embodiment 2.

[0124] Embodiment 5. The branching point group is given by the following formula: [ka] The compound according to any one of Embodiments 2 to 4.

[0125] Embodiment 6. The branching point group is given by the following formula: [ka] The compound according to any one of Embodiments 2 to 5.

[0126] Embodiment 7. The branching point group is given by the following formula: [ka] The compound according to any one of Embodiments 2 to 4.

[0127] Embodiment 8. The branching point group is given by the following formula: [ka] The compound according to any one of Embodiments 2 to 4 or 7.

[0128] Embodiment 9. The compound according to any one of Embodiments 1 to 8, wherein the metabolically stable hydrocarbon ligand comprises metabolically stable N-acetylgalactosamine.

[0129] Embodiment 10. A metabolically stable hydrocarbon is given by the following formula: [ka] And in the formula, X is CH2 or S, and [ka] The compound according to any one of Embodiments 1 to 9, wherein indicates a bonding site with the rest of the compound.

[0130] Embodiment 11. A metabolically stable hydrocarbon is given by the following formula: [ka] And in the formula, X is CH2 or S, and [ka] The compound according to any one of Embodiments 1 to 9, wherein indicates a bonding site with the rest of the compound.

[0131] Embodiment 12. The compound according to Embodiment 10 or 11, wherein X is CH2.

[0132] Embodiment 13. The compound according to Embodiment 10 or 11, wherein X is S.

[0133] Embodiment 14. The compound according to any one of Embodiments 1 to 6 or 9 to 13, wherein the metabolically stable hydrocarbon ligand comprises three metabolically stable N-acetylgalactosamine moieties.

[0134] Embodiment 15. The compound according to any one of Embodiments 2 to 6 or 9 to 13, wherein n is 3.

[0135] Embodiment 16. The linker is of the following formula: [ka] The compound according to any one of Embodiments 2 to 15.

[0136] Embodiment 17. Metabolically stable hydrocarbon ligands are as follows: [ka] [ka] The formula includes a structure selected from the group consisting of, [ka] The compound according to any one of Embodiments 1 to 16, wherein indicates a bonding site with the rest of the compound.

[0137] Embodiment 18. The oligonucleotide is a double-stranded RNAi agent comprising a sense strand and an antisense strand, where: (i) The sense strand of the double-stranded RNAi agent contains 19 to 23 nucleotides; and (ii) The antisense strand of the double-stranded RNAi agent contains 19 to 23 nucleotides, A compound according to any one of Embodiments 1 to 17.

[0138] Embodiment 19. The compound according to any one of Embodiments 1 to 18, wherein the sense chain comprises 19 to 21 nucleotides and the antisense chain comprises 19 to 21 nucleotides.

[0139] Embodiment 20. The compound according to any one of Embodiments 1 to 19, wherein the antisense strand of the double-stranded RNAi agent consists of 19 nucleotides.

[0140] Embodiment 21. The compound according to any one of Embodiments 1 to 20, wherein the antisense strand is at least partially complementary to the mRNA sequence encoded by a gene expressed in human hepatocytes.

[0141] Embodiment 22. The compound according to any one of Embodiments 1 to 21, wherein the antisense strand is completely complementary to the mRNA sequence encoded by a gene expressed in human hepatocytes.

[0142] Embodiment 23. The compound according to any one of Embodiments 1 to 22, wherein a metabolically stable hydrocarbon ligand is conjugated at the 3' end of the sense chain.

[0143] Embodiment 24. The compound according to any one of Embodiments 1 to 23, wherein a metabolically stable hydrocarbon ligand is conjugated at the 5' end of the sense chain.

[0144] Embodiment 25. The compound according to any one of Embodiments 1 to 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 at the 3' ends of both the first and second sense chains.

[0145] Embodiment 26. The compound according to Embodiment 25, wherein the end cap is an inverted base deletion portion or NH2-C6.

[0146] Embodiment 27. The following: [ka] [ka] A compound comprising a structure selected from the group consisting of the following, or a pharmaceutically acceptable salt thereof, wherein the formula is: [ka] The symbol indicates the bonding site with the rest of the compound, or a pharmaceutically acceptable salt thereof.

[0147] Embodiment 28. The compound is of the following formula: [ka] or a pharmaceutically acceptable salt thereof, in the formula, [ka] The compound according to Embodiment 27, where indicates a bonding site with the rest of the compound.

[0148] Embodiment 29. The compound is of the following formula: [ka] or a pharmaceutically acceptable salt thereof, in the formula, [ka] The compound according to embodiment 27, which indicates the bonding point with the remaining part of the compound.

[0149] Embodiment 30. The compound is of the following formula:

Chemical formula

Chemical formula

[0150] Embodiment 31. The compound is of the following formula:

Chemical formula

Chemical formula

[0151] Embodiment 32. The compound according to any one of embodiments 27 to 31, wherein the remaining part of the compound comprises an oligonucleotide chain having a length of 12 to 49 nucleotides.

[0152] Embodiment 33. 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 comprises 19 to 23 nucleotides; and (ii) the antisense strand of the double-stranded RNAi agent comprises 19 to 23 nucleotides, The compound according to embodiment 32.

[0153] Embodiment 34. The compound according to embodiment 33, wherein the sense strand comprises 19 to 21 nucleotides and the antisense strand comprises 19 to 21 nucleotides.

[0154] Embodiment 35. The compound according to Embodiment 33 or 34, wherein the antisense strand of the double-stranded RNAi agent consists of 19 nucleotides.

[0155] Embodiment 36. The compound according to any one of Embodiments 33 to 35, wherein the antisense strand is at least partially complementary to mRNA encoded by a gene expressed in human hepatocytes.

[0156] Embodiment 37. The compound according to any one of Embodiments 33 to 36, wherein the antisense strand is completely complementary to the mRNA encoded by a gene expressed in human hepatocytes.

[0157] Embodiment 38. The compound according to any one of Embodiments 33 to 37, wherein the end cap is located at the 3' end of the sense chain.

[0158] Embodiment 39. The compound according to any one of Embodiments 33 to 38, wherein the end cap is located at the 5' end of the sense chain.

[0159] Embodiment 40. A pharmaceutical composition comprising a compound described in any one of Embodiments 1 to 39 and a pharmaceutically acceptable excipient.

[0160] Embodiment 41. A method for inhibiting gene expression, comprising administering a compound described in any one of Embodiments 1 to 39 to a subject requiring it. [Examples]

[0161] The following embodiments are intended to illustrate, and not limit, specific embodiments disclosed herein.

