RNAi agent for inhibiting the expression of mitochondrial amidoxime reducing agent 1 (MARC1), the pharmaceutical composition thereof, and method of use.

Abstract

This disclosure relates to RNAi agents, such as double-stranded RNAi agents, that can inhibit the expression of the mitochondrial amidoxime reducing agent 1 (MARC1) gene. Pharmaceutical compositions containing MARC1 RNAi agents and methods of use thereof are also disclosed. The MARC1 RNAi agents disclosed herein may be conjugated to a targeted ligand, including a ligand containing N-acetyl-galactosamine, to facilitate delivery to hepatocytes. In vivo delivery of MARC1 RNAi agents results in inhibition of MARC1 gene expression. RNAi agents can be used in methods of treating diseases, disorders, or conditions partially mediated by MARC1 gene expression, including non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related disorders.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 490,694, filed on 16 March 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing (in accordance with standard ST26) submitted in XML format, which is incorporated herein by reference in its entirety. The XML sequence listing file is named 30716WO_SeqListing.xml, was created on March 5, 2024, and has a size of 6388KB.

[0003] Field of the present invention This disclosure relates to pharmaceutical compositions comprising MARC1 RNAi agents, such as RNA interference (RNAi) agents, for example, double-stranded RNAi agents, such as chemically modified small (short) interfering RNA (siRNA), for the inhibition of mitochondrial amidoxime reducing agent 1 (MARC1), and to methods of using the same for the treatment of MARC1-related diseases and disorders. [Background technology]

[0004] background Mitochondrial amidooxime reducing component (MARC) proteins were first discovered and described in 2006 as molybdenum cofactor-containing components of the mitochondrial benzamidoxime prodrug conversion system. The human genome contains two MARC gene types: MTARC1 and MTARC2 (commonly known and simply referred to as MARC1 and MARC2, respectively), which encode MARC1 and MARC2 proteins that share significant homology in sequence and function.

[0005] Researchers identified a very rare missense variant of the MARC1 protein (referred to as the p.A165T mutation) that causes loss of function in the MARC1 protein, and this mutation was associated with protection against cirrhosis, reduced liver fat, and reductions in various other biomarkers in liver disease (Emdin CA, et al., A missense variant in Mitochondrial Amidoxime Reducing Component 1 gene and protection against liver disease, PLoS Genet. (April 2020); 16(4):e1008629). Individual homozygotes for this loss-of-function mutation in MARC1 have shown low levels of liver fat and a low likelihood of fatty liver diagnosed by a physician. The loss-of-function mutation in MARC1 has also been associated with low blood levels of alanine transaminase, alkaline phosphatase, total cholesterol, and LDL-cholesterol.

[0006] While the precise mechanisms of MARC1's role in liver disease progression are not yet fully understood, the reported associations of MARC1 have been further validated by recent genome-wide association studies focusing on liver disease and autoimmune hepatitis, which further confirmed that missense mutations in MARC1 and resulting loss of function are protective against liver injury and cirrhosis (Janik et al., MARC1 p. A165T variant is associated with decreased markers of liver injury and enhanced antioxidant capacity in autoimmune hepatitis. Sci Rep (2021); 11:24407). These combined data suggest that reducing the MARC1 protein may lower blood cholesterol levels and protect against cirrhosis, and that inhibiting MARC1 is a potential therapeutic target for the treatment of liver disease. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] overview There is a need for novel RNA interference (RNAi) agents (also referred to as RNAi agents, RNAi triggers, or triggers), such as double-stranded RNAi agents like chemically modified siRNA, that can selectively and efficiently inhibit MARC1 gene expression. Furthermore, there is a need for novel MARC1-specific RNAi agent compositions for use as therapeutic agents or pharmacologics for the treatment of MARC1-related diseases or disorders, such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related disorders. [Means for solving the problem]

[0008] The nucleotide sequences and chemical modifications of the MARC1 RNAi agents disclosed herein differ from those previously disclosed or known in the art. The MARC1 RNAi agents disclosed herein provide highly specific, potent, and effective in vivo and / or in vitro inhibition of MARC1 gene expression.

[0009] In some embodiments, the sense strand comprises a nucleotide sequence of 15 consecutive nucleotides from any one of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6D, and a nucleotide sequence of at least 15 consecutive nucleotides differing by 0 or 1 nucleotide, wherein the sense strand has a region of at least 85% complementarity with respect to the antisense strand across the 15 consecutive nucleotides.

[0010] In some embodiments, the following: Antisense strand, where nucleotides 1-19 of the antisense strand include nucleotides 1-19 of the antisense strand sequence in Table 2, Table 3, or Table 6D, and A sense strand containing a nucleotide sequence that is at least partially complementary to the antisense strand, Includes, Here, all or substantially all of the nucleotides of the antisense strand and / or sense strand are modified nucleotides, and the RNAi agent is linked to a targeting ligand containing N-acetyl-galactosamine. RNAi agents for inhibiting the expression of the MARC1 gene are disclosed herein.

[0011] In some embodiments, the following: A sense strand comprising a nucleotide sequence of 15 consecutive nucleotides from any one of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6D, and a nucleotide sequence of at least 15 consecutive nucleotides differing by 0 or 1 nucleotide, wherein the sense strand has a region of at least 85% complementarity with respect to the antisense strand across the 15 consecutive nucleotides. RNAi agents for inhibiting the expression of the MARC1 gene, including [specific RNAi agent], are disclosed herein.

[0012] In some embodiments, at least one nucleotide of the MARC1 RNAi agent contains a modified nucleoside linkage.

[0013] In some embodiments, the modified nucleotides of the MARC1 RNAi agents disclosed herein are selected from the group consisting of: 2'-O-methylnucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-seconucleotide mimetic, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, debasalized nucleotide, ribitol, reversed nucleotide, reversed 2'-O-methylnucleotide, reversed 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide, vinyl phosphonate-containing nucleotide, cyclopropylphosphonate-containing nucleotide, and 3'-O-methylnucleotide.

[0014] In other embodiments, all or substantially all of the modified nucleotides of the RNAi agents disclosed herein are 2'-O-methyl nucleotides, 2'-fluoro nucleotides, or combinations thereof.

[0015] In some embodiments, the antisense strand consists of, consists essentially of, or comprises the nucleotide sequence of any one of the modified antisense strand sequences in Table 3 or Table 6D.

[0016] In some embodiments, the sense strand consists of, consists essentially of, or comprises the nucleotide sequence of any of the modified sense strand sequences in Table 4, Table 5, or Table 6D.

[0017] In some embodiments, the antisense strand comprises the nucleotide sequence of any one of the modified sequences in Table 3 or Table 6D, and the sense strand comprises the nucleotide sequence of any one of the modified sequences in Table 4 or Table 6D.

[0018] The RNAi agents disclosed herein are linked to a targeting ligand comprising N-acetyl-galactosamine. In further embodiments, the targeting ligand is linked to the sense strand. In some embodiments, the targeting ligand is linked to the 5' end of the sense strand.

[0019] In some embodiments, the sense strand is 15 to 30 nucleotides in length, and the antisense strand is 19 to 30 nucleotides in length. In other embodiments, the sense strand and the antisense strand are each 21 to 27 nucleotides in length. In other embodiments, the sense strand and the antisense strand are each 21 to 24 nucleotides in length. In still other embodiments, the sense strand and the antisense strand are each 21 nucleotides in length.

[0020] In some embodiments, the RNAi agent has two blunt ends.

[0021] In some embodiments, the sense strand includes one or two terminal caps. In other embodiments, the sense strand includes one or two reverse debase residues.

[0022] In some embodiments, the RNAi agent consists of a sense strand and an antisense strand that form a double-stranded sequence of the double-stranded structure shown in Tables 6A, 6B, 6C, or 6D.

[0023] In some embodiments, the sense strand further comprises a reverse debase residue at the 3' end of the nucleotide sequence, the 5' end of the nucleotide sequence, or both.

[0024] In further embodiments, the targeted ligand is as follows: [ka] It includes or consists of.

[0025] Also disclosed are compositions comprising the RNAi agent of the present disclosure, further comprising pharmaceutically acceptable excipients.

[0026] Furthermore, the following method is provided for inhibiting the expression of the MARC1 gene in human hepatocytes in vivo, comprising introducing an effective amount of the MARC1 RNAi agent or composition of the Disclosure into the subject.

[0027] A method for treating a MARC1-related disease, disorder, or symptom is further provided herein, comprising administering a therapeutically effective amount of the composition of this disclosure to a human subject in need thereof.

[0028] In some embodiments, the disease is non-alcoholic steatohepatitis (NASH), alcoholic and non-alcoholic fatty liver disease (NAFLD), fatty liver disease, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related diseases.

[0029] In some embodiments, the RNAi agent is administered at a dose of approximately 0.05 mg / kg to approximately 5.0 mg / kg per body weight of the human subject. In some embodiments, the MARC1 RNAi agents disclosed herein are administered in a fixed single dose containing approximately 50 mg, approximately 100 mg, approximately 200 mg, approximately 300 mg, or approximately 400 mg of the MARC1 RNAi agent.

[0030] The use of the RNAi agents or compositions of the disclosure for the treatment of diseases, disorders, or conditions mediated at least partially by MARC1 gene expression is also provided herein.

[0031] The use of the RNAi agents or compositions of the disclosure for the preparation of pharmaceutical compositions for treating diseases, disorders, or symptoms that are at least partially mediated by MARC1 gene expression is further provided herein. [Modes for carrying out the invention]

[0032] Detailed explanation The RNAi agents, compositions thereof, and methods of use of this disclosure can be more readily understood by referring to the following detailed description, which forms part of this disclosure. It should be understood that this disclosure is not limited to the methods specifically described and / or shown herein, and furthermore, that the terms used herein are intended solely to illustrate and not to limit specific embodiments.

[0033] Certain features of the Disclosure cited herein are described in the context of separate embodiments for clarity, but it should be understood that they may also be provided in combination of individual embodiments. Conversely, various features of the methods of the Disclosure described as a single embodiment for brevity may also be provided separately or in any partial combination.

[0034] definition

[0035] As used herein, “RNAi agent” means a chemical composition comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of reducing or inhibiting the translation of mRNA transcripts of a target messenger RNA (mRNA) in a sequence-specific manner. As used herein, RNAi agents may act via RNA interference mechanisms (i.e., by inducing RNA interference through interaction with the RNA interference pathway mechanism (RNA-induced silencing complex, RISC) in mammalian cells) or by any alternative mechanism or pathway. While the term RNAi agent as used herein is primarily understood to act via RNA interference mechanisms, the RNAi agents of this disclosure are not bound by, and are not limited to, any particular pathway or mechanism of action. The RNAi agents disclosed herein consist of a sense strand and an antisense strand, and include, but are not limited to, short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the targeted mRNA (i.e., MARC1 mRNA). The RNAi agents may include one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0036] As used herein, the terms “silence,” “reduction,” “inhibition,” “downregulate,” or “knockdown,” when relating to the expression of a given gene, mean that the expression of the gene is reduced when the cell, cell population, tissue, organ, or subject is treated with an oligomeric compound such as an RNAi agent described herein, compared to a second cell, cell population, tissue, organ, or subject that is not treated in the same way, as measured by the level of RNA transcribed from the gene or the level of polypeptides, proteins, or protein subunits translated from mRNA in the cell, cell population, tissue, organ, or subject from which the gene is transcribed.

[0037] As used herein, the terms “sequence” or “nucleotide sequence” mean a sequence or order of nucleic acid bases or nucleotides, and are written as a sequence of letters using standard nomenclature. Nucleic acid molecules may contain unmodified and / or modified nucleotides. Nucleic acid sequences may contain unmodified and / or modified nucleotides.

[0038] As used herein, “base,” “nucleotide base,” or “nucleic acid base” refers to a heterocyclic pyrimidine or purine compound that is a component of a nucleotide, including primary purine bases adenine and guanine, and primary pyrimidine bases cytosine, thymine, and uracil. Nucleic acid bases may be further modified to include, but are not limited to, universal bases, hydrophobic bases, indiscriminate bases, size-expanded bases, and fluorinated bases. (See, for example, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008). The synthesis of such modified nucleic acid bases (including phosphoramidite compounds containing modified nucleic acid bases) is known in the art.

[0039] As used herein, the term “nucleotide” has the same meaning as it is commonly understood in the art. Thus, as used herein, “nucleotide” refers to a glycoside comprising a sugar moiety, a base moiety, and a covalent bonding group (linking group), such as a phosphate or phosphorothioate nucleoside linking group, and encompasses both natural nucleotides, such as DNA or RNA, and non-natural nucleotides (also referred herein as nucleotide analogs) comprising modified sugars and / or base moieties. In this specification, a single nucleotide may also be referred to as a monomer or unit.

[0040] As used herein, and unless otherwise specified, the term “complementary” means, when used to describe a first nucleic acid base or nucleotide sequence (e.g., the sense strand of an RNAi agent or the targeted mRNA) in relation to a second nucleic acid base or nucleotide sequence (e.g., the antisense strand of an RNAi agent or a single-strand antisense oligonucleotide), that an oligonucleotide or polynucleotide containing the first nucleotide sequence can hybridize (form base-pair hydrogen bonds under mammalian physiological conditions (or otherwise preferred in vivo or in vitro conditions)) with an oligonucleotide or polynucleotide containing the second nucleotide sequence under specific standard conditions to form a double-stranded or double-helical structure. Those skilled in the art will be able to select the set of conditions best suited for hybridization testing. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and also include native nucleotides, modified nucleotides, or nucleotide mimetic compounds, at least insofar as the above hybridization requirements are met. Sequence identity or complementarity is independent of modification. For example, a and Af as defined herein are complementary to U (or T) and are identical to A for the purpose of determining identity or complementarity.

[0041] As used herein, “perfectly complementary” or “completely complementary” means that in a pair of hybridized nucleic acid bases or nucleotide sequence molecules, all (100%) of the bases in the sequence of the first oligonucleotide hybridize to the same number of bases in the sequence of the second polynucleotide. The sequence may consist of all or part of the first or second nucleotide sequence.

[0042] As used herein, “partially complementary” means that in a pair of hybridized nucleic acid bases or nucleotide sequence molecules, at least 70% but not all of the bases in the sequence of the first polynucleotide hybridize to the same number of bases in the sequence of the second polynucleotide. The sequence may comprise all or part of the first or second nucleotide sequence.

[0043] As used herein, “substantially complementary” means that in a pair of hybridized nucleic acid bases or nucleotide sequence molecules, at least about 85% of, but not all, of the bases in the sequence of the first polynucleotide hybridize to the same number of bases in the sequence of the second polynucleotide. The sequence may comprise all or part of the first or second nucleotide sequence.

[0044] 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 the sequence of MARC1 mRNA.

[0045] As used herein, the terms “substantially identical” or “substantially identical,” when applied to nucleic acid sequences, mean that a nucleic acid sequence (or a portion of a nucleotide sequence) has at least about 85% sequence identity, preferably at least 90%, at least 95%, or at least 99% identity, compared to a reference sequence. The percentage of sequence identity is determined by comparing two optimally aligned sequences across a comparison window. This percentage is calculated by determining the number of positions in both sequences where the same type of nucleic acid base occurs, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the window, and multiplying the result by 100 to obtain the percentage of sequence identity (%). The inventions disclosed herein encompass nucleotide sequences that are substantially identical to those disclosed herein.

[0046] As used herein, the terms “individual,” “patient,” and “subject” are used interchangeably to refer to birds, humans, and other primates, as well as members of any animal species, including but not limited to commercially relevant mammals or other mammals, including animal models such as mice, rats, monkeys, cattle, pigs, horses, sheep, cattle, and dogs. Preferably, the subject is human.

[0047] As used herein, the terms “to treat,” “treatment,” etc., mean a method or step taken to provide relief or mitigation of the number, severity, and / or frequency of one or more symptoms of a disease or condition in a subject. As used herein, “to treat” and “treatment” may also include prevention, 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.

[0048] As used herein, the phrase “introduced into cells” when referring to RNAi agents means functionally delivering the RNAi agent into cells. The phrase “functionally delivered” means delivering the RNAi agent to cells in a manner that enables the RNAi agent to have its expected biological activity, such as sequence-specific inhibition of gene expression.

[0049] Unless otherwise specified, symbols used herein [ka] The use of means any one of the bases which may be linked in accordance with the scope of the invention as described herein.

[0050] As used herein, the term "isomer" refers to a compound having the same molecular formula but differing in the nature or arrangement of its atomic bonds, or in the spatial arrangement of its atoms. Isomers that differ in the spatial arrangement of their atoms are called "stereoisomers." Stereoisomers that are not mirror images of each other are called "diastereomers," while stereoisomers that are mirror images that cannot be superimposed are called "enantiomers" or sometimes "optical isomers." A carbon atom bonded to four non-identical substituents is called a "chiral center."

[0051] When used herein, unless otherwise specified in the structure as having a particular conformation, for each structure in which an asymmetric center exists, resulting in an enantiomer, diastereomer, or other stereoisomer configuration, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure form and racemates. For example, the structures disclosed herein are intended to encompass mixtures of diastereomers, as well as individual stereoisomers.

[0052] When used in the claims of this application, the phrase "consists of" excludes elements, steps, or components not specified in the claims. When used in the claims of this application, the phrase "essentially consists of" limits the scope of the claims to specified materials or steps, and those that do not substantially affect the basic and novel features of the claimed patent.

[0053] It will be readily understood and appreciated by those skilled in the art that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound or composition is located. Therefore, when used herein, the structures disclosed herein are assumed to have certain functional groups (e.g., OH, SH, or NH) that can be protonated or deprotonated. The disclosure herein is intended to encompass the disclosed compounds and compositions regardless of the protonation state based on the environment (e.g., pH), as will be readily understood by those skilled in the art. Accordingly, compounds described herein having an unstable proton or basic atom should be understood as representing a salt form of the corresponding compound. The compounds described herein may be free acids, free bases, or salt forms. pharmaceutically acceptable salts of the compounds described herein should be understood to be within the scope of the invention.

[0054] When used herein, the terms “linked” or “conjugated” mean that the two compounds or molecules are joined by a covalent bond. Unless otherwise stated herein, the terms “linked” and “conjugated” may also mean a connection between a first compound and a second compound, with or without any intervening atoms or atomic groups.

[0055] As used herein, the term “including” means “including, but not limited to” and is used interchangeably. Unless otherwise explicitly stated by the context, the term “or” means “and / or” and is used interchangeably.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly interpreted by those skilled in the art. Methods and materials similar to or equivalent to those described herein may be used to carry out or test the present invention, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference in their entirety. In case of any inconsistency, this specification shall prevail, including definitions. In addition, materials, methods, and examples are illustrative and not intended to be limiting.