[0162] Example 1. Synthesis of RNAi agents and multimer RNAi agent conjugates The following describes specific RNAi agents, including the multimeric RNAi conjugates exemplified in the non-limiting examples described herein, and general methods for the synthesis of their conjugates.

[0163] Synthesis of RNAi agents. RNAi agents can be synthesized using methods commonly known in the art. In the synthesis of RNAi agents exemplified in the examples described herein, the sense and antisense strands of the RNAi agent are synthesized according to phosphoramidite techniques on a solid phase used in oligonucleotide synthesis. Depending on the scale, MerMade96E® (Bioautomation), MerMade12® (Bioautomation), or Oligopilot 100 (GE Healthcare) are used. Synthesis was carried out on solid supports made of control-pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston, PA, USA) or polystyrene (obtained from Kinovate, Oceanside, CA, USA). All RNA and 2'-modified RNA phosphoramidites are obtained from Thermo Fisher Scientific (Milwaukee, WI, USA), ChemGenes (Wilmington, MA, USA), or Hungry Purchased from Biotech (Morrisville, NC, USA). Specifically, the 2'-O-methylphosphoramidite used includes: (5'-O-dimethoxytrityl-N 6 -(benzoyl)-2'-O-methyladenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5'-O-dimethoxy-trityl-N 4 -(acetyl)-2'-O-methylcytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphoramidite, (5'-O-dimethoxytrityl-N 2-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite. 2'-deoxy-2'-fluoro-phosphoramidite and 2'-O-propargylphosphoramidite have the same protecting group as 2'-O-methylphosphoramidite. 5'-dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite was purchased from Glen Research (Virginia). The inverted base-deleted residue (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite was purchased from ChemGenes. The UNA phosphoramidites used included: 5'-(4,4'-dimethoxytrityl)-N6-(benzoyl)-2',3'-seco-adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-2',3'-seco-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N 5'-(4,4'-dimethoxytrityl)-N-isobutyryl-2',3'-seco-guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-(4,4'-dimethoxytrityl)-2',3'-seco-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. To introduce phosphorothioate linkages, a 100 mM anhydrous acetonitrile solution of 3-phenyl1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) or a 200 mM pyridine solution of xanthan hydride (TCI America, Portland, OR, USA) was used.

[0164] The TFA amino - linked phosphoramidite was also purchased commercially (ThermoFisher), and a (NH2 - C6) reactive group linker was introduced. The TFA amino - linked phosphoramidite was dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3 Å) were added. 5 - Benzoylthio - 1H - tetrazole (BTT, 250 mM in acetonitrile) or 5 - ethylthio - 1H - tetrazole (ETT, 250 mM in acetonitrile) was used as the activator solution. The coupling times were 10 minutes (RNA), 90 seconds (2’O - Me), and 60 seconds (2’F). The trialkyne - containing phosphoramidites were synthesized to introduce the respective (TriAlk#) linkers. When used in combination with the RNAi agents shown in the specific examples herein, the trialkyne - containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), and all other amidites were dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3 Å) were added. 5 - Benzoylthio - 1H - tetrazole (BTT, 250 mM in acetonitrile) or 5 - ethylthio - 1H - tetrazole (ETT, 250 mM in acetonitrile) was used as the activator solution. The coupling times were 10 minutes (RNA), 90 seconds (2’O - Me), and 60 seconds (2’F).

[0165] In some RNAi agents, linkers such as C6 - SS - C6 or 6 - SS - 6 groups, C6 - SS(Me) - C5 are introduced at the 3’ end of the sense strand. The pre - loaded resin was obtained commercially with each linker. Alternatively, for some sense strands, dT resin was used, and then the respective linkers were added via standard phosphoramidite synthesis.

[0166] Cleavage and deprotection of the carrier - bound oligomer. After completion of solid - phase synthesis, the dried solid support was treated with a 1:1 volume solution of a 40 wt% aqueous methylamine and a 28% - 31% ammonium hydroxide solution (Aldrich) at 30 °C for 1.5 hours. The solution was evaporated, and the solid residue was redissolved in water (see below).

[0167] Purification. Crude oligomers were purified by anion exchange HPLC using a TSKgel SuperQ-5PW 13 μm column and a Shimadzu LC-8 system. Buffer A contained 20 mM Tris, 5 mM EDTA, pH 9.0, and 20% acetonitrile, while Buffer B was the same as Buffer A with the addition of 1.5 M sodium chloride. UV traces were recorded at 260 nm. Appropriate fractions were pooled and then subjected to size exclusion HPLC using a GE Healthcare XK 16 / 40 column packed with Sephadex G-25 Fine, with a running buffer of 100 mM ammonium bicarbonate, pH 6.7, and 20% acetonitrile or sterile water.

[0168] Annealing. Complementary strands were mixed by combining equimolar amounts of RNA solution (sense and antisense) in 1×PBS (phosphate-buffered saline, 1×, Corning, Cellgro) to form RNAi agents. Several RNAi agents were lyophilized and stored at -15 to -25°C. Double-stranded concentrations were determined by measuring the solution absorbance in 1×PBS using a UV-Vis spectrophotometer. The solution absorbance at 260 nm was then multiplied by a conversion factor and a dilution factor to determine the double-stranded concentration. The conversion factor used was either 0.037 mg / (mL·cm) or one calculated from the experimentally determined extinction coefficient.

[0169] Example 2. Synthesis of target ligand Synthesis of NAG52 (Compound 9) [ka] [ka]

[0170] Compounds 1, 2, and 3 were synthesized according to previously published procedures (see, for example, U.S. Patent Application Publication No. 2002 / 0107224 A1), and Cbz-NH-Glu-Glu-OH: [ka] It was synthesized according to the procedure described in the international patent application publication (WO2017 / 156012) of Arrowhead Pharmaceuticals, Inc., and the contents of these references are incorporated herein by reference as being fully described herein. More specifically, 1 was synthesized via the following route: [ka]

[0171] 5 synthesis [ka]

[0172] 3 (4.61 g, 12.3 mmol) and Boc-N-amide-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 under reduced pressure to 30 mL and diluted with chloroform (300 mL). The resulting solution was washed first with saline / citric acid (1:1, 30 mL) and then with saline / saturated bicarbonate solution (1:1, 30 mL). The organic layer was dried over Na2SO4, concentrated under reduced pressure, and purified by silica column (100% DCM ~ 20% MeOH in DCM). The fractions containing the target product 4 were combined, and the solvent was removed under reduced pressure to obtain a foamy residue, which was redissolved in 1,4-dioxane in 4 M HCl (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 obtain the target product 5 as an HCl salt (5.30 g, 9.30 mmol, 76% yield). Calculated molecular weight: 533.26. Verified by ESI MS. + m / z = 534.23[M+H + ].