[0057] Where a value is explicitly stated, it should be understood that a value approximately equal to or the same as the stated value is also within the scope of this disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of this disclosure. Conversely, where different elements or groups of elements are disclosed individually, combinations of these are also disclosed. Where any element of this disclosure is disclosed as having multiple alternative forms, examples of that disclosure in which each alternative form is excluded, either alone or in any combination with other alternative forms, are also disclosed herein, and multiple elements of the disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0058] Other objects, features, embodiments, and advantages of the present invention will become apparent from the following detailed description, the accompanying drawings, and the claims.

[0059] Detailed explanation RNAi agents RNAi agents for inhibiting the expression of the MARC1 gene are described herein. Each MARC1 RNAi agent comprises a sense strand and an antisense strand. The sense strand is 15 to 49 nucleotides long. The antisense strand may be 19 to 49 nucleotides long. The sense strand and antisense strand may be the same length or different lengths. In some embodiments, the sense strand and antisense strand are each independently 19 to 27 nucleotides long. In some embodiments, both the sense strand and antisense strand are each 21 to 26 nucleotides long. In some embodiments, the sense strand and antisense strand are each 21 to 24 nucleotides long. In some embodiments, the sense strand is about 19 nucleotides long and the antisense strand is about 21 nucleotides long. In some embodiments, the sense strand is about 21 nucleotides long and the antisense strand is about 23 nucleotides long. In some embodiments, the sense strand is 23 nucleotides long and the antisense strand is 21 nucleotides long. In some embodiments, both the sense strand and antisense strand are each 21 nucleotides long. In some embodiments, the antisense strands of the RNAi agent are independently 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, the sense strands of the RNAi agent are independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides long. The sense and antisense strands are aligned to form a double helix, and in some embodiments, the double-stranded RNAi agent has a double-strand length of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0060] Examples of nucleotide sequences used to form MARC1 RNAi agents are provided in Tables 2, 3, 4, and 5. Examples of RNAi agent double-stranded forms containing the sense and antisense strand sequences from Tables 2, 3, 4, and 5 are shown in Tables 6A and 6B.

[0061] In some embodiments, the region of complete, substantial, or partial complementarity between the sense strand and the antisense strand is 15–26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides long and located at or near the 5' end of the antisense strand (e.g., this region may be separated from the 5' end of the antisense strand by 0, 1, 2, 3, or 4 nucleotides that are not completely, substantial, or partially complementary).

[0062] The sense strand of the MARC1 RNAi agent described herein contains at least 16 consecutive nucleotides having at least 85% identity with the core stretch sequence (also referred to herein as the “core stretch” or “core sequence”) of the same number of nucleotides in the MARC1 mRNA. In some embodiments, the sense strand core stretch sequence is 100% (completely) complementary to the antisense strand core stretch sequence, or at least about 85% (substantially) complementary, and therefore, the sense strand core stretch sequence is typically completely identical, or at least about 85% identical, to a nucleotide sequence of the same length present in the MARC1 mRNA target (which may also be referred to as the target sequence). In some embodiments, this sense strand core stretch is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides long. In some embodiments, this sense strand core stretch is 17 nucleotides long. In some embodiments, this sense strand core stretch is 19 nucleotides long. In some embodiments, this sense strand core stretch is 21 nucleotides long.

[0063] The antisense strand of the MARC1 RNAi agent described herein comprises a core stretch of the same number of nucleotides in the MARC1 mRNA and the same number of nucleotides in the corresponding sense strand, and at least 15 consecutive nucleotides having at least 85% complementarity. In some embodiments, the antisense strand core stretch is 100% (completely) complementary or at least about 85% (substantially) complementary to a nucleotide sequence of the same length present in the MARC1 mRNA target (e.g., the target sequence). In some embodiments, this antisense strand core stretch is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides long. In some embodiments, this antisense strand core stretch is 21 nucleotides long. In some embodiments, this antisense strand core stretch is 19 nucleotides long. The sense strand core stretch sequence may be the same length as the corresponding antisense core sequence, or it may be of a different length.

[0064] The sense and antisense strands of a MARC1 RNAi agent anneal to form a double helix. The sense and antisense strands of a MARC1 RNAi agent may be partially, substantially, or completely complementary to each other. Within the complementary double-stranded region, the sense strand core stretch sequence is at least about 85% or 100% complementary to the antisensor core stretch sequence. In some embodiments, the sense strand core stretch sequence includes a sequence of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides that is at least about 85% or 100% complementary to the corresponding sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides of the antisense strand core stretch sequence (i.e., the sense and antisense core stretch sequences of the MARC1 RNAi agent have a region of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides that are at least 85% or 100% base-paired).

[0065] In some embodiments, the antisense strand of the MARC1 RNAi agent disclosed herein differs from any of the antisense strand sequences in Table 2 or Table 3 by 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of the MARC1 RNAi agent disclosed herein differs from any of the sense strand sequences in Table 2, Table 4, or Table 5 by 0, 1, 2, or 3 nucleotides.

[0066] In some embodiments, the sense strand and / or antisense strand may optionally and independently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides (extensions) at the 3' end, 5' end, or both the 3' and 5' ends of the core stretch sequence. The additional nucleotides of the antisense strand, if present, may or may not be complementary to the corresponding sequence in the MARC1 mRNA. The additional nucleotides of the sense strand, if present, may or may not be identical to the corresponding sequence in the MARC1 mRNA. The additional nucleotides of the antisense strand, if present, may or may not be complementary to the corresponding additional nucleotides of the sense strand (if present).

[0067] When used herein, the extensions contain 1, 2, 3, 4, 5, or 6 nucleotides at the 5' and / or 3' ends of the sense strand core stretch sequence and / or antisense strand core stretch sequence. The sense strand extension nucleotides may or may not be complementary to the corresponding antisense strand nucleotides (either core stretch sequence nucleotides or extension nucleotides). Conversely, the antisense strand extension nucleotides may or may not be complementary to the corresponding sense strand nucleotides (either core stretch sequence nucleotides or extension nucleotides). In some embodiments, both the sense and antisense strands of the RNAi agent contain 3' and 5' extensions. In some embodiments, one or more 3' extension nucleotides of one strand are base-paired with one or more 5' extension nucleotides of the other strand. In other embodiments, one or more 3' extension nucleotides of one strand are not base-paired with one or more 5' extension nucleotides of the other strand. In some embodiments, the MARC1 RNAi agent has an antisense strand having a 3' extension and a sense strand having a 5' extension. In some embodiments, the extension nucleotides are unpaired and form an overhang. As used herein and in the art, “overhang” refers to the extent of a stretch of one or more unpaired nucleotides located at the end of either the sense strand or the antisense strand that does not form part of the hybridized or double-stranded portion of the RNAi agent disclosed herein.

[0068] In some embodiments, the MARC1 RNAi agent comprises an antisense strand having a 3' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In other embodiments, the MARC1 RNAi agent comprises an antisense strand having a 3' extension of 1, 2, or 3 nucleotides in length. In some embodiments, one or more nucleotides in the antisense strand extension contain nucleotides complementary to the corresponding MARC1 mRNA sequence. In some embodiments, one or more nucleotides in the antisense strand extension contain nucleotides that are not complementary to the corresponding MARC1 mRNA sequence.

[0069] In some embodiments, the MARC1 RNAi agent comprises a sense strand having a 3' extension of 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprise adenosine, uracil, or thymidine nucleotide, AT dinucleotide, or a nucleotide corresponding to or identical to a nucleotide in the MARC1 mRNA sequence. In some embodiments, the 3' sense strand extension comprises, but is not limited to, one of the following sequences: T, UT, TT, UU, UUT, TTT, or TTTT (each listed from 5' to 3').

[0070] The sense strand may have a 3' extension and / or a 5' extension. In some embodiments, the MARC1 RNAi agent includes a sense strand having a 5' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, one or more nucleotides in the sense strand extension include nucleotides that correspond to or are identical to nucleotides in the MARC1 mRNA sequence.

[0071] Examples of nucleotide sequences used to form MARC1 RNAi agents are provided in Tables 2, 3, 4, 5, and 6D. In some embodiments, the antisense strand of the MARC1 RNAi agent contains a nucleotide sequence from any of the sequences in Tables 2, 3, or 6D. In certain embodiments, the MARC1 RNAi agent antisense strand contains or consists of one of the modified sequences in Table 3 or Table 6D. In some embodiments, the antisense strand of the MARC1 RNAi agent contains the nucleotide (5'-to-3' end) sequences 1-17, 2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21 from any of the sequences in Tables 2, 3, or 6D. In some embodiments, the sense strand of the MARC1 RNAi agent contains a sequence from any of the sequences in Tables 2, 4, 5, or 6D. In some embodiments, the MARC1 RNAi agent sense strand contains the sequence of nucleotides (5' end to 3' end) 1-18, 1-19, 1-20, 1-21, 2-19, 2-20, 2-21, 3-20, 3-21, or 4-21 of any of the sequences in Table 2, 4, 5, or 6D. In certain embodiments, the MARC1 RNAi agent sense strand contains or consists of one of the modified sequences in Table 4, 5, or 6D.

[0072] In some embodiments, the sense and antisense strands of the RNAi agent described herein have the same number of nucleotides. In some embodiments, the sense and antisense strands of the RNAi agent described herein have different numbers of nucleotides. In some embodiments, the 5' end of the sense strand and the 3' end of the antisense strand of the RNAi agent form a blunt end. In some embodiments, the 3' end of the sense strand and the 5' end of the antisense strand of the RNAi agent form a blunt end. In some embodiments, both ends of the RNAi agent form a blunt end. In some embodiments, neither end of the RNAi agent is a blunt end. As used herein, “blunt end” refers to the end of a double-stranded RNAi gene where the terminal nucleotides of the two annealed strands are complementary (forming complementary base pairs).

[0073] In some embodiments, the 5' end of the sense strand and the 3' end of the antisense strand of the RNAi agent form a frayed end. In some embodiments, the 3' end of the sense strand and the 5' end of the antisense strand of the RNAi agent form a frayed end. In some embodiments, both ends of the RNAi agent form a frayed end. In some embodiments, neither end of the RNAi agent is a frayed end. As used herein, a frayed end refers to the end of a double-stranded RNAi agent where the terminal nucleotides of the two annealed strands form a pair (i.e., do not form an overhang) but are not complementary (i.e., form a non-complementary pair). In some embodiments, one or more unpaired nucleotides at the end of one strand of the double-stranded RNAi agent form an overhang. The unpaired nucleotides may be on the sense strand or the antisense strand and form either a 3' or 5' overhang. In some embodiments, the RNAi agent includes a blunt end and a frayed end, a blunt end and a 5' overhang end, a blunt end and a 3' overhang end, a frayed end and a 5' overhang end, a frayed end and a 3' overhang end, two 5' overhang ends, two 3' overhang ends, a 5' overhang end and a 3' overhang end, two frayed ends, or two blunt ends. Typically, if present, the overhangs are located at the 3' end of the sense strand, the antisense strand, or both the sense strand and the antisense strand.

[0074] The MARC1 RNAi agents disclosed herein may also consist of one or more modified nucleotides. In some embodiments, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand of the MARC1 RNAi agent are modified nucleotides. The MARC1 RNAi agents disclosed herein may further consist of one or more modified nucleoside linkages, for example, one or more phosphorothioate linkages. In some embodiments, the MARC1 RNAi agent comprises one or more modified nucleotides and one or more modified nucleoside linkages. In some embodiments, a 2'-modified nucleotide is combined with a modified nucleoside linkage.

[0075] In some embodiments, the MARC1 RNAi agent is prepared or provided as a salt, a mixed salt, or a free acid. In some embodiments, the MARC1 RNAi agent is prepared as a pharmaceutically acceptable salt. In some embodiments, the MARC1 RNAi agent is prepared as a pharmaceutically acceptable sodium salt. Such forms well known in the art are within the scope of the present invention as disclosed herein.

[0076] Modified nucleotides Modified nucleotides, when used in various oligonucleotide constructs, can maintain the activity of the compounds in cells, while simultaneously enhancing the serum stability of these compounds, and can also minimize the potential for activating interferon activity in humans when the oligonucleotide constructs are administered.

[0077] In some embodiments, the MARC1 RNAi agent comprises one or more modified nucleotides. As used herein, “modified nucleotide” is a nucleotide other than a ribonucleotide (2'-hydroxylnucleotide). 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. When used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimes, debasalized nucleotides, 2'-modified nucleotides, reversed nucleotides, modified nucleic acid base-containing nucleotides, cross-linked nucleotides, peptide nucleic acids (PNA), 2',3'-seconucleotide mimes (locked nucleic acid base analogs), locked nucleotides, 3'-O-methoxy(2'-nucleoside linkage) nucleotides, 2'-F-arabinonucleotides, 5'-Me, 2'-fluoronucleotides, morpholinonucleotides, vinylphosphonate deoxyribonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides. Examples of 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, but are not limited to, 2'-O-methylnucleotide (also referred to herein or in the art as 2'-methoxynucleotide), 2'-fluoronucleotide (also referred to herein or in the art as 2'-deoxy-2'-fluoronucleotide), 2'-deoxynucleotide, 2'-methoxyethyl (2'-O-2-methoxyethyl)) nucleotide (also referred to herein or in the art as 2'-MOE nucleotide), 2'-aminonucleotide, and 2'-alkylnucleotide. It is not necessary to uniformly modify all positions in a given compound. Conversely, two or more modifications can be incorporated into a single MARC1 RNAi agent, or even a single nucleotide thereof. The sense and antisense strands of a MARC1 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.

[0078] Modified nucleic acid bases include synthetic and natural nucleobases, such as 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 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) derivatives of guanine, and other alkyl derivatives, 2-thiouracil Examples include syl, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azouracil, 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.

[0079] In some embodiments, the 5' and / or 3' ends of the antisense strand may contain a debasic residue (Ab), which may also be referred to as a “debasic site” or “debasic nucleotide.” A debasic residue (Ab) is a nucleotide or nucleoside lacking a nucleic acid base at the 1' position of the sugar moiety. In some embodiments, the debasic residue may be located internally in the nucleotide sequence. In some embodiments, Ab or AbAb may be added to the 3' end of the antisense strand. In some embodiments, the 5' end of the sense strand may contain one or more additional debasic residues (e.g., (Ab) or (AbAb)). In some embodiments, UUAb, UAb, or Ab may be added to the 3' end of the sense strand. In some embodiments, a debasic (deoxyribose) residue may be replaced with a ribitol (debasic ribose) residue.

[0080] In some embodiments, all or substantially all of the nucleotides of the RNAi agent are modified nucleotides. As used herein, an RNAi agent in which substantially all of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand, which are ribonucleotides (i.e., unmodified). As used herein, a sense strand in which substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2) modified nucleotides in the sense strand, which is a ribonucleotide. As used herein, an antisense strand in which substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) modified nucleotides in the antisense strand, which is a ribonucleotide. In some embodiments, one or more nucleotides of the RNAi agent are unmodified ribonucleotides. The chemical structures of specific modified nucleotides are given in Table 7 of this specification.

[0081] Inter-modified nucleoside linkage In some embodiments, one or more nucleotides of the MARC1 RNAi agent are linked by non-standard linkages or skeletons (i.e., modified nucleoside linkages or modified skeletons). These modified nucleoside linkages or skeletons include 5'-phosphorothioate groups (represented herein by lowercase "s"), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkylphosphonates (e.g., methylphosphonate or 3'-alkylenephosphonate), chiral phosphonates, phosphinates, and phosphoramidates (e.g., 3'-aminophosphoramidates). Examples include, but are not limited to, aminoalkylphosphoromidates or thionophosphoromidates, thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having the usual 3'-5' linkages, 2'-5' linked analogues of boranophosphates, or boranophosphates having opposite polarity in which adjacent nucleoside pair units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. In some embodiments, the modified internucleoside linkages or skeletons do not contain phosphorus atoms. Examples of modified internucleoside linkages that do not contain phosphorus atoms include, but are not limited to, 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, but are not limited to, siloxane skeletons, sulfide skeletons, sulfoxide skeletons, sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamate skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and other skeletons having mixed N, O, S, and CH2 components.

[0082] In some embodiments, the sense strand of a MARC1 RNAi agent may contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, the antisense strand of a MARC1 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 a MARC1 RNAi agent may contain 1, 2, 3, or 4 phosphorothioate linkages, the antisense strand of a MARC1 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.

[0083] In some embodiments, the sense strand of the MARC1 RNAi agent contains at least two phosphorothioate nucleoside linkages. In some embodiments, the 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 nucleotide sequence, and another phosphorothioate linkage is located at the 3' end of the sense strand nucleotide sequence. 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 optionally includes a reverse debase residue terminal cap. In some embodiments, the targeted ligand is linked to the sense strand via phosphorothioate linkages.

[0084] In some embodiments, the antisense strand of the MARC1 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 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 1-4 from the 5' end of the antisense strand, and a fourth phosphorothioate nucleoside linkage is located between 20-21 from the 5' end of the antisense strand. In some embodiments, the MARC1 RNAi agent contains at least three or four phosphorothioate nucleoside linkages in the antisense strand.

[0085] Capping residue or portion In some embodiments, the sense strand may include one or more capping residues or portions, which may also be referred to in the art as “caps,” “terminal caps,” or “capping residues.” As used herein, “capping residues” are non-nucleotide compounds or other portions that can be incorporated into one or more ends of the nucleotide sequence of an RNAi agent disclosed herein. Capping residues can provide an RNAi agent that, in some cases, has certain beneficial properties, such as protection against exonuclease degradation. In some embodiments, reverse debase residues (invAb) (also referred to in the art as “reverse base sites”) are added as capping residues (see, for example, F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16; U.S. Patent No. 5,998,203). Capping residues are generally known in the art, and include, for example, reverse debase residues and terminal C3H7 (propyl), C6H 13 (Hexyl), or C 12 H 25The capping residue includes a carbon chain such as a (dodecyl) group. In some embodiments, the capping residue is located at either the 5' end, the 3' end, or both the 5' and 3' ends of the sense chain. In some embodiments, the 5' and / or 3' ends of the sense chain may contain two or more reverse debasing deoxyribose moieties as capping residues.

[0086] In some embodiments, one or more reverse debase residues (invAb) are added to the 3' end of the sense strand. In some embodiments, one or more reverse debase residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more reverse debase nucleosides may be inserted between the targeting ligand and the nucleic acid base sequence of the sense strand of the RNAi agent. In some embodiments, including one or more reverse debase residues or reverse debase sites at or near the end or both ends of the sense strand of the RNAi agent can enhance the activity of the RNAi agent or enable other desired properties.