[0173] 7 synthesis 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 reddish-brown and darkened over time. The reaction mixture was stirred at room temperature for 2 hours until no unreacted starting material was detected by LC-MS. The solvent was removed by co-evaporation with toluene, and the residue was redissolved in chloroform (400 mL). The resulting solution was washed first with saline / water (1:1, 60 mL), and then with saline / bicarbonate solution (1:1, 60 mL). The organic layer was dried over Na2SO4, concentrated under reduced pressure, and purified by silica column (100% DCM to 20% MeOH in DCM). The fraction containing the target product 6 was combined with the solvent removed under reduced pressure, and the resulting foamy 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 Celite pad and concentrated under reduced pressure to obtain the target product 7 for use in the next step (3.80 g, 2.09 mmol, 77% yield). Calculated molecular weight: 1821.84. Measured by ESI MS. + m / z = 912.13[M+2H + ].

[0174] 8 synthesis [ka]

[0175] 7 (3.80 g, 2.09 mmol) was dissolved in anhydrous DMF (30 mL) and slowly added to an anhydrous DMF (30 mL) solution 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 saline / 5% citric acid (1:1, 30 mL), dried over Na2SO4, concentrated under reduced pressure, and purified by silica column (100% DCM ~ 30% MeOH in DCM) to obtain the target product 8 (2.40 g, 1.23 mmol, 59% yield). Calculated molecular weight: 1947.90. Measured by ESI MS. + m / z = 975.46 [M + 2H + ].

[0176] 9 synthesis [ka]

[0177] Compound 8 (2.40 g, 1.23 mmol) was thoroughly dried by co-evaporation of DCM and toluene, and then dried in vacuum for 30 minutes. A stirring rod and pre-treated molecular sieves were placed in a round-bottom flask and filled with nitrogen. The flask was filled with DCM (100 mL) and the molecular sieves were gently stirred for 10 minutes. Diisopropylammonium tetrazolide (1.40 g, 8.19 mmol) was added to the solution, and the reaction mixture was stirred for a further 30 minutes. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (0.55 mL, 1.73 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour until no unreacted starting material was detected by LC-MS. The solution was filtered through a Celite pad to remove the molecular sieves, and diluted with saturated bicarbonate solution (100 mL) while stirring. After 15 minutes, the organic layer was separated, and the aqueous layer was extracted with chloroform (2 x 200 mL). The combined organic layers were dried over Na₂SO₄, concentrated under reduced pressure, and purified over silica gel (100% DCM (+0.1% triethylamine) ~ 10% MeOH in DCM (+0.1% triethylamine)). The fractions containing the target product 9 were combined and concentrated under reduced pressure. The product was evaporated twice with toluene to remove any remaining triethylamine, and the target product was obtained as an off-white solid (2.4 g, 1.16 mmol, 94% yield).

[0178] Compound 9 characterization: 1 H NMR (DMSO-d6): 1.14d (12H), 1.44m (7H), 1.62-1.90m (11H), 1.80s (9H), 1.94s (9H), 2. 00s(9H), 2.07s(9H), 2.03-2.16m(4H), 2.20-2.31m(6H), 2.76t(2H), 2.88-2.98m(3H), 3.12-3.23m(10H), 3.34-3.42m(6H), 3.46s(12H), 3.57t(8H), 3.62-3.76m(2H), 3.98- 4.20m(15H), 4.20-4.30m(3H), 4.96dd(3H), 5.28, d(3H), 7.56-8.00m(8H), 8.12d(3H). 31P NMR (DMSO-d6): 145.84, 146.01.

[0179] Synthesis of NAG42 (compound 9B) The synthesis of NAG42 follows the same synthetic route as NAG52 described above, differing only in that it employs a beta-anomeric stabilization linkage instead of an alpha-anomeric linkage. More specifically, compound 1B, which has a beta-anomeric linkage, can be synthesized as follows. [ka] The remaining synthesis follows the NAG52 synthesis described above, with 1 replaced by 1B to obtain compound 9B. [ka]

[0180] Characterization of compound 9B: 1 H NMR (DMSO-d6): 1.14d(12H), 1.36-1.54m(7H), 1.60-1.86m(11H), 1.79s(9H), 1.89s( 9H), 1.99s(9H), 2.10s(9H), 2-02-2.16m(4H), 2.24-2.30m(6H), 2.76t(2H), 2.98-3. 08m(3H), 3.12-3.24m(10H), 3.30-3.42m(8H), 3.47s(12H), 3.58t(8H), 3.62-3.76m( 2H), 3.80-4.06m(14H), 4.10-4.20m(2H), 4.88dd(3H), 5.26d(3H), 7.55-8.00m(11H). 31 P NMR (DMSO-d6): 145.84, 145.89.

[0181] Synthesis of NAG1008 phosphoramidite [ka]

[0182] 3 synthesis α-C-Nag-Peg-2-amine 1 (3.864 g, 6.79 mmol) hydrochloride was taken in anhydrous DMF (250 mL), glutamic acid derivative 2 (734 mg, 2.61 mmol), and DIEA (4 mL, 23 mmol), and TBTU (1.860 g, 5.8 mmol) was added. The pH was checked to confirm that it was basic. The reaction mixture was stirred for 1.5 hours, and all volatile substances were removed under vacuum for 40 minutes. o The product was removed with C, and the remaining DMF was removed by evaporating toluene twice. 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 80g column, eluate A=DCM; B=20% MeOH in DCM, 0-60% over 60 minutes. Yield 2.915g, 81%. Calculated molecular weight 1311.59. Measured by ESI MS. + m / z = 1312.42[M+H + ].

[0183] 4 synthesis Product 3 (2.910 g, 2.222 mmol) was hydrogenated with 10% Pd / C (330 mg) in MeOH (60 mL) for 16 hours using a hydrogen balloon. The product was filtered through Celite, concentrated, and dried under vacuum. The product was further dried by evaporation of toluene and used directly in the next step. Calculated molecular weight: 1177.55. Measured ESI MS. + m / z = 1179.02[M+H + ].

[0184] 5 synthesis Cis-4-hydroxycyclohexecarboxylic 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 minutes. Compound 4 (2.222 mmol) from the previous step was dissolved in anhydrous DMF (40 mL) and added to the solution containing the active acid. After stirring for 1.5 hours, the DMF was removed under vacuum at 40°C, and the residue DMF was removed by evaporating toluene twice. 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: Calculated molecular weight 1303.62. Measured ESI MS. + m / z = 1304.42[M+H + The crude product was used directly in the next step.

[0185] Synthesis of NAG1008 phosphoramidite Crude product precursor 5 (1.449 mmol, 1.89 g) was dried by two toluene evaporations and redissolved in anhydrous DCM (60 mL). NN,-diisopropylammonium tetrazolide (348 mg, 2 mmol) and molecular sieves (100 mg) were added, and the mixture was stirred for 45 minutes. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (611 mg, 2 mmol) was added, and the reaction was allowed to proceed for 4 hours. The mixture was filtered and stirred in DCM with cold NaHCO-3 (vv 10%) for 15 minutes. The aqueous layer was separated, extracted with CHCl3 (x2), dried to (Na2SO4), filtered, and concentrated. The crude product (2.02 g) was purified using Combiflash®. Column 24 g, liquid load. Elution: A = DCM; B = 20% MeOH in DCM, 0-25%, 30 minutes. Yield 1.357g, (62%). MS: Calculated molecular weight 1503.72 Actual ESI MS - m / z = 1502.42 [MH] + ]. NMR P-31, (DMSO, d6): 144.913, 144.944.

[0186] Synthesis of NAG55 phosphoramidite [ka]

[0187] Synthesis of compound 2: A solution of compound 1 (1 equivalent) in MeOH (12 volumes) was cooled to 0°C. To this stirred solution, TFA (0.8 volumes) and water (0.8 volumes) were added. The solution was warmed to room temperature and stirred for 2 hours. The reaction product was concentrated, and the crude product of compound 2 was used in the next step.

[0188] Synthesis of compound 3: The crude product of compound 2 (1 equivalent) was dissolved in acetonitrile (8 volumes) under a nitrogen atmosphere and cooled in an acetone / dry ice bath. DBU (1.5 equivalents) and Br-PEG3-NHBoc (1.05 equivalents) were added, and the reaction mixture 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.

[0189] Synthesis of compound 4: Compound 3 (1 equivalent) was added to a flask, and 3.7 volumes of HCl solution (4 M in 1,4-dioxane) were added. The reaction 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.

[0190] Synthesis of compound 6: The crude product of compound 4 (3.7 equivalents) and compound 5 (1 equivalent) were dissolved in DMF (50 vol). DIEA (15 equivalents) and TBTU (3.5 equivalents) were added to this solution and stirred at room temperature for 2 hours. The reaction mixture was concentrated, and the crude product was then dissolved in chloroform (3.5 vol) and washed with water / saline solution (1:1) and water / saturated NaHCO3 (1:1) solution. The organic layer was dried over Na2SO4 and concentrated. The crude product was purified by column chromatography to obtain compound 6.

[0191] Synthesis of compound 7: A solution of compound 6 (1 equivalent) and TFA (10 equivalents) were stirred in methanol under an H2 atmosphere at room temperature for 2 hours in the presence of Pd / C (50% by weight). The solution was filtered and concentrated to obtain compound 7.

[0192] Synthesis of compound 9: Compound 7 was dissolved in DCM (32 vol) under a nitrogen atmosphere and cooled to 0°C. In a separate flask, compound 8 (1.1 equivalents), TBTU (1.1 equivalents), and DIEA (3.5 equivalents) were stirred in DCM (20 vol) 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 in a separate flask for 10 minutes, and then 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 layer was washed with saturated NaHCO3 solution and brine. It was then dried over Na2SO4 and concentrated. The crude product was purified by column chromatography to obtain compound 9.

[0193] Synthesis of NAG55 phosphoramidite: Compound 9 was dissolved in DCM (15 vol) under a nitrogen atmosphere. 2.7 equivalents of 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite and 0.7 equivalents of diisopropylammonium tetrazolide were added, and the mixture was stirred for 2 hours. Next, 0.5 equivalents of 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite and 0.3 equivalents of diisopropylammonium tetrazolide were added, and the mixture was stirred for a further 1.5 hours. The reaction mixture was cooled to 0°C and washed with saturated NaHCO3 solution. The organic layer was dried over Na2SO4 and concentrated. The crude product was purified by column chromatography to obtain NAG55 phosphoramidite.

[0194] Example 3. Conjugation of linker and target ligand to RNAi agent A. Conjugation of active ester linkers One promising method for linker conjugation is by coupling with activated esters. In some embodiments, the following method may be used to conjugate a linker having a terminal propargyl group to an RNAi agent having an amino-functionalized sense chain, e.g., C6-NH2, NH2-C6, or (NH2-C6), as shown in Table 2 above. Lyophilized, annealed RNAi agents were dissolved at 25 mg / mL in DMSO and 10% water (v / v%). Then, 50-100 equivalents of TEA and 3 equivalents of activated ester linker were added to the solution. The solution was allowed to react for 1-2 hours and monitored by RP-HPLC-MS (XBridge C18 column, Waters Corp., mobile phase A: 100 mM HFIP, 14 mM TEA; mobile phase B: acetonitrile).

[0195] The product can then be precipitated by adding 12 mL of acetonitrile and 0.4 mL of PBS and centrifugating the solid into a pellet. The pellet was then redissolved in 0.4 mL of 1X PBS and 12 mL of acetonitrile. The resulting pellet was dried under high vacuum for 1 hour.

[0196] B. Conjugation of target ligands to propargyl linkers Similarly, another acceptable method for coupling the target ligands of the compounds disclosed herein is via their conjugation to propargyl linkers. In some embodiments, a 5' or 3' tridentate alkyne-functionalized sense chain can be conjugated to the NAG ligand either before or after annealing. Below is one promising method for the conjugation of a / b-anomers of metabolically stable NAG to annealed double chains: A storage 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 was prepared in deionized water. A 75 mg / mL DMSO solution of ligand azide was prepared. To a 1.5 mL centrifuge tube containing trialquine-functionalized double-chain (3 mg, 75 μL, 40 mg / mL in deionized water, approximately 15,000 g / mol), 25 μL of 1 M Hepes pH 8.5 buffer was added. After vortexing, 35 μL of DMSO was added and the solution was vortexed. The ligand could then be added to the reactant (e.g., 6 equivalents / double-chain, 2 equivalents / alkyne, approximately 15 μL), and the solution was vortexed. The pH was checked using pH test paper and confirmed to be approximately 8. In another 1.5 mL centrifuge tube, 50 μL of 0.5 M THPTA was mixed with 10 μL of 0.5 M copper(II) sulfate pentahydrate, vortexed, and incubated at room temperature for 5 minutes. After 5 minutes, THPTA / copper solution (7.2 μL, 6 equivalents 5:1 THPTA:copper) was added to the reaction vial and vortexed. Immediately thereafter, 2M ascorbic acid (5 μL, 50 equivalents per double chain, 16.7 per alkyne) was added to the reaction vial and vortexed. Once the reaction was complete (typically in 0.5 to 1 hour), the reaction mixture was immediately purified by non-denaturing anion exchange chromatography.