[0087] In some embodiments, one or more reverse debasing residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more reverse debasing nucleosides may be inserted between the targeting ligand and the nucleic acid base sequence of the sense strand of the RNAi agent. The reverse debasing residues may be linked via a phosphate, a phosphorothioate (e.g., indicated herein as (invAb)s), or other internucleoside linkages. In some embodiments, including one or more inverted debasing residues at the end or both ends of the sense strand of the RNAi agent, or near the end or both ends, may enable enhanced activity of the RNAi agent or other desired properties. In some embodiments, the reverse debasing (deoxyribose) residue may be replaced with a reverse ribitol (debasing ribose) residue. In some embodiments, the 3' end of the antisense strand core stretch sequence, or the 3' end of the antisense strand sequence, may contain a reverse debasing residue. The chemical structure of the reverse debasic deoxyribose residue is shown in Table 7 below.

[0088] MARC1 RNAi agent The MARC1 RNAi agents disclosed herein are designed to target specific locations in the MARC1 gene (e.g., SEQ ID NO: 1). Human mitochondrial amidoxime reducing component 1 (MARC1), mRNA transcript (SEQ ID NO: 1) (7287 bases), NCBI reference sequence: NM_022746.4: 1 cttgccgccg ccacctcgcg gagaagccag ccatgggcgc cgccggctcc tccgcgctgg 61 cgcgctttgt cctcctcgcg caatcccggc ccgggtggct cggggttgcc gcgctgggcc 121 tgaccgcggt ggcgctgggg gctgtcgcct ggcgccgcgc atggcccacg cggcgccggc 181 ggctgctgca gcaggtgggc acagtggcgc agctctggat ctaccctgtg aaatcctgca 241 aggggtgcc ggtgagcgag gcggagtgca cggccatggg gctgcgcagc ggcaacctgc 301 gggacaggtt ttggcttgtg atcaaccagg agggaaacat ggttactgct cgccaggaac 361 ctcgcctggt cctgatttcc ctgacctgcg atggtgacac cctgactctc agtgcagcct 421 acacaaagga cctactactg cctatcaaaa cgcccaccac aaatgcagtg cacaagtgca 481 gagtgcacgg cctggagata gagggcaggg actgtggcga ggccaccgcc cagtggataa 541 ccagcttcct gaagtcacag ccctaccgcc tggtgcactt cgagcctcac atgcgaccga 601 gacgtcctca tcaaatagca gacttgttcc gacccaagga ccagattgct tactcagaca 661 ccagcccatt cttgatcctt tctgaggcgt cgctggcgga tctcaactcc aggctagaga 721 781 cagaggattc ttgggatgag cttcttattg gtgacgtgga actgaaaagg gtgatggctt 841 gttccagatg cattttaacc acagtggacc cagacaccgg tgtcatgagc aggaggaac 901 961 aatcaccact ctttgggcag tattttgtgc tggaaaaccc agggaccatc aaagtgggag 1021 accctgtgta cctgctgggc cattaatggg aaccgtatgt cctggaatat tagatgcctt 1081 ttaaaaatgt tctcaaaaat gacaacactt gaagcatggt gtttcagaac tgagacctct 1141 acattttctt taaatttgtg attttcacat ttttcgtctt ttggacttct ggtgtctcaa 1201 1261 cagtaagtca cttaatgac agacaggat tctgaaact cccgtttaa ctgatttagg 1321 atagttctt tctcctgctt ctccgtttat ctaccaagag cgcagacttg catcctgtca 1381 ctaccactcg ttagagaag agagagaag aagaggaag agtggggtgg ctggaagaat 1441 atcctagaat gtgttattgc ccctgttcat gaggtacgca atgaaatta aattgcaccc 1501 caatatggc tggaatgcca cttcctttt cttctcaagc cccggggctag ctttgaat 1561 ggcataaga ctgaggtgac ctcaggag cactgcagat atttattc catagatctg 1621 gatctgccc tgctgctct cagacagcat tggattctt aaggtgctc aggaggatgg 1681 ttgtgtagtc atggaggacc cctggatcct tgccattccc ctcagctat gacggagtgc 1741 tccttcca gttccggttg aaaagttct gattctgtg gaggagaaga aaagtgatc 1801 agtgatttca gatagactac tgaaaacctt taaaggggga aaggaaagc atatgtcagt 1861 tgttaaac ccaatatcta tttttaact gattgtataa ctctaagatc tgatgaagta 1921 tattttt tgccatttg tcctttgatt atattgggaa gttgactaaa cttgaaaaat 1981 2041 ttggcacaaa gttagactgt gaaagctgac tgaggctgggg cacaggggct catgcctgta 2101 attccagcac tttgggaggc caaggtggga gaatggcttg agcccaggag tttgagacca 2161 gcccagaaaa tataatggga tcctgtcgct acaaaatgtt tttaaaatgc actcggtgtg 2221 gtggtgtgtg cctgcagtcc tggctatggc tactcgggag gatgaggtag areattggt 2281 tgagcccagg agcgggagat tgaggctgca gtgagttatg attgcaccac tacctccag 2341 cctgagtgat agagtgagac cctatctcta aaaaagaaac aggaaaaaaa aagaaagctg 2401 actgaggtga atgggcaaag ccagtaattc tgacacctga ccacagctgg gtcttctgca 2461 taatggacct cctcacccac agcctcccag gcaagcaccc atgtttgaag gactatcaag 2521 tcaacatgct ttttaccaaa agctgcacat ttttcacttt gattttataa aagaggtcag 2581 taatcgctga aatctagctg agccctgaag taaagttctg agcaaagagg tgcatgtgct 2641 tgttttatgg ttggtgaatt attacagttt gttttctgca tgcttggcat gaggtgaata 2701 attacatcaa ttttccagag aacctgggcc atcaccttcc ccaacaagtc cagttgatgt 2761 tgaaactaca gatagattga gacaaagcga agtgttcagc aagtagcatt actaatggga 2821 ccgggggacc cgtgggagag tgagtgtaca caggatttag gaaaccatgt gaatatgggc 2881 tctctgggaa tagccaatag gtagggagca atcagaaacc caaggtttgg tggctcttcc 2941 taggtattta taattagtgg caagtgaaag ccttagtcct gaatttctaa ccacttgtaa 3001 gaactaacag ccacttctct gtgccccgtc cgggcagtaa ccatcattct ccatggacag 3061 gctctcgggg tagctagctc tgcagggcag cacccacgtg gaagggagca cccagaaacc 3121 ctcctcactg ggcagacctg tccttctgtg cctcacagtg tgaggaagat tcctgtttga 3181 agagagaagt tccagtgacc tctagaatct cagagtagtt gccaagcttt ctgtcagtga 3241 gatttaaagg ccatttactt gtgtttattt tatatttaat gagttggtta atgccagaga 3301 caaagctgat atcccattta ttttggatac tgagcatttg cacactattc cacttgaaat 3361 atagaatcag gaatgtaggc catcccagac tttcagatct tacaacagca aatgacagat 3421 gtttgagatc aggccaaaat atccaccctc ggtgggcatc tcctctgtgt ggcaacttat 3481 gctgcagcca cagtggggag tcacaaactc agagctggag gtcttgaaaa ggacaatgtg 3541 ggccaggctc cggaggggct gcctaaaggc ttgcttttgt gactctcctg cagaaaatgt 3601 tagaaacttc caaccgaaag acgagggcag caacttatac acacgaaggc agaaagaaat 3661 tggggaaggg gaggctgttg gaattcaggc cgttgtccta tagggagaaa tactcctcct 3721 ctccttctcc ctttactgat aacggggcat ggtgaggaga tgagcttgtg agggtctgcc 3781 agtttggtaa gagtgcatgg ggaggttggg taaattagac tagccaaatg ggacttcggg 3841 aaaccattta tgaggctgtc accaacagtg atggcaggct gaaattccag gcaagtgctc 3901 ccagcattcc aagagtgtat caaattaaag caacccatga tggtggagaa cagatacatt 3961 aaagttcctt gaaaatgaca gagtggctct cagaccagac cttgattgtg ggtataatcg 4021 gagtgttgct accacaccct aacactgcat ttcccgtgtt ttattggtcc atggaattct 4081 gaaagtttgc ctttcgggat gcttctaaaa acaattccat ggaccagtaa gtttggaaag 4141 tcctgcgtgc ctcacttctc ttcaaaggca aaaggctctg gagaggcctt catgaagaca 4201 tctgtgttta atgctgccct tcccaaaggt ctgtttttga ctgtcttttg agaaatgatc 4261 ctctgatctc taggcagaat gccagtgagc caaggaatcc cagttagcag gaggggtgca 4321 4381 cacttccct tgtcctctcc cttgcccctc ttgctggagt aaaaggatgg aactgggact 4441 tgataggtta aaggaggtgt ggagaagtgt cttagaccag ctctcctgtt gtgggcctta 4501 gggagaagca ctctctttct tcgggatcat tttccaaaca tgcatttttg gatggatagg 4561 gtggatcagg gtgagggaag ggaaccaaa ctctctctaa ccttgccctt acagcaatac 4621 ctgtgatgta agttacaaaa ccacctgtga tgaaagtgct ccaggatgct tcatgcacca 4681 gggaggggtg ccctgtttct cttctgctag cttctccttt ctttttttt tttcttcttt 4741 tttttgagac agtgtctcac tctgttgcca ggctggagatg cagtggtgag atctcagctc 4801 actgcagcct ctgcctccca ggttcaagca attcttctgc ctcagcctcc cgagtagctg 4861 gtgtgtctgg agttggttcc ttctggtggg ttcttggtct cgctgacttc aagaatgaag 4921 ccacagacct tcgcagtgag tgttacagct cttaaaggtg gcacggaccc aaagtgagca 4981 gtagcaagat ttatgtgga gagcgaaaga acaaagcttc ggaaggggac ccaaatgggc 5041 tgctgctgct ggctgggggtg gccaccttt attccctttat ttgtccctgc ccatgtcctg 5101 ctgattgctc cattttacag agtgctgatt ggtccatttt acagagtgct gattggtgca 5161 tttacaatcc tttagctaga cacagagtgc cgattggtga gtttttacag tgctgattgg 5221 tgcatttaca atcctttagc tagacacaga acactgactg gtgcatttat aatcctctag 5281 ctagaaagaa aagttctcca agtccccact agacccagga agtccagctg gcttcacctc 5341 tcactgggac tacaggtgca caccaccaca cccagctaat ttttgtattt ttagtagaga 5401 cggggtttca ccatgttgtt caggatggtc tcgaactctt gatctcgtga tctgcccgcc 5461 tcggcctccc aaagtgctgg gattacagtt gtgagccacc acgcccggcc ctagcttttc 5521 ctttctgttg caagtcctct caactagtgt tgccttccac cctacaaagc agaattacct 5581 cagaagtcct atggccctga ctctatctat gtctgcacaa agcactactg tgcttgctg 5641 tctgcaagaa cagagattgt ttgctcaac cactttctct gatggatga atgagttg 5701 atgatatcta aagttaccca atttcaagca agaggaagaa tctggctcgg taccacagat 5761 gttcttggaa ttgggatagt aaaaagtcc ctgaggcatc ctttgtctg ctctgaccac 5821 actctctca caggaagagg cttgggccac agctctgact atactctgc tctcctcca 5881 aacacagctg aggaattggg tggtggggca cctgctccca tgctgtgg cctggctcag 5941 agagagagt tgccttaatt acattattat tctcctgga caggctgtag gttgtgtaaa 6001 gtaacaaaaa ggactgagaa gtgactccc attcagccctc tccaggcc attttgata 6061 ggcaggtcaa attcactcac atttggttt tgttggcca gtctagtgca ttcacctg 6121 ctgtcctca gtcatgctcc tttaccttta cagagcatcc tagactgctc ttcctcttac 6181 cttccttgtg aaacccacaa cccctagtcc ctcccttcc ctgcattg ttagccctc 6241 taccaatccc tgacctgta ttggtcagtc tccaatcctg gtggatccct gtgggaacta 6301 agttaagtct aacttttgtc tccctcttta gaatttactg ggagtactgt aaataacta 6361 ttgttgttat aattattct gattaacatt tttacaccta acaaagtctc agagagattg 6421 aatttactgg gttgaaggga ggagcacctt ccacatgacc tgcccagcaa ttaaagccgc 6481 ttgttagtcc gaggcccagg acggccgagg acagctggag agctcttcgt tgcaggcagc 6541 tctggttaac atcaaccggg aaagctcttt gtaaacacat gaataattga tcgtccagcg 6601 ctcacatagc taccgcggat ctgagcccgt atgactcatt tgcgagccat tcctgtcgtc 6661 tggatgccat aacattggag gaatgatgat cgtttcttgg aggttcttct gtggccagag 6721 ttgccaagac caaggctgta atggtttgtt atgatgacct ttgttattcc attaggctca 6781 attgctttaa aaaatgatgt gtgcatactt taggaacgtt tttacccttt atgttgacct 6841 gacatcatag tttatattat aaaatgtatt aatgacagaa gagtgttttc atgtcccaag 6901 gacaaatttt aacaaccata atctgccctc agtcatcata aatataaatg tattggtcaa 6961 7021 ctagagaata tatctggagc ttttgtgggg ctaagagatc ttgtatatat gctatcaaaa 7081 ggctgagaaa attaacatgt tcccccctct gattttgcat tggacagata taaatgtctt 7141 ggggatgtca agtaagattg ttcacatagt ttctggacac cattaatgcc tgatggggtg 7201 aatcttagtt cttaaagcta tattctgctc attatgctca cagggctttt gaaaagagaa 7261 caaaataaag atttcaagtc ttagcaa

[0089] As defined herein, an antisense strand sequence is designed to target the MARC1 gene at a given position on the gene when the 5' terminal nucleic acid base of the antisense strand aligns with a position 21 nucleotides downstream (towards the 3' end) from the position on the gene during base pairing. For example, as shown in Tables 1 and 2 herein, an antisense strand sequence designed to target the MARC1 gene at position 1275 requires that the 5' terminal nucleic acid base of the antisense strand align with position 1295 of the MARC1 gene during base pairing.

[0090] As provided herein, if there is at least 85% complementarity between the antisense strand and the gene across a core stretch sequence of at least 16 consecutive nucleotides (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity), then the MARC1 RNAi agent does not require that the nucleic acid base at position 1 (5'→3') of the antisense strand be complementary to the gene. For example, in the case of a MARC1 RNAi agent disclosed herein designed to target position 1275 of the MARC1 gene, the 5' terminal nucleic acid base of the antisense strand of the MARC1 RNAi agent needs to be aligned with position 1295 of the gene; however, the 5' terminal nucleic acid base of the antisense strand may be complementary to position 1295 of the MARC1 gene, but is not required if there is at least 85% complementarity between the antisense strand and the gene across a core stretch sequence of at least 15 consecutive nucleotides (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity). In particular, as shown in various examples disclosed herein and known in the art, the specific binding site of the antisense strand of a MARC1 RNAi agent to the gene (e.g., whether the MARC1 RNAi agent is designed to target the MARC1 gene at position 1275, position 1190, or some other position) is important to the level of inhibition achieved by the MARC1 RNAi agent and the toxicity profile achieved by the molecule. (See, for example, Kamola et al., The siRNA Non-seed Region and Its Target Sequences are Auxiliary Determinants of Off-Target Effects, PLOS Computational Biology, 11(12), Figure 1 (2015)).

[0091] In some embodiments, the MARC1 RNAi agents disclosed herein target the MARC1 gene at or near the MARC1 location shown in Table 1. In some embodiments, the antisense strand of the MARC1 RNAi agent disclosed herein includes a core stretch sequence that is fully, substantially, or at least partially complementary to the target MARC1 19mer sequence disclosed in Table 1. Table 1. MARC1 19mer mRNA target sequence (Homo sapiens MARC1, mRNA, obtained from GenBank NM_022746.4 (Sequence ID 1)) [Table 1-1] [Table 1-2] [Table 1-3]

[0092] In some embodiments, the MARC1 RNAi agent includes an antisense strand (5'→3') whose 19th position can form a base pair with position 1 of the 19-mer target sequence disclosed in Table 1.

[0093] In some embodiments, the MARC1 RNAi agent includes an antisense strand (5'→3') whose 2 position can base-pair with the 18 position of the 19mer target sequence disclosed in Table 1. In some embodiments, the MARC1 RNAi agent includes an antisense strand (5'→3') whose 2-18 positions can base-pair with each of the complementary bases located at positions 18-2 of the 19mer target sequence disclosed in Table 1.

[0094] In the MARC1 RNAi agents disclosed herein, the nucleotide at position 1 of the antisense strand (5' end → 3' end) may be either perfectly complementary to the MARC1 gene or incompletely complementary to the MARC1 gene. In some embodiments, the nucleotide at position 1 of the antisense strand (5' end → 3' end) is U, A, or dT. In some embodiments, the nucleotide at position 1 of the antisense strand (5' end → 3' end) forms an A:U or U:A base pair with the sense strand.

[0095] In some embodiments, the antisense strand of the MARC1 RNAi agent contains the sequence of nucleotides (5' end to 3' end) 2-18, 2-19, 2-20, or 2-21 from any of the antisense strand sequences in Table 2 or Table 3. In some embodiments, the MARC1 RNAi sense strand contains the sequence of nucleotides (5' end to 3' end) 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 from any of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6D.

[0096] In some embodiments, the MARC1 RNAi antisense strand contains a sequence of nucleotides (5' end to 3' end) 2-18, 2-19, 2-20, or 2-21 from any of the antisense strand sequences in Table 2 or Table 3. In some embodiments, the MARC1 RNAi sense strand contains a sequence of nucleotides (5' end to 3' end) 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 from any of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6D.

[0097] In some embodiments, the MARC1 RNAi agent comprises (i) an antisense strand containing the sequence of nucleotides (5' end to 3' end) 2-18 or 2-19 from any of the antisense strand sequences in Table 2, Table 3, or Table 6D, and (ii) a sense strand containing the sequence of nucleotides (5' end to 3' end) 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 from any of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6D.

[0098] In some embodiments, the MARC1 RNAi agent comprises (i) an antisense strand containing a sequence of nucleotides (5' end to 3' end) 2-18 or 2-19 from any of the antisense strand sequences in Table 2, Table 3, or Table 6D, and (ii) a sense strand containing a sequence of nucleotides (5' end to 3' end) 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 from any of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6D.