[0197] C. Conjugation of target ligands to amine-functionalized sense chains In some embodiments, the following method may be used to conjugate activated ester-functionalized target ligands, such as metabolically stable hydrocarbon ligands, to amine-functionalized RNAi agents containing amines such as C6-NH2, NH2-C6, or (NH2-C6), as shown in Table 2: Annealed lyophilized RNAi agents were dissolved at 25 mg / mL in DMSO and 10% water (v / v%). Next, 50-100 equivalents of TEA and 3 equivalents of activated ester-target ligand were added to the mixture. The reaction mixture was stirred for 1-2 hours and monitored by RP-HPLC-MS (mobile phase A: 100 mM HFIP, 14 mM TEA; mobile phase B: acetonitrile; column: XBridge C18). After completion of the reaction mixture, 12 mL of acetonitrile was added, followed by 0.4 mL of PBS, and the mixture was centrifuged. The solid pellet was collected, dissolved in 0.4 mL of 1xPBS, and then 12 mL of acetonitrile was added. The resulting pellets were collected and dried under reduced pressure for 1 hour.

[0198] D. Addition of target ligand by phosphoramidite synthesis or on resin. Other acceptable methods for coupling the target ligand include preparing the desired ligand as a phosphoramidite compound and adding it to the 5' end using standard solid-phase synthesis of the chain, or preparing the target ligand on a resin and placing it at the 3' end of the cleaved chain using standard solid-phase oligonucleotide synthesis.

[0199] Example 4. In vivo administration of metabolically stable RNAi agent conjugates in cynomolgus monkeys. Multimeric (dimeric) RNAi conjugates containing metabolically stable NAG target ligands were evaluated for gene silencing activity in cynomolgus monkeys (Macaca fascicularis) (hereinafter referred to as "cynos") of the primate genus Macaca. Each multimeric RNAi conjugate evaluated contained one RNAi agent of the conjugate that was well complemented by the mouse angiopoietin-like 3 (ANGPTL3) gene transcript, and a second RNAi agent that was well complemented by the mouse factor 12 (FXII) gene transcript. Gene expression levels of both FXII and ANGPTL3 were evaluated, as discussed in further detail below.

[0200] On day 1, male cynomolgus monkeys were given a single subcutaneous dose of either 6.0 mg / kg (mpk) of a multimerized RNAi conjugate formulated in isotonic saline, or 0.3 mL / kg of animal body weight (20 mg / mL concentration) containing 3.0 mg / kg of two separate RNAi conjugates, according to Table 3 below.

[0201] [Table 3]

[0202] The multimeric RNAi conjugates and individual monomeric RNAi conjugates of Group 1 each contain a modified nucleotide. The RNAi conjugates are synthesized according to phosphoramidite techniques on a solid phase, following common methods known in the art and commonly used in oligonucleotide synthesis, as described in Example 1 of this specification. In all RNAi conjugates, the target ligand is positioned at the 5' end of the sense strand, as shown in Table 4 below.

[0203] [Table 4]

[0204] 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 a phosphorothioate linkage; invAb represents a deoxyribose residue with an inverted base deletion (see Table 2); sp18 represents a spacer 18 polyethylene glycol (PEG) linker as described in Table 2; C6-NH2 represents an amino-linked linker or end cap as described in Table 2, and NAG37 has the structure shown below: [ka] It shows an acetylgalactosamine trimer consisting of; NAG42s shows a metabolically stable NAG trimer having the structure shown in Table 2; and NAG52s shows a metabolically stable NAG trimer having the structure shown in Table 2.

[0205] The NAG37 structure is added to the sense chain as a phosphoramidite compound and is commonly synthesized according to Arrowhead Pharmaceuticals, Inc.'s International Patent Application Publication WO2018 / 044350, which is incorporated by reference as fully described herein. The NAG42s and NAG52s structures are also added to the sense chain according to Examples 1, 2, and 3 herein.

[0206] In Table 4 above, AS refers to the antisense strand and SS refers to the sense strand. The individual nucleotides of the strands, shown separated by commas for convenience in the table above, are linked by phosphodiester linkages, and where "s" is present, the phosphodiester linkage is replaced by a phosphorothioate linkage, which links to the nucleotide or non-nucleotide component of each strand. The antisense strand then anneals to the respective sense strand. When used throughout this specification 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 multimeric RNAi conjugate complex of the sense strand. Each additional RNAi agent added to the multimeric conjugate (e.g., AS(2), AS(3), etc.) is located further toward the 5' end of the sense strand.

[0207] As described above and discussed herein, the multimeric RNAi agent conjugate of group 3 (AD14217) contains a metabolically stable NAG of formula II (more specifically, formula IIa), and the multimeric RNAi agent conjugate of group 4 (AD14218) contains a metabolically stable NAG of formula I (more specifically, formula Ia). The multimeric RNAi agent conjugate of group 2 (AD14216) and the monomeric RNAi agents of group 1 (AD14219 + AD14220) each contain an N-acetylgalactosamine target ligand that is metabolically unstable and has the structure described above for NAG37. Each target ligand was linked to the respective RNAi agent via phosphorothioate linkage.

[0208] Three (3) cynomolgus monkeys were administered to each group (n=3). Serum samples were collected on day -14, day -7, and day 1 (before administration). The monkeys were then administered according to the respective groups described in Table 5. Serum was then collected on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 98, 106, 120, 133, 148, and 162.

[0209] Serum ANGPTL3 protein levels were measured by ELISA assay (R&D Systems) according to the manufacturer's recommendations. Serum FXII protein levels were also measured by ELISA assay (R&D Systems) according to the manufacturer's recommendations. The ANGPTL3 and FXII protein levels of each animal were normalized. In normalization, the level of ANGPTL3 or FXII protein at each time point in each animal was divided by the geometric mean of the pre-treatment expression levels of the animals (in this case, -14 days, -7 days, and day 1 (pre-administration)) to determine the "normalized to pre-treatment" expression rate. The expression at a specific time point was then normalized to the saline control group by dividing the "normalized to pre-treatment" ratio in each individual animal by the mean of the "normalized to pre-treatment" ratio of all mice in the saline control group. This normalized the expression at each time point to that of the control group.

[0210] The data from the study described in this example are shown in Tables 5 and 6 below.

[0211] [Table 5-1] [Table 5-2]

[0212] [Table 6-1] [Table 6-2]

[0213] This embodiment illustrates the usefulness of the delivery platform of the present invention.

[0214] Regarding ANGPTL3 protein levels in cynomolgus monkeys, for example, on day 22 (i.e., 3 weeks post-administration), co-administration of monomeric conjugates in group 1 achieved approximately 76% cANGPTL3 knockdown (0.244), group 2 (using dimeric RNAi agent conjugates with NAG targeting moiety) achieved approximately 66% cANGPTL3 knockdown (0.339), while metabolically stable multimeric RNAi conjugates in group 3 (dimeric RNAi agent conjugates with metabolically stable NAG having beta-anomeric linkages) and group 4 (dimeric RNAi agent conjugates with metabolically stable NAG having alpha-anomeric linkages) showed approximately 81% (0.194) and 86% (0.137) knockdowns of cANGPTL3, respectively. On day 106 (i.e., more than 3 months after administration), both the co-administered monomeric conjugate in group 1 and the dimerized RNAi agent conjugate with a NAG target region in group 2 essentially returned to baseline and did not show significant knockdown of cANGPTL3. On the other hand, at day 106, the dimerized RNAi agent conjugates with metabolically stable NAG target ligands in group 3 (approximately 33% knockdown (0.777)) and group 4 (approximately 45% knockdown (0.565)) still showed gene silencing activity.

[0215] Regarding cynomolgus monkey FXII protein levels, for example, on day 22 (i.e., 3 weeks post-administration), the co-administered monomer conjugate in group 1 achieved approximately 71% cFXII knockdown (0.295); both group 2 (using a dimer RNAi agent conjugate with a NAG targeting moiety) and group 3 (a dimer RNAi agent conjugate with a metabolically stable NAG having an alpha-anomeric linkage) achieved approximately 78% cFXII knockdown; and group 4 (a dimer RNAi agent conjugate with a metabolically stable NAG having a beta-anomeric linkage) showed approximately 85% knockdown (0.156). At day 98 (i.e., more than 3 months post-administration), the co-administered monomer conjugate in group 1 returned to showing only 25% cFXII knockdown, and similarly, the dimer RNAi agent conjugate with a NAG targeting moiety in group 2 showed only approximately 41% knockdown (0.592). On the other hand, dimerized RNAi conjugates with metabolically stable NAG target ligands in Group 3 (approximately 69% knockdown (0.316)) and Group 4 (approximately 86% knockdown (0.139)) provided substantially more significant gene knockdown at day 98, demonstrating longer-lasting silencing activity in this study. In fact, even at day 162 (i.e., more than 5 months after administration), the dimerized RNAi conjugate with metabolically stable NAG target ligand in Group 4 (AD14218) continued to show over 70% (0.285) cFXII inhibition.

[0216] While this particular example involves RNAi agents for ANGPTL3 and FXII inhibition, the same multimeric RNAi agent delivery platform can be used to inhibit gene expression of other genes present in the liver, including hepatocytes.

[0217] Example 5. In vivo administration of RNAi agents in cynomolgus monkeys APOC3-PCSK9 RNAi agents were tested for inhibition of APOC3 and PCSK9 in cynomolgus monkeys.

[0218] On day 1, three (n=3) male cynomolgus monkey test animals in each test group were administered APOC3-PCSK9 RNAi preparation (6.0 mg / kg) formulated in physiological saline at a dose of 20.0 mL / kg via subcutaneous (SQ) injection using a syringe and needle in the mid-scapular region.

[0219] The cynomolgus macaques were allowed to acclimate for at least one day. The animals were 2 to 7 years old. To monitor any effects of the test substance, the animals were not mixed for at least 24 hours after administration of the test substance (RNAi agent). The animals were fed Certified Primate Diet $5048 (PMI, Inc.) and given free access to water from Greenfield City. The animals were maintained at a temperature of 20–26°C, a relative humidity of 50+ / -20%, and in a 12-hour light / 12-hour dark cycle.

[0220] The treatment regimen followed the guidelines in Table 7 below.

[0221] [Table 7]

[0222] The multimeric RNAi conjugates and individual monomeric RNAi conjugates were each synthesized using phosphoramidite techniques on a solid phase, following common methods known in the field and typically used in oligonucleotide synthesis. In all RNAi conjugates, the target ligand is positioned at the 5' end of the sense strand, as shown in Table 8 below.

[0223] [Table 8]

[0224] 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 a phosphorothioate linkage; invAb represents an inverted base-deleted deoxyribose residue (see Table 2); sp18 represents a spacer 18 polyethylene glycol (PEG) linker as described in Table 2; C6-NH2 represents an amino-linked linker or end cap as described in Table 2, and NAG52s represents a metabolically stable NAG trimer having the structure shown in Table 2.

[0225] Prior to each SQ injection, the test animals were first sedated. Sedation was achieved by administering ketamine hydrochloride (10 mg / kg) or terazole (5-8 mg / kg) as an intramuscular (IM) injection, with ketamine (5 mg / kg) supplementation as needed.

[0226] The test animals were administered subcutaneously via syringe and needle in the scapular region (upper left, upper right, lower left, or lower right scapular region). The administration site was shaved at least one day prior to each dose. Individual doses of APOC3-PCSK9 RNAi were calculated based on body weight measured on each day of administration. On each day of dose administration, the APOC3-PCSK9 RNAi was warmed to approximate ambient room temperature at least 30 minutes prior to dose administration. The animals were fasted overnight prior to administration.

[0227] Serum blood (approximately 5.0 mL) was collected from any animal that was mortally injured or sacrificed at unscheduled intervals on days 6, 8, 15, 22, 29, 36, 43, 50, 57, and 63, prior to liver biopsy sample collection or dose administration (if applicable). The collection site was the femoral vein, with the saphenous vein as an alternative collection site.