[0099] In some embodiments, the MARC1 RNAi agent contains the core 19mer nucleotide sequence shown in Table 2 below. Table 2. Antisense and sense strand corestretch sequences of MARC1 RNAi agents (N = arbitrary nucleic acid bases) [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Table 2-16] [Table 2-17] [Table 2-18] [Table 2-19]

[0100] The sense and antisense strands of a MARC1 RNAi agent containing or comprising the sequences in Table 2 may be modified or unmodified nucleotides. In some embodiments, a MARC1 RNAi agent having sense and antisense strand sequences containing or comprising the sequences in Table 2 consists entirely or substantially entirely of modified nucleotides.

[0101] In some embodiments, the antisense strand of the MARC1 RNAi agent disclosed herein differs from any of the antisense strand sequences in Table 2 by 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of the MARC1 RNAi agent disclosed herein differs from any of the sense strand sequences in Table 2 by 0, 1, 2, or 3 nucleotides.

[0102] When used herein, each N listed in the sequences disclosed in Table 2 may be independently selected from any and all nucleic acid bases (including those found in both modified and unmodified nucleotides). In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have nucleic acid bases complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have nucleic acid bases that are not complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have the same nucleic acid bases as the N nucleotide at the corresponding position on the other strand. In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have different nucleic acid bases from the N nucleotide at the corresponding position on the other strand.

[0103] Table 3 provides specific modified MARC1 RNAi agent antisense strand sequences and their underlying unmodified sequences. Table 4 provides specific modified MARC1 RNAi agent sense strands and their underlying unmodified sequences. In the formation of MARC1 RNAi agents, each nucleotide in each underlying unmodified sequence listed in Tables 3 and 4, as well as Table 2 and above, may be a modified nucleotide.

[0104] The MARC1 RNAi agents described herein are formed by annealing an antisense strand with a sense strand. A sense strand containing the sequences listed in Table 2, Table 4, or Table 6D can be hybridized with any antisense strand containing the sequences listed in Table 2, Table 3, or Table 6D, provided that it has a region of at least about 85% complementarity across a sequence of 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides.

[0105] In some embodiments, the antisense strand of the MARC1 RNAi agent contains one of the nucleotide sequences shown in Table 2, Table 3, or Table 6D.

[0106] In some embodiments, the MARC1 RNAi agent comprises or consists of a double-stranded sequence having the sense strand and antisense strand nucleic acid base sequences of any of the sequences in Table 2, Table 3, Table 4, or Table 6D. In some embodiments, the MARC1 RNAi agent comprises or consists of a double-stranded sequence prepared or provided as a sodium salt, a mixed salt, or a free acid.

[0107] Examples of antisense strands containing modified nucleotides are provided in Tables 3 and 6D. Examples of sense strands containing modified nucleotides are provided in Tables 4, 5, and 6D.

[0108] When used in Tables 3, 4, and 5, the following nomenclature is used to indicate modified nucleotides and linking groups: A = Adenosine-3'-phosphate C = cytidine-3'-phosphate G = Guanosine-3'-phosphate U = uridine-3'-phosphate I = Inosine-3'-phosphate a=2'-O-methyladenosine-3'-phosphate as=2'-O-methyladenosine-3'-phosphorothioate c=2'-O-methylcytidine-3'-phosphate cs = 2'-O-methylcytidine-3'-phosphorothioate g = 2'-O-methylguanosine-3'-phosphate gs = 2'-O-methylguanosine-3'-phosphorothioate t=2'-O-methyl-5-methyluridine-3'-phosphate ts = 2'-O-methyl-5-methyluridine-3'-phosphorothioate u = 2'-O-methyluridine-3'-phosphate us=2'-O-methyluridine-3'-phosphorothioate i=2'-O-methylinosine-3'-phosphate is=2'-O-methylinosine-3'-phosphorothioate Af = 2'-fluoroadenosine-3'-phosphate Afs = 2'-fluoroadenosine-3'-phosphorothioate Cf = 2'-fluorocytidine-3'-phosphate Cfs = 2'-fluorocytidine-3'-phosphorothioate Gf = 2'-fluoroguanosine-3'-phosphate Gfs = 2'-fluoroguanosine-3'-phosphorothioate Tf = 2'-fluoro-5'-methyluridine-3'-phosphate Tfs = 2'-fluoro-5'-methyluridine-3'-phosphorothioate Uf = 2'-fluorouridine-3'-phosphate Ufs = 2'-fluorouridine-3'-phosphorothioate A UNA = 2',3'-seco-adenosine-3'-phosphate, see Table 7. A UNA s = 2',3'-seco-adenosine-3'-phosphorothioate; see Table 7. C UNA =2',3'-secocytidine-3'-phosphate, see Table 7. C UNAs = 2’,3’-seco-cytidine-3’-phosphorothioate, see Table 7 G UNA = 2’,3’-seco-guanosine-3’-phosphate, see Table 7 G UNA s = 2’,3’-seco-guanosine-3’-phosphorothioate, see Table 7 U UNA = 2’,3’-seco-uridine-3’-phosphate, see Table 7 U UNA s = 2’,3’-seco-uridine-3’-phosphorothioate, see Table 7 a_2N = 2’-O-methyl-2-aminoadenosine-3’-phosphate, see Table 7 a_2Ns = 2’-O-methyl-2-aminoadenosine-3’-phosphorothioate, see Table 7 (invAb) = reverse abasic deoxyribonucleotide, see Table 7 (invAb)s = reverse abasic deoxyribonucleotide-5’-phosphorothioate, see Table 7 cPrpa = 5’-cyclopropylphosphonate-2’-O-methyladenosine-3’-phosphate (see Table 7) cPrpas = 5’-cyclopropylphosphonate-2’-O-methyladenosine-3’-phosphorothioate (see Table 7) cPrpu = 5’-cyclopropylphosphonate-2’-O-methyluridine-3’-phosphate (see Table 7) cPrpus = 5’-cyclopropylphosphonate-2’-O-methyluridine-3’-phosphorothioate (see Table 7)

[0109] As will be readily apparent to those skilled in the art, unless otherwise indicated by the sequence (e.g., by a phosphorothioate linkage "s"), nucleotide monomers in oligonucleotides are linked to each other by a 5'-3'-phosphodiester bond. As will be clearly apparent to those skilled in the art, the inclusion of phosphorothioate linkages shown in the modified nucleotide sequences disclosed herein replaces the phosphodiester bond typically present in oligonucleotides. Furthermore, those skilled in the art will readily understand that the terminal nucleotide at the 3' end of a given oligonucleotide sequence typically has a hydroxyl (-OH) group at the 3' position of each given monomer in ex vivo, instead of a phosphate moiety. Furthermore, in the embodiments disclosed herein, when viewing each chain from 5' to 3', the reverse debasing residue is inserted such that the 3' position of the deoxyribose is linked to the 3' end of the preceding monomer on each chain (see, for example, Table 7). Furthermore, as will be readily understood and recognized by those skilled in the art, the phosphorothioate chemical structures shown herein typically represent an anion on a sulfur atom, and the present invention disclosed herein encompasses all phosphorothioate tautomers and resonance structures (e.g., those with a double bond on the sulfur atom and an anion on an oxygen atom). Unless otherwise expressly stated herein, such understanding of those skilled in the art is used when describing the MARC1 RNAi agents and MARC1 RNAi agent compositions disclosed herein.

[0110] Specific examples of targeted ligands, targeting groups, and linking groups used with the MARC1 RNAi agents disclosed herein are provided in Table 7 below. More specifically, the targeting groups and linking groups (which together can form a targeted ligand) include (NAG37) and (NAG37)s, whose chemical structures are provided in Table 7 below. Each sense strand and / or antisense strand may have any of the targeted ligands, targeting groups, or linking groups listed herein, as well as other groups, conjugated at the 5' and / or 3' ends of the sequence. Table 3. Antisense strand sequences of MARC1 RNAi agents [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] Table 4. Sense strand sequences of MARC1 RNAi agents (shown without linkers, conjugates, or capping regions) [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] Table 5. Sense strand sequences of MARC1 RNAi agents (shown with (NAG37) targeting ligands (see Table 7 for structural information)). [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

[0111] The MARC1 RNAi agents described herein are formed by annealing an antisense strand and a sense strand. A sense strand containing sequences listed in Table 2, Table 4, or the tables listed in Table 3 can be hybridized to any antisense strand containing sequences listed in Table 2 or Table 3, provided that the two sequences have a region of at least about 85% complementarity across a sequence of 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides.

[0112] In some embodiments, the antisense strand of the MARC1 RNAi agent disclosed herein differs from any of the antisense strand sequences in Table 3 by 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of the MARC1 RNAi agent disclosed herein differs from any of the sense strand sequences in Table 4 or Table 5 by 0, 1, 2, or 3 nucleotides.

[0113] In some embodiments, the antisense strand of the MARC1 RNAi agent contains a nucleotide sequence from either Table 2 or Table 3. In some embodiments, the MARC1 RNAi agent antisense strand contains a nucleotide sequence (5' end → 3' end) 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21 from either Table 2 or Table 3. In some embodiments, the MARC1 RNAi agent antisense strand contains or consists of one of the modified sequences from Table 3.

[0114] In some embodiments, the sense strand of the MARC1 RNAi agent contains one of the nucleotide sequences in Table 2, Table 4, or Table 5. In some embodiments, the antisense strand of the MARC1 RNAi agent contains the nucleotide (5' end → 3' end) sequences 1-17, 2-17, 3-17, 4-17, 1-18, 2-18, 3-18, 4-18, 1-19, 2-19, 3-19, 4-19, 1-20, 2-20, 3-20, 4-20, 1-21, 2-21, 3-21, or 4-21. In some embodiments, the sense strand of the MARC1 RNAi agent contains or consists of one of the modified sequences in Table 4 or Table 5.

[0115] In the MARC1 RNAi agents disclosed herein, the nucleotide at position 1 of the antisense strand (5' end → 3' end) may be either perfectly complementary to the MARC1 gene or incompletely complementary to the MARC1 gene. In some embodiments, the nucleotide at position 1 of the antisense strand (5' end → 3' end) is U, A, or dT. In some embodiments, the nucleotide at position 1 of the antisense strand (5' end → 3' end) forms an A:U or U:A base pair with the sense strand.

[0116] A sense strand containing sequences listed in Table 2, Table 4, or Table 5 can be hybridized with any antisense strand containing sequences listed in Table 2 or Table 3, provided that the two sequences have a region of at least approximately 85% complementarity across a sequence of 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides. In some embodiments, the MARC1 RNAi agent has a sense strand consisting of a modified sequence from one of the modified sequences in Table 4 or Table 5, and an antisense strand consisting of a modified sequence from one of the modified sequences in Table 3. Specific representative sequence pairs are exemplified by the double-stranded ID numbers shown in Table 6A or 6B.

[0117] In some embodiments, the MARC1 RNAi agent comprises, consists of, or essentially consists of a double strand represented by any one of the double-stranded ID numbers presented herein. In some embodiments, the MARC1 RNAi agent comprises a sense strand and an antisense strand nucleotide sequence of either of the double strands represented by any of the double-stranded ID numbers presented herein. In some embodiments, the MARC1 RNAi agent comprises a sense strand and an antisense strand nucleotide sequence of either of the double strands represented by any of the double-stranded ID numbers presented herein, as well as a targeting group and / or linking group, the targeting group and / or linking group being covalently linked (i.e., conjugated) to the sense strand or antisense strand. In some embodiments, the MARC1 RNAi agent comprises a sense strand and an antisense strand modified nucleotide sequence of any of the double-stranded ID numbers presented herein. In some embodiments, the MARC1 RNAi agent comprises a modified nucleotide sequence of a sense strand and / or antisense strand of any of the double-stranded ID numbers presented herein, and a targeting group and / or linking group, the targeting group and / or linking group being covalently linked to the sense strand or antisense strand.

[0118] In some embodiments, the MARC1 RNAi agent comprises an antisense strand and a sense strand having a nucleotide sequence of any of the antisense / sense strand double-stranded sequences of Table 2 or Tables 6A and 6B, and further comprises a targeting group or a targeting ligand. In some embodiments, the MARC1 RNAi agent comprises an antisense strand and a sense strand having a nucleotide sequence of any of the antisense / sense strand double-stranded sequences of Table 2 or Tables 6A and 6B, and further comprises an asialoglycoprotein receptor ligand targeting group.

[0119] The targeting group may be linked to the 5' or 3' end of any sense chain and / or antisense chain disclosed in Tables 2, 3, 4, or 5, with or without a linker. The linker may be attached to the 5' or 3' end of any sense chain and / or antisense chain disclosed in Tables 2, 3, 4, or 5, with or without a targeting group.

[0120] In some embodiments, the MARC1 RNAi agent comprises an antisense strand and a sense strand having one of the nucleotide sequences of the antisense / sense strand double helix shown in Table 2, Table 6A, or Table 6B, and further comprises a targeting ligand selected from the group consisting of (NAG37) and (NAG37)s, as defined in Table 7.

[0121] In some embodiments, the MARC1 RNAi agent comprises an antisense strand and a sense strand having a modified nucleotide sequence in either of the antisense strand and / or sense strand nucleotide sequences shown in Table 3 or Table 4.

[0122] In some embodiments, the MARC1 RNAi agent comprises an antisense strand and a sense strand having a modified nucleotide sequence of either of the double-stranded antisense strands and / or sense strand nucleotide sequences of Tables 6A and 6B, and further comprising an asialoglycoprotein receptor ligand targeting group.

[0123] In some embodiments, the MARC1 RNAi agent comprises, consists of, or essentially consists of, one of the double strands shown in Tables 6A and 6B. Table 6A. MARC1 RNAi double-stranded nucleotide sequences with corresponding sense and antisense strand ID numbers and sequence ID numbers for modified and unmodified nucleotide sequences. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] Table 6B. MARC1 RNAi double-stranded nucleotide sequences with corresponding sense and antisense strand ID numbers and sequence ID numbers for modified and unmodified nucleotide sequences. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] Table 6C. MARC1 RNAi double strands with corresponding sense and antisense strand ID numbers referencing the targeted location on the MARC1 gene (SEQ ID NO: 1). [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] Table 6D. MARC1 RNAi double-stranded drugs showing chemically modified antisense and sense strand sequences. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8] [Table 9-9] [Table 9-10] [Table 9-11] [Table 9-12] [Table 9-13] [Table 9-14] [Table 9-15]

[0124] In some embodiments, the MARC1 RNAi agent is prepared or provided as a salt, mixed salt, or free acid. The RNAi agents described herein inhibit the expression of one or more MARC1 genes in vivo and / or in vitro when delivered to cells expressing MARC1.

[0125] Targeting ligand or targeting group, linking group, and delivery vehicle In some embodiments, the MARC1 RNAi agent is conjugated to one or more non-nucleotide groups including, but not limited to, a targeting group, a linking group, a targeting ligand, a delivery polymer, or a delivery vehicle. This non-nucleotide group can enhance the targeting, delivery, or conjugation of the RNAi agent. Examples of targeting groups and linking groups are provided in Table 7. The non-nucleotide group can be covalently linked to either the 3' and / or 5' end of either the sense strand and / or the antisense strand. In some embodiments, the MARC1 RNAi agent comprises a non-nucleotide group linked to the 3' and / or 5' end of the sense strand. In some embodiments, the non-nucleotide group is linked to the 5' end of the sense strand of the MARC1 RNAi agent. The non-nucleotide group can be linked to the RNAi agent directly or indirectly via a linker / linking group. In some embodiments, the non-nucleotide group is linked to the RNAi agent via a labile cleavage ability, or a reversible bond or linker.

[0126] In some embodiments, the non-nucleotide group improves the pharmacokinetic or biodistribution properties of the RNAi agent or conjugate, which is attached to improve its cell-specific or tissue-specific distribution and cell-specific uptake of the RNAi agent or conjugate. In some embodiments, the non-nucleotide group improves the endocytosis of the RNAi agent.

[0127] Targeting groups or moieties are attached to improve the pharmacokinetic or intracellular distribution characteristics of conjugates or RNAi agents, thereby enhancing their cell-specific (and in some cases, organ-specific) distribution and cell-specific (or organ-specific) uptake. Targeting groups can be monovalent, divalent, trivalent, tetravalent, or have a higher valency for the target they are directed at. Typical targeting groups include, but are not limited to, compounds with affinity for cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimetic compounds with affinity for cell surface molecules.

[0128] In some embodiments, the targeting group is linked to the RNAi agent using a linker, for example, a PEG linker that can function as a linker in some cases, or one, two, or three debase and / or ribitol (debase-ribose) residues. In some embodiments, the targeting ligand comprises a galactose derivative cluster.

[0129] The MARC1 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 the 5' and / or 3' ends. This reactive group can then be used to attach a targeting moiety using methods typical in the art.

[0130] In some embodiments, the targeting group comprises an asialoglycoprotein receptor ligand. As used herein, an asialoglycoprotein receptor ligand is a ligand comprising a moiety having affinity for the asialoglycoprotein receptor. As described herein, the asialoglycoprotein receptor is highly expressed on hepatocytes. In some embodiments, the asialoglycoprotein receptor ligand comprises or consists of one or more galactose derivatives. As used herein, the term galactose derivative includes both galactose and galactose derivatives having an affinity greater than or equal to that of galactose for the asialoglycoprotein receptor. Examples of galactose derivatives include, but are not limited to, galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, Nn-butanoylgalactosamine, and N-isobutanoylgalactosamine (see, for example, ST. Iobst and K. Drickamer, JBC, 1996, 271, 6686). Galactose derivatives and a group of galactose derivatives useful for in vivo targeting of oligonucleotides and other molecules against the liver are a base in this art (see, for example, Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945).

[0131] Galactose derivatives have been used in vivo to target molecules to hepatocytes by binding to asialoglycoprotein receptors expressed on the surface of hepatocytes. Binding of asialoglycoprotein receptor ligands to asialoglycoprotein receptors facilitates cell-specific targeting to hepatocytes and endocytosis of molecules to 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 attached to the 3' or 5' end of the sense or antisense strand of an RNAi agent using methods known in the art.

[0132] The preparation of targeted ligands, such as galactose derivative clusters, is described, for example, in International Patent Application Publication No. 2018 / 044350 and International Patent Application Publication No. 2017 / 156012 to Arrowhead Pharmaceuticals, Inc., both of which are incorporated herein by reference in their entirety.