[0228] Liver biopsies and serum samples collected from test animals were used to analyze APOC3 and PCSK9 expression and additional biological parameters. Liver biopsies were collected on days -6, 15, 36, 50, and 64 (postmortem).

[0229] Liver biopsies were collected under sedation. Animals were fasted overnight (at least 12 hours, but less than 18 hours) prior to each liver biopsy. Each liver biopsy sample collected from an animal was approximately 100 mg (80–120 mg).

[0230] Liver biopsies were analyzed for APOC3 and PCSK9 expression and additional biological parameters. Liver mRNA expression levels of APOC3 and PCSK9 were quantified by qPCR using cARL1 as an endogenous control gene and normalized to day 6 (pre-administration). The qPCR APOC3 and PCSK9 expression data are shown in Tables 9 and 10 below.

[0231] [Table 9]

[0232] APOC3-PCSK9 RNAi agents achieved APOC3 transcript knockdown for at least 64 days when administered as a subcutaneous SQ injection of 6.0 mg / kg on day 1. Groups 1 and 2 achieved APOC3 knockdown. More specifically, AC003791 achieved approximately 66% inhibition (0.336) at day 50 with a single dose of 6.0 mg / kg. At day 64, AC003791 achieved approximately 50% inhibition (0.500) with a single dose of 6.0 mg / kg.

[0233] [Table 10]

[0234] APOC3-PCSK9 RNAi agents achieved PCSK9 transcript knockdown for at least 64 days when administered as a single subcutaneous SQ injection of 6.0 mg / kg on day 1. Groups 1 and 2 achieved PCSK9 knockdown. More specifically, AC003791 achieved approximately 64% inhibition (0.351) on day 64 with a single 6.0 mg / kg dose.

[0235] Serum PCSK9 was quantified by ELISA (R&D Systems, catalog #DPC900) according to the manufacturer's instructions. Relative PCSK9 levels were normalized from pre-administration to day 6. The data are shown in Table 11 below.

[0236] [Table 11]

[0237] APOC3-PCSK9 RNAi agents achieved knockdown of serum PCSK9 for at least 64 days when administered as a single subcutaneous SQ injection of 6.0 mg / kg on day 1. Groups 1 and 2 achieved PCSK9 knockdown. More specifically, AC003791 achieved approximately 67% inhibition (0.323) at day 36 (lowest point) with a single 6.0 mg / kg dose. At day 64, a single 6.0 mg / kg dose of AC003791 achieved approximately 46% inhibition (0.538).

[0238] Serum APOC3 was quantified using the Roshcobas® assay for APOC3, according to the manufacturer's instructions. The data are shown in Table 12 below.

[0239] [Table 12]

[0240] APOC3-PCSK9 RNAi agents achieved knockdown of serum APOC3 for at least 64 days when administered as a single subcutaneous SQ injection of 6.0 mg / kg on day 1. Groups 1 and 2 achieved APOC3 knockdown. More specifically, AC003791 achieved approximately 51% inhibition at day 36 (lowest point) with a single 6.0 mg / kg dose (2.96 mg / dL APOC3 at day 36 compared to 6.15 mg / dL APOC3 at day -6). Furthermore, AC005898 achieved approximately 40% inhibition at day 22 (lowest point) with a single 6.0 mg / kg dose (3.50 mg / dL APOC3 at day 22 compared to 5.84 mg / dL APOC3 at day -6). On day 64, AC003791 achieved approximately 24% inhibition with a single dose of 6.0 mg / kg (4.65 mg / dL compared to 6.15 mg / dL on day 6). On day 64, AC005898 achieved approximately 23% inhibition with a single dose of 6.0 mg / kg (4.47 mg / dL compared to 5.84 mg / dL on day 6).

[0241] Example 6. In vivo administration of a multimer FXII-gene X RNAi agent in cynomolgus monkeys. Multimeric RNAi agents targeting FXII and another gene target (denoted "gene X") produced in human hepatocytes were tested for inhibition of factor XII (FXII) and the gene in cynomolgus monkeys. The AS(1) of each dimer used in this example is identical to the AS(1) of AD14217 and AD14218 shown in Example 4 above. The AS(2) of each dimer used in this example is identical in all of 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 example contain the same sense strand sequence as AD14217 and AD14218 as shown in Example 4 above, except for the metabolically stable hydrocarbon ligands shown in Table 13, and the sense strand portion that is complementary to ANGPTL3 AS(2) in AD14217 and AD14218, which in this case is a modified nucleotide complementary to the antisense sequence of gene X's AS(2).

[0242] On day 1, three male cynomolgus monkeys (n=3) in each test group were administered a multimerized FXII-gene X RNAi agent (6.0 mg / kg) formulated in physiological saline at a dose of 0.3 mL / kg via subcutaneous (SQ) injection using a syringe and needle into the mid-scapular region.

[0243] The test animals were male non-naive cynomolgus monkeys. The RNAi test substance was administered subcutaneously (SQ) via syringe and needle into the mid-scapular region.

[0244] The dosage therapy was determined according to Table 13 below.

[0245] [Table 13]

[0246] Each multimeric RNAi agent conjugate was synthesized using phosphoramidite technology on a solid phase, following a well-known method in the field and commonly used in oligonucleotide synthesis.

[0247] The (NAG52)s, (NAG55)s, and (NAG1008)s structures were also added to the sense chain according to Examples 1, 2, and 3 of this specification.

[0248] As described above and discussed herein, the multimeric RNAi agent conjugates of Group 1 contain metabolically stable target ligands (NAG52)s (see Table 1), the multimeric RNAi agent conjugates of Group 2 contain metabolically stable target ligands (NAG55)s (see Table 1), and the multimeric RNAi agent conjugates of Group 3 contain metabolically stable target ligands (NAG1008)s (see Table 1).

[0249] Prior to each SQ injection, the test animals were first sedated. Sedation was achieved using ketamine hydrochloride (10 mg / kg), which was administered as an intramuscular (IM) injection.

[0250] Individual doses of the multimerized FXII-gene X RNAi agent were calculated based on body weight measured each day of administration. Animals were fasted overnight for at least 12 hours, but less than 24 hours, prior to administration.

[0251] Serum blood (approximately 5.0 mL) was collected from any animal that was mortally injured or sacrificed at unscheduled intervals on days -14, -7, 1 (pre-administration), 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, and 99. The collection site was the femoral vein, with the saphenous vein being used as an alternative collection site.