[0133] When used herein, a galactose derivative cluster comprises a molecule having 2 to 4 terminal galactose derivatives. The terminal galactose derivatives are attached to the molecule via their C-1 carbon. In some embodiments, the galactose derivative cluster is a galactose derivative trimer (also called a triply branched galactose derivative or trivalent galactose derivative). In some embodiments, the galactose derivative cluster contains an N-acetyl-galactosamine moiety. In some embodiments, the galactose derivative cluster contains three N-acetyl-galactosamine moieties. In some embodiments, the galactose derivative cluster is a galactose derivative tetramer (also called a tetra-branched galactose derivative or tetravalent galactose derivative). In some embodiments, the galactose derivative cluster contains four N-acetyl-galactosamine moieties.

[0134] As used herein, a galactose derivative trimer comprises three galactose derivatives linked to a central branch point. As used herein, a galactose derivative tetramer comprises four galactose derivatives linked to a central branch point. The galactose derivatives may be attached to the central branch point via the C-1 carbon of the sugar. In some embodiments, the galactose derivatives are linked to the branch point via a linker or spacer. In some embodiments, the linker or spacer is a flexible hydrophilic spacer such as a PEG group (see, for example, U.S. Patent No. 5,885,968; Biessen et al. J. Med. Chem. 1995 Vol. 39 p. 1538–1546). In some embodiments, the PEG spacer is a PEG3 spacer. This branch point can be any small molecule to which three galactose derivatives can be attached, and further, to which the branch point can be attached to an RNAi agent. Examples of branch point groups are dyridine or diglutamate salts. The branching point can be attached to the RNAi agent via a linker or spacer. In some embodiments, the linker or spacer includes a flexible hydrophilic spacer (e.g., a PEG spacer). In some embodiments, the linker includes a rigid linker (e.g., a cyclic group). In some embodiments, the galactose derivative includes or consists of N-acetyl-galactosamine. In some embodiments, the galactose derivative cluster consists of a galactose derivative tetramer, which may be, for example, an N-acetyl-galactosamine tetramer.

[0135] Embodiments of the present disclosure include pharmaceutical compositions for delivering MARC1 RNAi agents to hepatocytes in vivo. Such pharmaceutical compositions may, for example, include MARC1 RNAi agents conjugated to a group of galactose derivatives. In some embodiments, the galactose derivative cluster consists of a galactose derivative trimer (which may be, for example, an N-acetyl-galactosamine trimer) or a galactose derivative tetramer (which may be, for example, an N-acetyl-galactosamine tetramer).

[0136] A targeted ligand or targeting group may be ligated to the 3' or 5' end of the sense or antisense strand of a MARC1 RNAi agent disclosed herein.

[0137] Targeted ligands include, but are not limited to, (NAG37) and (NAG37)s as defined in Table 7. Other targeted groups and targeted ligands, including galactose cluster targeted ligands, are known in the art.

[0138] In some embodiments, a linking group is conjugated to the RNAi agent. The linking group facilitates covalent linkage of the drug to a targeting group or a delivery polymer or delivery vehicle. The linking group can be linked to the 3' or 5' end of the sense or antisense strand of the RNAi agent. In some embodiments, the linking group is linked to the sense strand of the RNAi agent. In some embodiments, the linking group is conjugated to the 5' or 3' end of the sense strand of the RNAi agent. In some embodiments, the linking group is conjugated to the 5' end of the sense strand of the RNAi agent. Examples of linking groups include, but are not limited to, reactive groups such as primary amines and alkynes, alkyl groups, debased nucleosides, ribitols (debased ribose), and / or PEG groups.

[0139] In some embodiments, the targeting group is internally linked to nucleotides on the sense and / or antisense strands of the RNAi agent. In some embodiments, the targeting group is linked to the RNAi agent via a linker.

[0140] A linker or linking group is a connection between two atoms that, by one or more covalent bonds, links one chemical group (such as an RNAi agent) or segment of interest to another chemical group (e.g., a targeting group or a delivery polymer) or segment of interest. Unstable linkages include unstable bonds. Linkages may optionally include spacers that increase the distance between the two bonding atoms. Spacers can further add flexibility and / or length to the linkage. Examples of spacers include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkylnyl groups. Each of these may include one or more heteroatoms, heterocycles, amino acids, nucleotides, and sugars. Spacer groups are well known in the art, and the above list is not intended to limit the scope of this specification.

[0141] In some embodiments, when two or more RNAi agents are contained in a single composition, each RNAi agent may be linked to the same targeting group or to two different targeting groups (i.e., targeting groups having different chemical structures). In some embodiments, the targeting groups are linked to the MARC1 RNAi agents disclosed herein without the use of additional linkers. In some embodiments, the targeting groups themselves are designed to have linkers or other sites to facilitate the presence of conjugation. In some embodiments, when two or more MARC1 RNAi agents are contained in a single molecule, each RNAi agent may utilize the same linker or different linkers (i.e., linkers having different chemical structures).

[0142] Any of the MARC1 RNAi agent nucleotide sequences listed in Tables 2, 3, 4, 5, or 6D can include 3' and / or 5' targeting groups or linking groups, whether modified or unmodified. Alternatively, any of the MARC1 RNAi agent sequences listed in Tables 3, 4, or 6D or described herein that include a 3' or 5' targeting group or linking group may not include a 3' or 5' targeting group or linking group, or can include different 3' or 5' targeting groups or linking groups including but not limited to those shown in Table 7. Any of the MARC1 RNAi agent double strands listed in Tables 6A, 6B, 6C, or 6D can further include targeting groups or linking groups including but not limited to those shown in Table 7, whether modified or unmodified, and the targeting groups or linking groups can be attached to the 3' or 5' end of either the sense strand or the antisense strand of the MARC1 RNAi agent double strand.

[0143] Examples of targeting groups and linking groups (which can form a targeting ligand when combined) are provided in Table 7. Tables 5 and 6D provide specific embodiments of the sense strand of the MARC1 RNAi agent having a targeting group or linking group linked to the 5' or 3' end. Table 7. Structures showing various modified nucleotides, targeting ligands or targeting groups, capping residues, and linking groups

Table 10-1

Table 10-2

Table 10-3

[0144] In each of the structures shown in Table 7, NAG comprises N-acetyl-galactosamine. In some embodiments, the NAG shown in Table 7 above may comprise another galactose derivative having affinity for asialoglycoprotein receptors present in hepatocytes, which would be understood by those skilled in the art to be attached in consideration of the above structures and the descriptions provided herein. Other linking groups known in the art may also be used.

[0145] In some embodiments, a delivery vehicle can be used to deliver RNAi agents to cells or tissues. The delivery vehicle is a compound that improves the delivery of RNAi agents to cells or tissues. Examples of delivery vehicles include, but are 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. In some embodiments, the RNAi agent 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 to targeting groups, lipids (including but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example, International Publications 2000 / 053722, 2008 / 0022309, 2011 / 104169, and 2012 / 083185, 2013 / 032829, and 2013 / 158141, each of which is incorporated herein by reference), hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, protein vectors, or other delivery systems known and available in the art that are suitable for the delivery of nucleic acids or oligonucleotides.

[0146] Pharmaceutical composition The MARC1 RNAi agents disclosed herein can be prepared as pharmaceutical compositions or formulations (also referred to herein as “pharmaceuticals”). In some embodiments, the pharmaceutical compositions comprise at least one MARC1 RNAi agent. These pharmaceutical compositions are particularly useful for inhibiting the expression of target mRNA in target cells, cell populations, tissues, or in vivo.

[0147] The pharmaceutical composition can be used to treat subjects having a disease, disorder, or condition in which they would benefit from a reduction in the level of a target MARC1 mRNA or inhibition of the expression of a target gene. The pharmaceutical composition can be used to treat subjects having a risk of developing a disease, disorder, symptom, or condition in which they would benefit from a reduction in the level of a target mRNA or inhibition of the expression of a target gene. In one embodiment, the method comprises administering a MARC1 RNAi agent linked to a targeted ligand as described herein to a subject to be treated. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) are added to the pharmaceutical composition containing the MARC1 RNAi agent to form a pharmaceutical formulation or drug suitable for in vivo delivery to subjects including humans.

[0148] Pharmaceutical compositions and methods comprising MARC1 RNAi agents disclosed herein involve administering a therapeutically effective amount of the MARC1 RNAi agent described herein to a subject, thereby reducing the level of target mRNA in cells, cell populations, tissues, organs, or the subject by inhibiting the expression or translation of MARC1 in the subject. In some embodiments, the subject has been previously identified as having upregulation of the pathogenicity of the target gene in hepatocytes. In some embodiments, the subject has been previously identified or diagnosed with non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related disorders. In some embodiments, the subject will benefit from reduced MARC1 gene expression in the liver of the subject.

[0149] In some embodiments, the described pharmaceutical compositions comprising a MARC1 RNAi agent are used to treat or manage clinical features associated with non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related disorders. In some embodiments, one or more therapeutic (including prophylactic) doses of the pharmaceutical compositions are administered to a subject in need of such treatment. In some embodiments, administration of any of the MARC1 RNAi agents of this disclosure can be used to reduce the number, severity, and / or frequency of disease symptoms in a subject.

[0150] The pharmaceutical compositions described, comprising MARC1 RNAi agents, can be used to treat at least one symptom in subjects with a disease or disorder in which a reduction or inhibition of MARC1 mRNA expression and / or a reduction in MARC1 protein levels would be beneficial. Measurement of MARC1 levels can be carried out by established methods known in the art.

[0151] In some embodiments, a subject is administered one or more therapeutically effective doses of pharmaceutical compositions containing a MARC1 RNAi agent to treat the symptoms. In other embodiments, a subject is administered one or more prophylactically effective doses of a MARC1 RNAi agent to prevent or inhibit at least one symptom.

[0152] The route of administration is the route through which the MARC1 RNAi agent comes into contact with the body. Generally, methods for administering drugs, oligonucleotides, and nucleic acids for the treatment of mammals are well known in the art and can be applied to the administration of the compositions described herein. The MARC1 RNAi agents disclosed herein can be administered via any suitable route in preparations tailored to specific routes. Thus, the pharmaceutical compositions described herein can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intra-articularly, or intraperitoneally. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection.

[0153] Pharmaceutical compositions comprising the MARC1 RNAi agent described herein can be delivered to cells, cell populations, tissues, or subjects using oligonucleotide delivery techniques known in the art. In general, any preferred method recognized in the art for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the compositions described herein. For example, delivery may be by parenteral routes, including local administration (e.g., direct injection, implantation, local administration), systemic administration, subcutaneous administration, intravenous, intraperitoneal, or intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or local (including oral and sublingual) administration. In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.

[0154] In some embodiments, the pharmaceutical compositions described herein comprise one or more pharmaceutically acceptable excipients. The pharmaceutical compositions described herein are formulated for administration to a subject.

[0155] When used herein, a pharmaceutical composition or drug comprises at least one of the described therapeutic compounds in a pharmacologically effective amount and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than the active pharmaceutical ingredient (API, therapeutic product, e.g., MARC1 RNAi agent) that is intentionally included in a drug delivery system. An excipient does not exert, or is not intended to exert, a therapeutic effect at the intended dose. An excipient 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 the overall safety, efficacy, or any other attributes of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0156] Excipients include, but are not limited to, absorption enhancers, anti-adhesion agents, antifoaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, surfactants (detergents), dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, fragrances, lubricants, humectants, lubricants, oils, polymers, preservatives, physiological saline, salts, solvents, sugars, surfactants, suspending agents, sustained-release matrices, sweeteners, thickeners, isotonic agents, vehicles, water repellents, and wetting agents.

[0157] Suitable pharmaceutical compositions for use by injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). Suitable carriers must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained by coating, for example, lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. In many cases, it is preferable that the composition contains, for example, sugars, polyhydric alcohols such as mannitol and sorbitol, and isotonic agents such as sodium chloride. By including substances that slow down absorption, such as aluminum monostearate and gelatin, in the composition, the long-term absorption of the injectable composition can be achieved.

[0158] Sterile injection solutions can be prepared by adding the required amount of the active compound to a suitable solvent, along with one or a combination of the components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a basic dispersion medium and a sterile excipient containing the other necessary components listed above. In the case of sterile powders for the preparation of sterile injection solutions, preparation methods include vacuum drying and freeze-drying, which yield powders of the active ingredient and any further desired components from a pre-sterilized and filtered solution.

[0159] In some embodiments, pharmaceutical formulations containing the MARC1 RNAi agents disclosed herein, suitable for subcutaneous administration, can be prepared in aqueous sodium phosphate buffer (e.g., MARC1 RNAi agents formulated in 0.5 mM mononucleotide sodium phosphate or 0.5 mM dibasic sodium phosphate in water). In some embodiments, pharmaceutical formulations containing the MARC1 RNAi agents disclosed herein, suitable for subcutaneous administration, can be prepared in sterile water for injection. MARC1 RNAi agents disclosed herein, suitable for subcutaneous administration, can be prepared in isotonic saline (0.9%).

[0160] Formulations suitable for intra-articular administration may be in the form of sterile aqueous preparations of drugs that may be in microcrystalline form, such as aqueous microcrystalline suspensions. Liposome formulations or biodegradable polymer systems can also be used to administer drugs for both intra-articular and intraocular administration.

[0161] Formulations of the MARC1 RNAi agents disclosed herein that are suitable for oral administration can also be prepared. In some embodiments, the MARC1 RNAi agents disclosed herein are administered orally. In some embodiments, the MARC1 RNAi agents disclosed herein are formulated in capsules for oral administration.

[0162] The active compound can be prepared with a carrier that prevents the compound from being rapidly eliminated from the body, such as a controlled-release formulation including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The methods for preparing such formulations will be obvious to those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared, for example, by methods well known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0163] MARC1 RNAi agents can be formulated in dose unit form for ease of administration and dose uniformity. A dose unit form refers to a physically distinct unit suitable as a unit dose for the target of treatment, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications for dose unit forms in this disclosure are determined and directly depend on the inherent properties of the active compound, the therapeutic effect to be achieved, and the limitations inherent in the art to formulate such an active compound for individual treatments.

[0164] Pharmaceutical compositions may include other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to, antipruritics, astringents, topical anesthetics, analgesics, antihistamines, or anti-inflammatory agents (e.g., acetaminophen, NSAIDs, diphenhydramine). It is also conceivable that cells, tissues, or isolated organs expressing or containing RNAi agents as defined herein may be used as a “pharmaceutical composition.” As used herein, “pharmacologically effective amount,” “therapeutic effective amount,” or simply “effective amount” refers to the amount of RNAi agent that produces a pharmacological, therapeutic, or prophylactic effect.

[0165] In some embodiments, the methods disclosed herein further include the step of administering a second therapeutic agent or treatment in addition to administering the RNAi agent disclosed herein. In some embodiments, the second therapeutic agent is another MARC1 RNAi agent (e.g., a MARC1 RNAi agent that targets a different sequence within the MARC1 target). In other embodiments, the second therapeutic agent may be a small molecule drug, an antibody, an antibody fragment, or an aptamer.

[0166] In some embodiments, the described MARC1 RNAi agent is optionally combined with one or more additional therapeutic agents. The MARC1 RNAi agent and the additional therapeutic agents may be administered in a single composition, or they may be administered separately. In some embodiments, one or more additional therapeutic agents are administered separately from the RNAi agent in separate dosage forms (for example, the MARC1 RNAi agent is administered by subcutaneous injection, and the additional therapeutic agents used in the therapeutic administration regimen are administered orally). In some embodiments, the described MARC1 RNAi agent is administered by subcutaneous injection to the subject in need, while one or more optional additional therapeutic agents are administered orally, and together they provide a therapeutic regimen for diseases and conditions associated with non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related diseases. In some embodiments, the described MARC1 RNAi agent is administered by subcutaneous injection to a subject in need, and one or more additional therapeutic agents, if desired, are administered by separate subcutaneous injection. In some embodiments, the MARC1 RNAi agent and one or more additional therapeutic agents are combined in a single dosage form (e.g., a “cocktail” formulated in a single composition for subcutaneous injection). The MARC1 RNAi agent can be combined with one or more excipients, with or without the additional therapeutic agents, to form a pharmaceutical composition.

[0167] Generally, the effective dose of a MARC1 RNAi agent is in the range of about 0.1 to about 100 mg / kg body weight / dose, for example, about 1.0 to about 50 mg / kg body weight / dose. In some embodiments, the effective dose of the active compound is in the range of about 0.25 to about 5 mg / kg body weight per single dose. In some embodiments, the effective dose of the active ingredient is in the range of about 0.5 to about 4 mg / kg body weight per single dose. In some embodiments, the effective dose of a MARC1 RNAi agent may be a fixed dose. In some embodiments, the fixed dose is in the range of about 5 mg to about 1,000 mg of MARC1 RNAi agent. In some embodiments, the fixed dose is in the range of 10 to 400 mg of MARC1 RNAi agent. In some embodiments, the fixed dose is in the range of 50 to 400 mg of MARC1 RNAi agent. Administration may be weekly, bi-weekly, monthly, quarterly, or at any other interval, depending on the dose of the MARC1 RNAi agent administered, the activity level of the specific MARC1 RNAi agent, and the desired level of inhibition against the specific target. The examples herein show suitable levels for inhibition in specific animal species. The dosage will depend on variables such as the overall health of the patient or target, the relative biological potency of the compound being delivered, the formulation of the drug, the presence and type of excipients in the formulation, and the route of administration. It will also be understood that the initial dose administered may be increased beyond the upper limit above to rapidly achieve the desired blood or tissue level, or the initial dose may be less than the optimal dose.

[0168] For the treatment of a disease, or for the formation of a pharmaceutical or composition for the treatment of a disease, the pharmaceutical compositions described herein, comprising a MARC1 RNAi agent, may be combined with excipients or a second therapeutic agent or treatment (including, but not limited to, a second or other RNAi agent, small molecule drug, antibody, antibody fragment, peptide and / or aptamer).

[0169] The MARC1 RNAi agents described herein may be packaged in kits, containers, packs, or dispensers when added to pharmaceutically acceptable excipients or adjuvants. The pharmaceutical compositions described herein may be packaged in pre-filled syringes, pen syringes, auto-injectors, infusion bags / devices, or vials.

[0170] Treatment methods and methods for inhibiting expression The MARC1 RNAi agents disclosed herein can be used to treat subjects (e.g., humans or mammals) having a disease or disorder for which administration of the compound would be beneficial. In some embodiments, the RNAi agents disclosed herein can be used to treat subjects (e.g., humans) who have been diagnosed with or are suffering from conditions associated with non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related diseases.