[0252] The collected serum samples were analyzed for the expression of FXII and gene X, as well as additional biological parameters. Serum protein expression levels of FXII and gene X were quantified by ELISA according to the manufacturer's instructions, using normalized relative expression levels before administration for each test group. The quantified protein levels of FXII and gene X are shown in Tables 14 and 15 below.

[0253] [Table 14-1] [Table 14-2]

[0254] When FXII-gene X multimer RNAi agents were administered as a single subcutaneous SQ injection of 6.0 mg / kg on day 1, FXII inhibition was achieved for at least 99 days. At the lowest point, a single dose of 6.0 mg / kg of NAG55s conjugate dimer achieved ~76% FXII inhibition (0.238) on day 36. On day 99, a single dose of 6.0 mg / kg of NAG52s conjugate dimer achieved ~63% FXII inhibition (0.363).

[0255] [Table 15-1] [Table 15-2]

[0256] The FXII-gene X multimer RNAi agent achieved gene X inhibition for at least 99 days when administered as a single subcutaneous SQ injection of 6.0 mg / kg on day 1. At its lowest point, a single dose of 6.0 mg / kg of NAG52s conjugate dimer achieved ~87% gene X inhibition (0.129) on day 22. On day 99, a single dose of 6.0 mg / kg of NAG52s conjugate dimer achieved ~71% gene X inhibition (0.284).

[0257] Other Embodiments The present invention is described in conjunction with its detailed description, but the above description is intended to be illustrative and does not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Compounds that inhibit the expression of one or more genes, the following: a. Oligonucleotides containing chains of 12 to 49 nucleotides; and b. Metabolically stable hydrocarbon ligands; A compound or a pharmaceutically acceptable salt thereof, comprising the oligonucleotide and the metabolically stable hydrocarbon ligand, wherein the oligonucleotide and the metabolically stable hydrocarbon ligand are covalently linked by a linkage more metabolically stable than phosphorothioate linkage, phosphorodithioate linkage, or phosphodiester linkage.

2. The aforementioned metabolically stable hydrocarbon ligand has the following formula: 【Chemistry 1】 During the ceremony, Each of the aforementioned examples of metabolically stable hydrocarbons is, independently, a chemically modified hydrocarbon moiety; Each of the above tether examples is independently expressed by the following formula: 【Chemistry 2】 In the formula, m is an integer selected from 1 to 20; The aforementioned branching point group is as follows: 【Transformation 3】 It is a structure selected from the group consisting of; The aforementioned linker is as follows: 【Chemistry 4】 It is a structure selected from the group consisting of; n is an integer between 1 and 4, as long as its valence allows; and 【Transformation 5】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound shows a bonding site with the rest of the compound.

3. The aforementioned tether is given by the following formula: 【Transformation 6】 The compound according to claim 2.

4. The aforementioned tether is given by the following formula: 【Transformation 7】 The compound according to claim 2.

5. The aforementioned group of branching points is given by the following equation: 【Transformation 8】 The compound according to any one of claims 2 to 4.

6. The aforementioned group of branching points is given by the following equation: 【Chemistry 9】 The compound according to any one of claims 2 to 5.

7. The aforementioned group of branching points is given by the following equation: 【Chemistry 10】 The compound according to any one of claims 2 to 4.

8. The aforementioned group of branching points is given by the following equation: 【Chemistry 11】 The compound according to any one of claims 2 to 4 or 7.

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

10. The aforementioned metabolically stable hydrocarbon is given by the following formula: 【Chemistry 12】 And in the formula, X is CH ２ or S, 【Chemistry 13】 The compound according to any one of claims 1 to 9, wherein indicates a bonding site with the rest of the compound.

11. The aforementioned metabolically stable hydrocarbon is given by the following formula: 【Chemistry 14】 And in the formula, X is CH ２ or S, 【Chemistry 15】 The compound according to any one of claims 1 to 9, wherein indicates a bonding site with the rest of the compound.

12. X is CH ２ The compound according to claim 10 or 11.

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

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

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

16. The linker is given by the following formula: 【Chemistry 16】 The compound according to any one of claims 2 to 15.

17. The metabolically stable hydrocarbon ligands are as follows: 【Chemistry 17-1】 【Chemistry 17-2】 The formula includes a structure selected from the group consisting of, [Chemistry 18] The compound according to any one of claims 1 to 16, wherein indicates a bonding site with the rest of the compound.

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

19. The compound according to any one of claims 1 to 18, wherein the sense chain comprises 19 to 21 nucleotides, and the antisense chain comprises 19 to 21 nucleotides.

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

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

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

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

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

25. The compound according to any one of claims 1 to 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 at the 3' ends of both the first and second sense chains.

26. The compound according to claim 25, wherein the end cap is an inverted base deletion portion or NH2-C6.

27. below: 【Chemistry 19-1】 【Chemistry 19-2】 A compound comprising a structure selected from the group consisting of or a pharmaceutically acceptable salt thereof, wherein the formula is: 【Chemistry 20】 The symbol indicates the bonding site with the rest of the compound.

28. The aforementioned compound is given by the following formula: 【Chemistry 21】 or a pharmaceutically acceptable salt thereof, in the formula, 【Chemistry 22】 The compound according to claim 27, wherein indicates a bonding site with the rest of the compound.

29. The aforementioned compound is given by the following formula: 【Chemistry 23】 or a pharmaceutically acceptable salt thereof, in the formula, 【Chemistry 24】 The compound according to claim 27, wherein indicates a bonding site with the rest of the compound.

30. The aforementioned compound is given by the following formula: 【Chemistry 25】 or a pharmaceutically acceptable salt thereof, in the formula, 【Chemistry 26】 The compound according to claim 27, wherein indicates a bonding site with the rest of the compound.

31. The aforementioned compound is given by the following formula: 【Chemistry 27】 or a pharmaceutically acceptable salt thereof, in the formula, 【Chemistry 28】 The compound according to claim 27, wherein indicates a bonding site with the rest of the compound.

32. The compound according to any one of claims 27 to 31, wherein the remaining portion of the compound comprises an oligonucleotide chain having a nucleotide length of 12 to 49 nucleotides.

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

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

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

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

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

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

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

40. A pharmaceutical composition comprising a compound according to any one of claims 1 to 39, and a pharmaceutically acceptable excipient.

41. A method for inhibiting gene expression, comprising administering a compound according to any one of claims 1 to 39 to a subject requiring it.

42. A method for inhibiting gene expression, comprising administering the pharmaceutical composition described in claim 40 to a subject requiring it.

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