[0171] In some embodiments, a subject is administered a therapeutically effective dose of any one or more MARC1 RNAi agents. Treatment of the subject may include therapeutic and / or prophylactic treatment. A subject is administered a therapeutically effective dose of any one or more MARC1 RNAi agents described herein. The subject may be an adult, adolescent, child, or infant. Administration of the pharmaceutical compositions described herein may be to humans or animals.

[0172] The MARC1 RNAi agents described herein may be used to treat at least one symptom in a subject having a MARC1-related disease or disorder, or a disease or disorder at least partially mediated by MARC1 gene expression. In some embodiments, the MARC1 RNAi agents are used to treat or manage clinical symptoms in a subject having a disease or disorder that benefits from a reduction in MARC1 mRNA or MARC1 protein levels, or that is at least partially mediated by a reduction in MARC1 mRNA. The subject is administered a therapeutically effective dose of one or more of the MARC1 RNAi agents described herein or a MARC1 RNAi agent-containing composition. In some embodiments, the methods disclosed herein include administering a composition containing the MARC1 RNAi agents described herein to the subject to be treated. In other embodiments, the subject is administered a prophylactically effective dose of one or more of the described MARC1 RNAi agents to treat the subject by preventing or inhibiting at least one symptom.

[0173] In certain embodiments, the Disclosure provides a method for treating a disease, disorder, condition, or pathological condition at least partially mediated by MARC1 gene expression, the method comprising administering to a patient one of the MARC1 RNAi agents described herein.

[0174] In some embodiments, the gene expression level and / or mRNA level of the MARC1 gene in subjects administered with the described MARC1 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 administration of the MARC1 RNAi agent or subjects that have not received the MARC1 RNAi agent. MARC1 mRNA levels in subjects may be reduced in the cells, cell populations, and / or tissues of the subjects. In some embodiments, MARC1 gene expression is inhibited by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or more than 65% in the cytoplasm of hepatocytes compared to subjects before administration of the MARC1 RNAi agent or subjects that have not received the MARC1 RNAi agent.

[0175] In some embodiments, MARC1 protein levels in subjects administered with the described MARC1 RNAi agents are reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% compared to subjects before administration of the MARC1 RNAi agent or subjects not receiving the MARC1 RNAi agent. Protein levels within the subjects may be reduced in the subjects' cells, cell populations, tissues, blood, and / or other bodily fluids.

[0176] The reduction of MARC1 mRNA levels and MARC1 protein levels can be evaluated by any method known in the art. As used herein, the reduction or decrease of MARC1 mRNA levels and / or protein levels are collectively referred to herein as the reduction or decrease of MARC1 or the inhibition or reduction of MARC1 gene expression. The examples described herein illustrate known methods for evaluating the inhibition of MARC1 gene expression. Those skilled in the art will be able to learn of further preferred methods for evaluating the inhibition of MARC1 gene expression in vivo and / or in vitro.

[0177] In some embodiments, methods for treating (including prophylactic or preventative treatment) diseases, disorders, or symptoms caused by non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related diseases are disclosed herein, comprising administering to a subject in need thereof a therapeutically effective amount of a MARC1 RNAi agent comprising an antisense strand at least partially complementary to a portion of MARC1 mRNA having the sequence of Table 1. In some embodiments, methods for treating (including prophylactic or preventative treatment) diseases or symptoms caused by non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related diseases are disclosed herein, comprising administering to a subject in need of such treatment a therapeutically effective amount of a MARC1 RNAi agent comprising an antisense strand containing a sequence of any of Table 2, 3, or 6D, and a sense strand containing a sequence of any of Table 2, 4, 5, or 6D that is at least partially complementary to the antisense strand. In some embodiments, methods for treating (including prophylactic or preventative treatment) diseases or symptoms caused by non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related diseases are disclosed herein, comprising administering to a subject in need of such treatment a therapeutically effective amount of a MARC1 RNAi agent comprising a sense strand comprising one of the sequences in Table 2, 4, 5, or 6D, and an antisense strand comprising one of the sequences in Table 2, 3, or 6D that is at least partially complementary to the sense strand.

[0178] In some embodiments, methods for inhibiting the expression of the MARC1 gene in cells are disclosed herein, comprising administering the cells a MARC1 RNAi agent comprising an antisense strand at least partially complementary to a portion of the MARC1 mRNA having the sequence of Table 1. In some embodiments, methods for inhibiting the expression of the MARC1 gene in cells are disclosed herein, comprising administering the cells a MARC1 RNAi agent comprising an antisense strand comprising a sequence of any of Table 2, 3, or 6D, and a sense strand comprising a sequence of any of Table 2, 4, 5, or 6D that is at least partially complementary to the antisense strand. In some embodiments, methods for inhibiting the expression of the MARC1 gene in cells are disclosed herein, comprising administering the cells a MARC1 RNAi agent comprising a sense strand comprising a sequence of any of Table 2, 4, 5, or 6D, and an antisense strand comprising a sequence of any of Table 2, 3, or 6D that is at least partially complementary to the sense strand.

[0179] The use of MARC1 RNAi agents provides methods for the therapeutic (including prophylactic) treatment of diseases / disorders associated with non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related disorders. The described MARC1 RNAi agents inhibit the expression of one or more genes necessary for the production of the MARC1 protein by mediating RNA interference. MARC1 RNAi agents may also be used to treat or prevent a variety of diseases, disorders, or conditions, including non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related disorders. Furthermore, a composition for delivering MARC1 RNAi agents to hepatocytes in vivo is described.

[0180] Cells, tissues, organs, and non-human organisms Cells, tissues, organs, and non-human organisms comprising at least one of the MARC1 RNAi agents described herein are intended. These cells, tissues, organs, or non-human organisms are generated by delivering the RNAi agent to cells, tissues, organs, or non-human organisms.

[0181] Additional exemplary embodiments Specific additional exemplary embodiments relating to the inventions of the present disclosure are provided herein. These embodiments are for illustrative purposes only and do not limit the scope of the present disclosure or the claims appended thereto. Embodiment 1. An RNAi agent for inhibiting the expression of the MARC1 gene, wherein the following: An antisense strand containing a nucleotide sequence of at least 15 consecutive nucleotides, each differing from one of the antisense strand sequences in Table 2, Table 3, or Table 6D by 0 or 1 nucleotide; and A sense strand comprising a nucleotide sequence at least partially complementary to the antisense strand, RNAi agents, including those mentioned above. Embodiment 2. The RNAi agent according to Embodiment 1, wherein the antisense strand comprises nucleotides 2 to 18 of any one sequence provided in Table 2, Table 3, or Table 6D. Embodiment 3. The RNAi agent according to Embodiment 1 or Embodiment 2, wherein the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides, each differing by 0 or 1 nucleotide from one of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6D, and the sense strand has a region of at least 85% complementarity with the antisense strand over at least 15 consecutive nucleotides. Embodiment 4. The RNAi agent according to any one of Embodiments 1 to 3, wherein at least one nucleotide of the RNAi agent contains a modified nucleoside linkage. Embodiment 5. The RNAi agent according to any one of Embodiments 1 to 4, wherein all or substantially all of the nucleotides are modified nucleotides. Embodiment 6. An RNAi agent according to any one of Embodiments 4 to 5, wherein the modified nucleotide is independently selected from the group consisting of: 2'-O-methylnucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-seconucleotide mimetic, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, debasic nucleotide, ribitol, reverse nucleotide, reverse 2'-O-methylnucleotide, reverse 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide, vinyl phosphonate-containing nucleotide, cyclopropyl phosphonate-containing nucleotide, and 3'-O-methylnucleotide. Embodiment 7. The RNAi agent according to Embodiment 5, wherein all or substantially all of the modified nucleotides are 2'-O-methylnucleotides, 2'-fluoronucleotides, or a combination thereof. Embodiment 8. The RNAi agent according to any one of Embodiments 1 to 7, wherein the antisense strand consists of or essentially consists of one nucleotide sequence from the modified antisense strand sequences of Table 3. Embodiment 9. The RNAi agent according to any one of Embodiments 1 to 8, wherein the sense strand consists of or essentially consists of one nucleotide sequence from among the modified sense strand sequences of Table 4, Table 5, or Table 6D. Embodiment 10. The RNAi agent according to Embodiment 1, wherein the antisense strand contains one nucleotide sequence from any of the modified sequences in Table 3 or Table 6D, and the sense strand contains one nucleotide sequence from any of the modified sequences in Table 4, Table 5, or Table 6D. Embodiment 11. The RNAi agent according to any one of Embodiments 1 to 10, wherein the sense strand is 18 to 30 nucleotides in length and the antisense strand is 18 to 30 nucleotides in length. Embodiment 12. The RNAi agent according to Embodiment 11, wherein the sense strand and antisense strand are each 18 to 27 nucleotides in length. Embodiment 13. The RNAi agent according to Embodiment 12, wherein the sense strand and antisense strand are each 18 to 24 nucleotides in length. Embodiment 14. The RNAi agent according to Embodiment 13, wherein the sense strand and the antisense strand are each 21 nucleotides long. Embodiment 15. The RNAi agent according to Embodiment 14, wherein the RNAi agent has two blunt ends. Embodiment 16. The RNAi agent according to any one of Embodiments 1 to 15, wherein the sense strand includes one or two terminal caps. Embodiment 17. The RNAi agent according to any one of Embodiments 1 to 16, wherein the sense strand comprises one or two reverse debase residues. Embodiment 18. The RNAi agent according to Embodiment 1, wherein the RNAi agent consists of a sense strand and an antisense strand that form a double helix having one of the double helix structures shown in Tables 6A and 6B. Embodiment 19. The RNAi agent according to Embodiment 18, wherein all or substantially all of the nucleotides are modified nucleotides. Embodiment 20. The following nucleotide sequence (5'→3'): UGAAAGAACUAUUCCAUAAUC(Sequence ID 1608); ACAGAAUCCUGUCUUGUCGUU(Sequence ID 1657); UCCUUUAAAGGUUUUCAGUAG(Sequence ID 1580); or UAUUGAAGCAUUGAGACACCG (Sequence ID 1659) The RNAi agent according to Embodiment 1, comprising an antisense strand consisting of, essentially consisting of, or containing one of the nucleotide sequences that differ from 0 or 1 nucleotide. Embodiment 21. The RNAi agent according to any one of Embodiments 1 to 20, wherein the nucleotides located at the 2nd and 14th positions from the 5' end of the antisense strand are 2'-fluoromodified nucleotides. Embodiment 22. The RNAi agent according to Embodiment 21, wherein the nucleotide at position 2 of the antisense strand is 2'-fluorouridine, and the nucleotide at position 14 of the antisense strand is 2'-fluorocytidine, and the antisense strand comprises three or four phosphorothioate nucleoside linkages. Embodiment 23. The sense strand has the following nucleotide sequence (5'→3'): GAUUAUGGAAUAGUUCUUUCA(Sequence ID 1734); AACGACAAGACAGGAUUCUGU (Sequence ID 1783); CUACUGAAAACCUUUAAAIGA(sequence number 1774); or CGGUGUCUCAAUGCUUCAAUA (Sequence ID 1784), An RNAi agent according to any one of Embodiments 1 to 22, comprising, essentially consisting of, or including, a nucleotide sequence in which one of the following differs by 0 or 1 nucleotide. Embodiment 24. The RNAi agent according to any one of Embodiments 20 to 23, wherein all or substantially all of the nucleotides are modified nucleotides. Embodiment 25. The following nucleotide sequence (5'→3'): cPrpusGfaaaGfaacuaUfuCfcAfuaausc(Sequence ID 1143); asCfagAfauccugUfcUfuGfucgusu(sequence code 1228); isCfagAfauccugUfcUfuGfucgusu(Sequence ID 1229); cPrpusCfscsUfuUfaaaggUfuUfuCfaGfuasg(SEQ ID NO: 1200); or usAfsusugaAfgcauUfgAfgAfcaccsg (Sequence ID 1235), {In the formula, a represents 2'-O-methyladenosine, c represents 2'-O-methylcytidine, g represents 2'-O-methylguanosine, i represents 2'-O-methylinosine; and u represents 2'-O-methyluridine; Af represents 2'-fluoroadenosine, Cf represents 2'-fluorocytidine, Gf represents 2'-fluoroguanosine, and Uf represents 2'-fluorouridine; cPrpu represents 5'-cyclopropylphosphonate-2'-O-methyluridine; and s represents a phosphorothioate linkage} It includes, consists of, or is essentially composed of, an antisense strand containing one of the modified nucleotide sequences and zero or one nucleotide that differs from them; and, The RNAi agent according to Embodiment 1, wherein all or substantially all of the nucleotides of the sense strand are modified nucleotides. Embodiment 26. The sense strand has the following nucleotide sequence (5'→3'): gauuauggAfAfUfaguucuuuca(sequence number 1319); aacgacaaGfAfCfaggauucugu(Sequence ID 1376); cuacugaaAfAfCfcuuuaaaiga(sequence number 1361); or cggugucuCfAfAfugcuucaaua (Sequence ID 1377), {In the formula, a represents 2'-O-methyladenosine, c represents 2'-O-methylcytidine, g represents 2'-O-methylguanosine, u represents 2'-O-methyluridine, and i represents 2'-O-methylinosine; Af represents 2'-fluoroadenosine, Cf represents 2'-fluorocytidine, Gf represents 2'-fluoroguanosine, and Uf represents 2'-fluorouridine; s represents a phosphorothioate linkage} A modified nucleotide sequence containing, consisting of, or essentially consisting of, one of the nucleotides and 0 or 1 nucleotides that are different from each other; and, The RNAi agent according to Embodiment 1, wherein all or substantially all of the nucleotides of the antisense strand are modified nucleotides. Embodiment 27. The RNAi agent according to any one of Embodiments 20 to 26, wherein the sense strand further comprises a reverse debase residue at the 3' end of the nucleotide sequence, at the 5' end of the nucleotide sequence, or both. Embodiment 28. The RNAi agent according to any one of Embodiments 1 to 27, wherein the RNAi agent is linked to a targeted ligand. Embodiment 29. The targeted ligand is as follows: [ka] An RNAi agent according to any one of Embodiments 1 to 28, including the above. Embodiment 30. The RNAi agent according to any one of Embodiments 1 to 29, wherein the targeted ligand is linked to the sense strand. Embodiment 31. The RNAi agent according to Embodiment 30, wherein the targeted ligand is ligated to the 5' end of the sense strand. Embodiment 32. A composition comprising an RNAi agent according to any one of Embodiments 1 to 31, wherein the composition further comprises a pharmaceutically acceptable excipient. Embodiment 33. The composition according to Embodiment 32, further comprising a second RNAi agent capable of inhibiting the expression of the MARC1 gene. The composition according to any one of Embodiments 32 to 33, further comprising Embodiment 34.1 or a plurality of additional therapeutic agents. Embodiment 35. The composition according to any one of Embodiments 32 to 34, wherein the composition is formulated for administration. Embodiment 36. The composition according to Embodiment 35, wherein the composition is delivered by subcutaneous injection. Embodiment 37. The composition according to any one of Embodiments 32 to 36, wherein the pharmaceutically acceptable excipient is a sodium phosphate buffer. Embodiment 38. The composition according to any one of Embodiments 32 to 36, wherein the pharmaceutically acceptable excipient is isotonic saline or water for injection. Embodiment 39. A method for inhibiting the expression of the MARC1 gene in hepatocytes, comprising introducing an effective amount of an RNAi agent according to any one of Embodiments 1 to 31 or a composition according to any one of Embodiments 32 to 38 into the target cells. Embodiment 40. The method according to Embodiment 39, wherein the subject is a human subject. Embodiment 41. The method according to any one of Embodiments 39 to 40, wherein the MARC1 mRNA level is reduced by at least about 50% in hepatocytes or subjects. Embodiment 42. The method according to any one of Embodiments 39 to 41, wherein the MARC1 protein level is reduced by at least about 50% in hepatocytes or subjects. Embodiment 43. A method for treating a MARC1-related disease, disorder, or symptom, comprising administering a therapeutically effective amount of any one of Embodiments 32 to 38 to a human subject in need thereof. Embodiment 44. The method according to Embodiment 43, wherein the disease is hypertriglyceridemia, non-alcoholic steatohepatitis (NASH), alcoholic and non-alcoholic fatty liver disease (NAFLD), fatty liver disease, cirrhosis, high blood cholesterol levels, liver disease, autoimmune hepatitis, and / or other MARC1-related diseases. Embodiment 45. The method according to any one of Embodiments 39 to 44, wherein the level of serum MARC1 protein is reduced in the subject. Embodiment 46. The method according to any one of Embodiments 39 to 45, wherein the RNAi agent is administered to a human subject at a dose of approximately 0.05 mg / kg to approximately 5.0 mg / kg per body weight of the human subject. Embodiment 47. Use of an RNAi agent according to any one of Embodiments 1 to 31 or a composition according to any one of Embodiments 32 to 38 for the treatment of a disease, disorder, or symptom at least partially mediated by a reduction in MARC1 gene expression. Embodiment 48. The use according to Embodiment 47, wherein the disease is non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related diseases. Embodiment 49. Use of an RNAi agent according to any one of Embodiments 1 to 31 or a composition according to any one of Embodiments 32 to 38 for the preparation of a pharmaceutical composition for treating a disease, disorder, or symptom that is at least partially mediated by a reduction in MARC1 gene expression.

[0182] Herein, the embodiments and items described above are illustrated in the following non-limiting embodiments. [Examples]

[0183] Example 1. Synthesis of MARC1 RNAi agent The MARC1 RNAi double-stranded agents shown in Tables 6A and 6B above were synthesized according to the following general procedure:

[0184] A.Synthesis Based on solid-phase phosphoramidite techniques used for oligonucleotide synthesis, sense and antisense strands of RNAi agents were synthesized. Such standard synthesis is generally known in the art. Depending on the scale, MerMade96E® (Bioautomation), MerMade12® (Bioautomation), or OP Pilot 100 (GE Healthcare) were used. Synthesis was carried out on solid supports made of controlled porous glass (CPG, 500 Å or 600 Å, available from Prime Synthesis, Aston, PA, USA). Monomers located at the 3' end of each strand were attached to the solid support as starting points for synthesis. All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA) or Hongene Biotech (Shanghai, PRC). Examples of 2'-O-methylphosphoramidites include: (5'-O-dimethoxytrityl-N 6 -(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5'-O-dimethoxytrityl-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 had the same protecting group as 2'-O-methylamidite. 5'-(4,4'-dimethoxytrityl)-2',3'-secor-uridine and 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite were also purchased from Thermo Fisher Scientific or Hongene Biotech. 5'-Dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite was purchased from Glen Research (Virginia) or Hongene Biotech. Cyclopropylphosphonate phosphoramidite was synthesized according to International Publication No. WO2017 / 214112 (see also Altenhofer et. al., Chem. Communications (Royal Soc. Chem.), 57(55): 6808-6811 (July 2021)). Reverse debase (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite) was purchased from ChemGenes (Wilmington, MA, USA) or SAFC (St Louis, MO, USA). 2 ,N 6 -(phenoxyacetate)-2'-O-methyl-diaminopurine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite was obtained from ChemGenes or Hongene Biotech.

[0185] Phosphoamidites containing the targeting ligand were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), and all other amidites were dissolved in anhydrous acetonitrile (50 mM) or anhydrous dimethylformamide, and molecular sieves (3 Å) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) were used as activator solutions. Binding times were 12 minutes (RNA), 15 minutes (targeting ligand), 90 seconds (2'OMe), and 60 seconds (2'F). To introduce phosphorothioate linkage, a 100 mM solution of 3-phenyl1,2,4-dithiazolin-5-one (POS, PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used. Each MARC1 RNAi double-stranded agent synthesized and tested in the following examples utilized N-acetyl-galactosamine as "NAG" in the targeted ligand chemical structure shown in Table 7. The (NAG37) and (NAG37)s targeted ligand phosphoramidite compounds can be synthesized according to International Publication No. WO2018 / 044350 to Arrowhead Pharmaceuticals, Inc.

[0186] B. Cleavage and deprotection of support-bound oligomers After completing the solid-phase synthesis, the dried solid support was treated with a 1:1 solution of 40% by weight methylamine in water and 28% ammonium hydroxide solution (Aldrich) at 30°C for 1.5 hours. This solution was evaporated, and the solid residue was reconstituted in water (see below).

[0187] C. 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 consisted of 20 mM Tris, 5 mM EDTA, pH 9.0, and contained 20% acetonitrile. Buffer B was the same as Buffer A, with the addition of 1.5 M sodium chloride. UV tracing was recorded at 260 nm. After pooling appropriate fractions, the samples were subjected to size exclusion HPLC using a GE Healthcare XK 26 / 40 column packed with Sephadex G-25 fine, with filtered DI water or 100 mM ammonium bicarbonate (pH 6.7) and 20% acetonitrile used as running buffers.

[0188] D. Annealing The complementary strands were mixed with equimolar amounts of RNA solution (sense and antisense) in 1× phosphate-buffered saline (Corning, Cellgro) to form RNAi agents. A portion of the RNAi agents were lyophilized and stored at -15 to -25°C. The double-stranded concentration was determined by measuring the solution absorbance using a UV-Vis spectrometer in 1× phosphate-buffered saline. Next, the double-stranded concentration was determined by multiplying the solution absorbance at 260 nm by a conversion factor and a dilution factor. The conversion factor used was either 0.050 mg / (mLcm) or calculated from experimentally determined extinction coefficients.

[0189] Example 2. hMARC1 SEAP mouse model To evaluate MARC1 RNAi agents, a MARC1-SEAP mouse model was used. Plasmids were transiently transfected in vivo into C57bl6 / albino mice via hydrodynamic tail vein injection (HTV). The mice were injected via HTV with plasmid pMIR1015, containing the 33-2500 region of the human MARC1 cDNA sequence (NCBI reference sequence: NM_022746.4 (SEQ ID NO: 1)) inserted into the 3'UTR of the SEAP (secreted human placental alkaline phosphatase) reporter gene. MARC1-SEAP model mice were created by injecting 50 μg of plasmid containing hMARC1 cDNA in a total volume of Ringer's solution equivalent to 10% of the animal's body weight via HTV. Following MARC1-SEAP transfection, the mice were subsequently administered MARC1 RNAi agents. Inhibition of MARC1 expression by MARC1 RNAi agents resulted in associated inhibition of SEAP expression. SEAP expression levels were measured using the Phospha-Light® SEAP Reporter Gene Assay System (ThermoFisher Cat#T1016). Before treatment, serum SEAP expression levels were measured, and mice were grouped according to their mean SEAP levels. Analysis: SEAP levels can be measured at various points in time, both before and after administration of MARC1 RNAi agents. i) Serum collection: Mice were anesthetized with 2-3% isoflurane, and blood samples were collected from the submandibular region into serum separation tubes (Sarstedt AG & Co., Numbrecht, Germany). The blood was allowed to coagulate at ambient temperature for 20 minutes. The tubes were centrifuged at 8,000 × g for 3 minutes to separate the serum, which was then stored at 4°C. ii) Serum SEAP levels: Serum was collected and measured using the Phospha-Light® SEAP Reporter Gene Assay System (ThermoFisher) according to the manufacturer's instructions. The serum SEAP levels of each animal were normalized to a control group injected with saline to account for the untreated-related decrease in MARC1 sequence expression in this model. First, the SEAP level of each animal at a given time point was divided by the pre-treatment expression level in that animal ("pre-treatment") to determine the "normalized to pre-treatment" expression ratio. Then, the "normalized to pre-treatment" ratio of each animal was divided by the mean "normalized to pre-treatment" ratio of all mice in the normal saline control group to normalize the expression at a specific time point to the control group. Alternatively, in some examples described herein, the serum SEAP levels of each animal were evaluated by normalizing them to pre-treatment levels only.

[0190] Example 3. In vivo administration of MARC1 RNAi agents in hMARC1-SEAP mice The hMARC1-SEAP model described in Example 2 above was used. On day 1, four female C57bl / 6 albino mice (n=4) were administered either saline or a MARC1 RNAi agent prescribed in saline (at 2 mg / kg) by subcutaneous (SQ) injection. The administration regimen followed Table 8 below.

[0191] Table 8. Administration of the method used in Example 3 to mice [Table 11-1] [Table 11-2]

[0192] Serum was collected on days -3, 1, 8, 15, 22, and 29. SEAP expression levels were measured according to the procedure described in Example 2 above. The experimental data, along with the mean SEAP reflecting the normalized mean SEAP, are shown in Table 9 below.

[0193] Table 9. Mean SEAP before treatment and normalized to saline control in hMARC1-SEAP mice of Example 3. [Table 12]

[0194] Groups 5, 7, 8, 12-16, 18, 22-25, 30, and 31 showed a reduction in SEAP on day 8. Groups 5, 7, 8, and 10-33 showed a reduction in SEAP on day 15. Groups 5, 7, 8, 10, 12, 13, 15-18, 20-26, 31, and 32 showed a reduction in SEAP on day 22.

[0195] Example 4. In vivo administration of MARC1 RNAi agents in hMARC1-SEAP mice The hMARC1-SEAP model described in Example 2 above was used. On day 1, four female C57bl / 6 albino mice (n=4) were administered either physiological saline or a MARC1 RNAi agent prescribed in physiological saline (at 2 mg / kg, 4 mg / kg, or 6 mg / kg) by subcutaneous (SQ) injection at an injection volume of 200 μL / 20 g body weight (10 mL / kg). The administration regimen followed the one shown in Table 10 below.

[0196] Table 10. Administration of Example 4 to Mice [Table 13]

[0197] Serum was collected on days -5, 1, 8, 15, 22, and 29. SEAP expression levels were measured according to the procedure described in Example 2 above. The experimental data, along with the mean SEAP reflecting the normalized mean SEAP, are shown in Table 11 below.

[0198] Table 11. Mean SEAP before treatment and normalized to saline control in MARC1-SEAP mice of Example 4. [Table 14]

[0199] Groups 2-11 showed a reduction in SEAP at all time points.

[0200] Example 5. In vivo administration of MARC1 RNAi agents in hMARC1-SEAP mice The hMARC1-SEAP model described in Example 2 above was used. On day 1, four female C57bl / 6 albino mice (n=4) were administered either saline or a MARC1 RNAi agent prescribed in saline (at 2 mg / kg) by subcutaneous (SQ) injection at an injection volume of 200 μl / 20 g body weight (10 mL / kg). The administration regimen followed Table 12 below.

[0201] Table 12. Administration of Example 5 to Mice [Table 15]

[0202] Serum was collected on days -9, 1, 8, 15, and 22. SEAP expression levels were measured according to the procedure described in Example 2 above. The experimental data, along with the mean SEAP reflecting the normalized mean SEAP, are shown in Table 13 below.

[0203] Table 13. Mean SEAP before treatment and normalized to saline control in MARC1-SEAP mice of Example 5. [Table 16]

[0204] Groups 2-12 showed a reduction in SEAP at all time points.

[0205] Example 6. In vivo administration of M ARC1 RNAi agents in hMARC1-SEAP mice The hMARC1-SEAP model described in Example 2 above was used. On day 1, four female C57bl / 6 albino mice (n=4) were administered either saline or a MARC1 RNAi agent prescribed in saline (at 2 mg / kg) by subcutaneous (SQ) injection at an injection volume of 200 μL / 20 g body weight (10 mL / kg). The administration regimen followed Table 14 below.

[0206] Table 14. Administration of Example 6 to Mice [Table 17]

[0207] Serum was collected on days -14, -7, 1, 8, 15, 22, and 29. SEAP expression levels were measured according to the procedure described in Example 2 above. The data from the experiment, along with the mean SEAP reflecting the normalized mean SEAP, are shown in Table 15 below.

[0208] Table 15. Mean SEAP in MARC1-SEAP mice of Example 6, normalized to pre-treatment and saline control. [Table 18]

[0209] Groups 2-10 showed a reduction in SEAP at all time points.

[0210] Example 7. In vivo administration of MARC1 RNAi agents in hMARC1-SEAP mice The hMARC1-SEAP model described in Example 2 above was used. On day 1, four female C57bl / 6 albino mice (n=4) were administered either saline or a MARC1 RNAi agent prescribed in saline (at 2 mg / kg) by subcutaneous (SQ) injection at an injection volume of 200 μL / 20 g body weight (10 mL / kg). The administration regimen followed Table 16 below.

[0211] Table 16. Administration of Example 7 to Mice [Table 19]

[0212] Serum was collected on days -14, -7, 1, 8, 15, 22, and 29. SEAP expression levels were measured according to the procedure described in Example 2 above. The data from the experiment, along with the mean SEAP reflecting the normalized mean SEAP, are shown in Table 17 below.

[0213] Table 17. Mean SEAP before treatment and normalized to saline control in MARC1-SEAP mice of Example 7. [Table 20]

[0214] Groups 2, 4, 5, 7, and 8 showed a reduction in SEAP on day 8. Groups 2, 5, 7, and 8 showed a reduction in SEAP on day 15. Groups 2, 4, 5, 7, and 8 showed a reduction in SEAP on day 22.

[0215] Example 8. In vivo administration of MARC1 RNAi agents in hMARC1-SEAP mice The hMARC1-SEAP model described in Example 2 above was used. On day 1, four female C57bl / 6 albino mice (n=4) were administered either saline or a MARC1 RNAi agent prescribed in saline (at 2 mg / kg) by subcutaneous (SQ) injection at an injection volume of 200 μL / 20 g body weight (10 mL / kg). The administration regimen followed Table 18 below.

[0216] Table 18. Administration of Example 8 to mice [Table 21]

[0217] Serum was collected on days -14, -7, 1, 8, 15, 22, and 29. SEAP expression levels were measured according to the procedure described in Example 2 above. The experimental data, along with the mean SEAP reflecting the normalized mean SEAP, are shown in Table 19 below.

[0218] Table 19. Mean SEAP before treatment and normalized to saline control in MARC1-SEAP mice of Example 8. [Table 22]

[0219] Groups 2-12 showed a reduction in SEAP at all time points.

[0220] Example 9. In vivo administration of MARC1 RNAi agents in hMARC1-SEAP mice The hMARC1-SEAP model described in Example 2 above was used. On day 1, four female C57bl / 6 albino mice (n=4) were administered either saline or a MARC1 RNAi agent prescribed in saline (at 2 mg / kg) by subcutaneous (SQ) injection at an injection volume of 200 μL / 20 g body weight (10 mL / kg). The administration regimen followed Table 20 below.

[0221] Table 20. Administration of Example 9 to Mice [Table 23]

[0222] Serum was collected on days -7, 1, 8, 15, and 22. SEAP expression levels were measured according to the procedure described in Example 2 above. The experimental data, along with the mean SEAP reflecting the normalized mean SEAP, are shown in Table 21 below.

[0223] Table 21. Mean SEAP before treatment and normalized to saline control in MARC1-SEAP mice of Example 9. [Table 24]

[0224] Groups 2-10 showed a reduction in SEAP at all time points.

[0225] Example 10. In vivo administration of MARC1 RNAi agents in hMARC1-SEAP mice The hMARC1-SEAP model described in Example 2 above was used. On day 1, four female C57bl / 6 albino mice (n=4) were administered either saline or a MARC1 RNAi agent prescribed in saline (at 2 mg / kg) by subcutaneous (SQ) injection at an injection volume of 200 μL / 20 g body weight (10 mL / kg). The administration regimen followed Table 22 below.

[0226] Table 22. Administration of Example 10 to Mice [Table 25]

[0227] Serum was collected on days -7, 1, 8, 15, and 22. SEAP expression levels were measured according to the procedure described in Example 2 above. The experimental data, along with the mean SEAP reflecting the normalized mean SEAP, are shown in Table 23 below.

[0228] Table 23. Mean SEAP before treatment and normalized to saline control in MARC1-SEAP mice of Example 10. [Table 26]

[0229] Groups 2-12 showed a reduction in SEAP at all time points.

[0230] Example 11. In vivo administration of MARC1 RNAi agents in hMARC1-SEAP mice The hMARC1-SEAP model described in Example 2 above was used. On day 1, four female C57bl / 6 albino mice (n=4) were administered either saline or an RNAi agent prescribed in saline (at 2 mg / kg) by subcutaneous (SQ) injection at an injection volume of 200 μL / 20 g body weight (10 mL / kg). The administration regimen followed Table 24 below.

[0231] Table 24. Administration of the method in Example 11 to mice [Table 27]

[0232] Serum was collected on days -7, 1, 8, 15, and 22. SEAP expression levels were measured according to the procedure described in Example 2 above. The experimental data, along with the mean SEAP reflecting the normalized mean SEAP, are shown in Table 25 below.

[0233] Table 25. Mean SEAP before treatment and normalized to saline control in MARC1-SEAP mice of Example 11. [Table 28]

[0234] Groups 2-12 showed a reduction in SEAP at all time points.

[0235] Example 12. In vivo administration of MARC1 RNAi agents in hMARC1-SEAP mice The hMARC1-SEAP model described in Example 2 above was used. On day 1, four female C57bl / 6 albino mice (n=4) were administered either saline or an RNAi agent prescribed in saline (at 2 mg / kg) by subcutaneous (SQ) injection at an injection volume of 200 μL / 20 g body weight (10 mL / kg). The administration regimen followed Table 26 below.

[0236] Table 26. Administration of the method in Example 12 to mice [Table 29]

[0237] Serum was collected on days -7, 1, 8, 15, and 22. SEAP expression levels were measured according to the procedure described in Example 2 above. The experimental data, along with the mean SEAP reflecting the normalized mean SEAP, are shown in Table 27 below.

[0238] Table 27. Mean SEAP before treatment and normalized to saline control in MARC1-SEAP mice of Example 12. [Table 30]

[0239] Groups 2-10 showed a reduction in SEAP at all time points.

[0240] Example 13. In vivo administration of MARC1 RNAi agents in hMARC1-SEAP mice The hMARC1-SEAP model described in Example 2 above was used. On day 1, four female C57bl / 6 albino mice (n=4) were administered either saline or an RNAi agent prescribed in saline (at 2 mg / kg) by subcutaneous (SQ) injection at an injection volume of 250 μL / 25 g body weight. The administration regimen followed Table 28 below.

[0241] Table 28. Administration of the method in Example 13 to mice [Table 31]

[0242] Serum was collected on days -7, 1, 8, 15, and 22. SEAP expression levels were measured according to the procedure described in Example 2 above. The experimental data, along with the mean SEAP reflecting the normalized mean SEAP, are shown in Table 29 below.

[0243] Table 29. Mean SEAP before treatment and normalized to saline control in MARC1-SEAP mice of Example 13. [Table 32]

[0244] Groups 2-9 showed a reduction in SEAP on day 8 and day 15. Groups 2-4 and 6-9 showed a reduction in SEAP on day 22.

[0245] Example 14. MARC1-GLuc AAV mouse model To evaluate specific MARC1 RNAi agents, a MARC1-GLuc (Gaussia Luciferase) AAV (adeno-associated virus) mouse model was used. Male C57BL / 6 mice aged 6–8 weeks were transduced with MARC1-GLuc AAV serotype 8, administered at least 14 days prior to administration of the MARC1 RNAi agent or control. The MARC1-GLuc AAV genome contains regions 33–2500 of the human MARC1 cDNA sequence (GenBank NM_022746.4 (SEQ ID NO: 1)) inserted within the 3'UTR of the GLuc reporter gene sequence. MARC1-GLuc AAV model mice were created by injecting mice via the tail vein with either 5E12 or 1E13 GC / kg of the respective virus in a total volume of PBS at 250 μL / 25 g per animal body weight. Inhibition of MARC1 expression by MARC1 RNAi agents resulted in associated inhibition of GLuc expression, which was measured. Before administering the treatment (before administration on day 7 to day 1), serum GLuc expression levels were measured using the Pierce® Gaussia Luciferase Glow Assay Kit (Thermo Fisher Scientific, Catalog #16161), and mice were then grouped according to their average GLuc levels.

[0246] Mice were anesthetized with 2-3% isoflurane, and blood samples were collected from the submandibular region into serum separation tubes (Sarstedt AG & Co., Numbrecht, Germany). The blood was allowed to coagulate at ambient temperature for 20 minutes. The tubes were centrifuged at 8,000 × g for 3 minutes to separate the serum, which was then stored at 4°C. The serum was collected and measured using the Pierce® Gaussia Luciferase Glow Assay Kit according to the manufacturer's instructions. The serum GLuc level of each animal could be normalized to a control mouse group injected with a vehicle control to account for the untreated-related changes in MARC1 expression in this model. To do so, the GLuc level of each animal at a given time point was first divided by the pre-treatment expression level in that animal (-1 day) to determine the "normalized to pre-treatment" expression ratio. Next, the expression at a specific time point was normalized relative to the control group by dividing the "normalized relative to pre-treatment" ratio of each animal by the mean "normalized relative to pre-treatment" ratio of all mice in the normal vehicle control group. Alternatively, the serum GLuc levels of each animal were evaluated by normalizing them to pre-treatment levels only.

[0247] Example 15. In vivo study of MARC1 RNAi agents in MARC1-GLuc AAV mice The MARC1-GLUC AAV mouse model described in Example 14 above was used, which utilized MARC1-GLuc AAV containing the 33-2500 region of the human MARC1 cDNA sequence. On day 1, four male C57bl / 6 mice (n=4) were administered either saline or an RNAi agent prescribed in saline (at 2 mg / kg) by subcutaneous (SQ) injection at an injection volume of 250 μL / 25 g body weight. The injection was administered between the skin and muscle (i.e., subcutaneously). The administration regimen followed the one shown in Table 30 below.

[0248] Table 30. Administration of Example 15 to Mice [Table 33]

[0249] Each MARC1 RNAi agent contained a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand containing three N-acetyl-galactosamine groups (tridentate ligands) having the modified sequences described herein in the double-stranded structure. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structural information related to MARC1 RNAi agents containing (NAG37)s ligands.) MARC1 RNAi agent AD12363 (group 2) contains a nucleotide sequence designed to inhibit the expression of the MARC1 gene at position 305; MARC1 RNAi agent AD12364 (group 3) contains a nucleotide sequence designed to inhibit the expression of the MARC1 gene at position 761; MARC1 RNAi agent AD12365 (group 4) contains a nucleotide sequence designed to inhibit the expression of the MARC1 gene at position 956; MARC1 RNAi agent AD12366 (group 5) contains a nucleotide sequence designed to inhibit the expression of the MARC1 gene at position 1109; MARC1 RNAi agent AD12367 (group 6) contains a nucleotide sequence designed to inhibit the expression of the MARC1 gene at position 1275; MARC1 RNAi agent AD12368 (group 7) contains a nucleotide sequence designed to inhibit the expression of the MARC1 gene at position 1633; MARC1 RNAi agent AD12369 (group 8) contains a nucleotide sequence designed to inhibit the expression of the MARC1 gene at position 1817; MARC1 RNAi agent AD12370 (group 9) contains a nucleotide sequence designed to inhibit the expression of the MARC1 gene at position 1900; MARC1 RNAi agent AD12371 (group 10) contains a nucleotide sequence designed to inhibit the expression of the MARC1 gene at position 1954; and MARC1 RNAi agent AD11786 (group 11) contains a nucleotide sequence designed to inhibit the expression of the MARC1 gene at position 1313 (see, for example, Sequence ID 1 and Table 2 for the referenced MARC1 gene).

[0250] Serum samples were collected on days 1, 8, 15, and 22. GLuc expression levels were measured according to the procedure described in Example 14. The experimental data, along with the mean GLuc reflecting the normalized mean value, are shown in Table 31 below.

[0251] Table 31. Mean GLuc in MARC1-GLuc-AAV mice of Example 15, normalized to pre-treatment and saline control. [Table 34]

[0252] Groups 2-11 showed a reduction in GLuc on day 8. Groups 4, 6, 9, and 11 showed a reduction in GLuc on day 15. Group 2 showed a reduction in GLuc on day 22.

[0253] Example 16. In vivo administration of MARC1 RNAi agents in rats Regarding MARC1 inhibition, MARC1 RNAi agents were tested in Sprague Dawley rats. On day 1, four male Sprague Dawley rats (n=4) were administered either physiological saline or the RNAi agent prescribed in physiological saline (at a dose of 3 mg / kg) by subcutaneous (SQ) injection at an injection volume of 1000 μL / 25 g body weight (4 mL / kg). The administration regimen followed the one shown in Table 32 below.

[0254] Table 32. Administration of the method used in Example 16 to mice [Table 35]

[0255] The target gene sites of MARC1 RNAi agents in groups 2-15 are cross-reactive with human MARC1.

[0256] Eight days after administration, the animals were sacrificed and liver tissue was collected. Rat MARC1 expression was measured using qPCR, with rat β-actin as a control. The mean MARC1 expression in the liver tissue of each animal was normalized to pre-administration and control group 1 (physiological saline). The experimental data are shown in Table 33 below.

[0257] Table 33. Mean relative MARC1 expression in rat liver on day 8 of Example 16 [Table 36]

[0258] Groups 3, 4, 8-10, 14, and 15 showed a reduction in rMARC1 on day 8.

[0259] Example 17. In vivo administration of MARC1 RNAi agents in rats Regarding MARC1 inhibition, MARC1 RNAi agents were tested in Sprague Dawley rats. On day 1, four male Sprague Dawley rats (n=4) were administered either physiological saline or the RNAi agent prescribed in physiological saline (at a dose of 3 mg / kg) by subcutaneous (SQ) injection at an injection volume of 1000 μL / 25 g body weight (4 mL / kg). The administration regimen followed the one shown in Table 34 below.

[0260] Table 34. Administration of the method in Example 17 to mice [Table 37]

[0261] The target gene sites of MARC1 RNAi agents in groups 2-10 are cross-reactive with human MARC1.

[0262] Eight days after administration, the animals were sacrificed and liver tissue was collected. Rat MARC1 expression was measured using qPCR, with rat β-actin as a control. The mean MARC1 expression in the liver tissue of each animal was normalized to pre-administration and control group 1 (physiological saline). The experimental data are shown in Table 35 below.

[0263] Table 35. Mean relative MARC1 expression in rat liver on day 8 of Example 17 [Table 38]

[0264] Groups 2-10 showed a reduction in rMARC1 on day 8.

[0265] Example 18. In vivo administration of MARC1 RNAi agents in cynomolgus monkeys. MARC1 RNAi agents were tested for MARC1 inhibition in cynomolgus monkeys. Liver biopsies were taken from all animals on day 7 and used as internal standard samples for normalization. On day 1, four groups of female cynomolgus monkeys (treated animals), each consisting of 3 animals (n=3), were administered the RNAi agent prescribed in saline (3 mg / kg) by subcutaneous (SQ) injection using a syringe and needle at a volume of 0.3 mL / kg body weight in the midscapular region. Additional liver biopsies were taken on days 15 and 43. All animals were sedated and fasted for at least 12 hours but less than 18 hours prior to liver biopsy. The administration regimens followed Table 36 below.

[0266] Table 36. Administration of Example 18 to Mice [Table 39]

[0267] Prior to each SQ injection, the test animals were first sedated. Sedation was achieved using ketamine HCl (10 mg / kg) administered as an intramuscular (IM) injection (no injections were given into the quadriceps femoris muscle). The individual doses of the MARC1 RNAi agent were calculated based on the body weight recorded on each administration day.

[0268] For each animal, liver biopsy samples (approximately 200 mg (160-240 mg; ±10%)) were collected for preliminary gene knockdown analysis.

[0269] Serum blood was collected on days -7, 1, 15, 29, and 43, and, where applicable, before liver biopsy sample collection or dose administration, as well as from any animals found in a mortally injured state or slaughtered at unscheduled intervals.

[0270] Cyno MARC1 expression was measured using qPCR with cyno ARL1 as a control. The mean MARC1 expression in liver tissue of each animal was normalized to the sample level at day -7 for each individual test animal. Next, the mean relative expression was calculated for each group from the individual normalized values. The experimental data are shown in Table 37 below.

[0271] Table 37. Normalized mean MARC1 for pre-treatment cynomolgus monkeys in Example 18. [Table 40]

[0272] Groups 1-4 showed a reduction in MARC1 between day 15 and day 43 after administration.

[0273] MARC1 protein levels were quantified by a planned LC-MS / MS assay. For this purpose, cynomolgus monkey liver samples were homogenized using RIPA Lysis and extraction buffer (Thermo Scientific). Proteins were extracted using a magnetic bead protocol and trypsin digestion over 20 hours in the presence of an internal standard. LC-MS / MS-based peptide quantification was performed to quantify the area under the reaction curve for two MARC1-specific peptides (sequences listed below), their corresponding internal standards, and the SLC25A3 protein-specific peptide. SLC25A3 is a phosphate carrier protein selected as the proximal normalization protein for the MARC1 protein in the mitochondrial membrane. The SLC25A3 normalized MARC1 protein concentrations in the livers of cynomolgus monkey test animals are shown in Table 38 below.

[0274] Table 38. MARC1 protein levels in cynomolgus monkey liver before administration and in relation to SLC25A3 expression in Example 18. [Table 41]

[0275] Group 4 (AD12369 at 3 mg / kg) showed time-dependent >50% MARC1 protein knockdown.

[0276] Example 19. In vivo administration of MARC1 RNAi agents in cynomolgus monkeys. MARC1 RNAi agents were tested for MARC1 inhibition in cynomolgus monkeys. On days 1 and 29, four groups of male cynomolgus monkeys (recipients), each containing three animals (n=3), were administered the RNAi agent prescribed in saline (at 3 mg / kg) by subcutaneous (SQ) injection at an injection volume of 0.3 mL / kg body weight. The administration regimens followed Table 39 below.

[0277] Table 39. Administration of Example 19 to Mice [Table 42]

[0278] On days 1 and 29, the test animals were weighed, and the drug was administered subcutaneously (SQ) using a syringe and needle in the mid-scapular region.

[0279] Liver biopsies were taken on days 7 (pre-administration), 15, 29, and 43. Liver biopsies were taken as a sedation measure. Animals were fasted overnight (at least 12 hours, but less than 18 hours) before each liver biopsy. For each animal, liver biopsy samples (~40 mg and 30-60 mg, ±10%, respectively) were taken for gene knockdown analysis. An additional liver biopsy (~40 mg and 30-60 mg, ±10%, respectively) was taken on day 15 only for animals in group 2. Liver biopsies were also taken from any animals found in a mortal state or sacrificed at unscheduled intervals (internal), where applicable.

[0280] Blood was collected on days -7 (pre-administration), 1, 15, 29, and 43, and, where applicable, before liver biopsy sample collection or RNAi drug dose administration. Blood was also collected, where applicable, from any animals found in a mortally injured state or slaughtered at unscheduled intervals (internal). Animals were fasted overnight (at least 12 hours, but less than 18 hours) for scheduled collections. Fasting periods were consistent between collections (±1 hour). Animals were not fasted for unscheduled collections. The blood collection site was the femoral vein with the saphenous vein as an alternative collection site.

[0281] Sedation was administered using ketamine HCl (10 mg / kg) via intramuscular (IM) injection (no injections were given into the quadriceps femoris muscle).

[0282] cMARC1 expression was measured using qPCR with cyno ARL1 as an endogenous control. The mean MARC1 expression in liver tissue of each animal was normalized to the sample level at day -7 for each individual test animal. Next, the mean relative expression was calculated for each group from the individual normalized values. The experimental data are shown in Table 40 below.

[0283] Table 40. Normalized mean MARC1 for pre-treatment cynomolgus monkeys in Example 19. [Table 43]

[0284] Groups 1-4 showed a reduction in MARC1 between day 15 and day 43 after administration.

[0285] MARC1 protein levels were quantified by a planned LC-MS / MS assay. For this purpose, cynomolgus monkey liver samples were homogenized using RIPA Lysis and extraction buffer (Thermo Scientific). Proteins were extracted using a magnetic bead protocol and trypsin digestion over 20 hours in the presence of an internal standard. LC-MS / MS-based peptide quantification was performed to quantify the area under the reaction curve for two MARC1-specific peptides (sequences listed below), their corresponding internal standards, and the SLC25A3 protein-specific peptide. SLC25A3 is a phosphate carrier protein selected as the proximal normalization protein for the MARC1 protein in the mitochondrial membrane. The SLC25A3 normalized MARC1 protein concentrations in the livers of cynomolgus monkey test animals are shown in Tables 41 and 42 below. Tables 41 and 42 show the MARC1 protein levels quantified by their respective peptide sequences according to the LC-MS / MS assay.

[0286] Table 41. MARC1 protein levels in cynomolgus monkey liver (quantified using peptide sequence: DLLLPIK) before administration and in relation to SLC25A3 expression in Example 19. [Table 44]

[0287] MARC1 RNAi agents showed complete MARC1 protein inhibition for at least 43 days after administration. Groups 1, 2, and 4 showed a reduction in MARC1 at all time points, while group 3 showed only a slight reduction at all time points. More specifically, AD13805 achieved approximately 71% MARC1 inhibition (0.281) at day 43 after administration of 2 × 3.0 mg / kg.

[0288] Table 42. MARC1 protein levels in cynomolgus monkey liver (quantified using peptide sequence: SPLFGQYFVLENPGTIK) before administration and in relation to SLC25A3 expression in Example 19. [Table 45]

[0289] MARC1 RNAi agents showed complete MARC1 protein inhibition for at least 43 days after administration. Groups 1, 2, and 4 showed a reduction in MARC1 at all time points, while group 3 showed only a non-significant reduction at all time points (a small reduction on day 15). More specifically, AD13805 achieved approximately 75% MARC1 inhibition (0.245) at day 43 after administration of 2 × 3.0 mg / kg.

[0290] Other Embodiments Although the present invention has been described in connection with its detailed description, the foregoing description is illustrative of the scope of the invention and is not intended to limit the scope of the invention, and the scope of the invention is understood to be defined by the appended claims. Other aspects, advantages, and modifications are within the following claims.

Claims

1. An RNAi agent for inhibiting the expression of the MARC1 gene, wherein the following: An antisense strand containing a nucleotide sequence of at least 15 consecutive nucleotides, each differing from one of the antisense strand sequences in Table 2, Table 3, or Table 6D by 0 or 1 nucleotide; and A sense strand comprising a nucleotide sequence at least partially complementary to the antisense strand, RNAi agents, including those mentioned above.

2. The RNAi agent according to claim 1, wherein the antisense strand comprises nucleotides 2 to 18 of any one sequence provided in Table 2, Table 3, or Table 6D.

3. The RNAi agent according to claim 1 or 2, wherein the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides, each differing by 0 or 1 nucleotide from any one of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6D, and the sense strand has a region of at least 85% complementarity with the antisense strand over at least 15 consecutive nucleotides.

4. The RNAi agent according to any one of claims 1 to 3, wherein at least one nucleotide of the RNAi agent comprises a modified nucleoside linkage.

5. The RNAi agent according to any one of claims 1 to 4, wherein all or substantially all of the nucleotides are modified nucleotides.

6. The RNAi agent according to any one of claims 4 to 5, wherein the modified nucleotide is independently selected from the group consisting of: 2'-O-methylnucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-seconucleotide mimetic, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, debasic nucleotide, ribitol, reverse nucleotide, reverse 2'-O-methylnucleotide, reverse 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide, vinyl phosphonate-containing nucleotide, cyclopropyl phosphonate-containing nucleotide, and 3'-O-methylnucleotide.

7. The RNAi agent according to claim 5, wherein all or substantially all of the modified nucleotides are 2'-O-methylnucleotides, 2'-fluoronucleotides, or a combination thereof.

8. The RNAi agent according to any one of claims 1 to 7, wherein the antisense strand consists of or essentially consists of one nucleotide sequence from any one of the modified antisense strand sequences in Table 3 or Table 6D.

9. The RNAi agent according to any one of claims 1 to 8, wherein the sense strand consists of or essentially consists of one nucleotide sequence from the modified sense strand sequences of Table 4, Table 5, or Table 6D.

10. The RNAi agent according to claim 1, wherein the antisense strand contains one nucleotide sequence from any of the modified sequences in Table 3, and the sense strand contains one nucleotide sequence from any of the modified sequences in Table 4, Table 5, or Table 6D.

11. The RNAi agent according to any one of claims 1 to 10, wherein the sense strand is 18 to 30 nucleotides long and the antisense strand is 18 to 30 nucleotides long.

12. The RNAi agent according to claim 11, wherein the sense strand and the antisense strand each have a length of 18 to 27 nucleotides.

13. The RNAi agent according to claim 12, wherein the sense strand and the antisense strand are each 18 to 24 nucleotides in length.

14. The RNAi agent according to claim 13, wherein the sense strand and the antisense strand each have a length of 21 nucleotides.

15. The RNAi agent according to claim 14, wherein the RNAi agent has two blunt ends.

16. The RNAi agent according to any one of claims 1 to 15, wherein the sense strand comprises one or two terminal caps.

17. The RNAi agent according to any one of claims 1 to 16, wherein the sense strand comprises one or two reverse debase residues.

18. The RNAi agent according to claim 1, wherein the RNAi agent comprises a sense strand and an antisense strand that form a double helix having one of the double helix structures shown in Table 6A and Table 6B.

19. The RNAi agent according to claim 18, wherein all or substantially all of the nucleotides are modified nucleotides.

20. The following nucleotide sequence (5'→3'): UGAAAGAACUAUUCCAUAAUC (Sequence ID 1608); ACAGAAUCCUGUCUUGUCGUU (Sequence ID 1657); UCCUUUAAAGGUUUUCAGUAG (Sequence ID 1580); or UAUUGAAGCAUUGAGACACCG (Sequence ID 1659) The RNAi agent according to claim 1, comprising an antisense strand consisting of, essentially consisting of, or containing one of the nucleotide sequences that differ from 0 or 1 nucleotide.

21. The RNAi agent according to any one of claims 1 to 20, wherein the nucleotides located at the 2nd and 14th positions from the 5' end of the antisense strand are 2'-fluoromodified nucleotides.

22. The RNAi agent according to claim 21, wherein the nucleotide at position 2 of the antisense strand is 2'-fluorouridine, and the nucleotide at position 14 of the antisense strand is 2'-fluorocytidine, and the antisense strand comprises three or four phosphorothioate nucleoside linkages.

23. The sense strand has the following nucleotide sequence (5'→3'): GAUUAUGGAAUAGUUCUUUCA (Sequence ID 1734); AACGACAAGACAGGAUUCUGU (Sequence ID 1783); CUACUGAAAACCUUUAAAIGA (Sequence ID 1774); or CGGUGUCUCAAUGCUUCAAUA (Sequence ID 1784), An RNAi agent according to any one of claims 1 to 22, comprising, essentially consisting of, or including, a nucleotide sequence in which one of the following is different by 0 or 1 nucleotide.

24. The RNAi agent according to any one of claims 20 to 23, wherein all or substantially all of the nucleotides are modified nucleotides.

25. The following nucleotide sequence (5'→3'): cPrpusGfaaaGfaacuaUfuCfcAfuaausc(SEQ ID NO: 1143); asCfagAfauccugUfcUfuGfucgusu(Sequence ID 1228); isCfagAfauccugUfcUfuGfucgusu(Sequence ID 1229); cPrpusCfscsUfuUfaaaggUfuUfuCfaGfuasg(SEQ ID NO: 1200); or usAfsusugaAfgcauUfgAfgAfcaccsg (Sequence ID 1235), {In the formula, a represents 2'-O-methyladenosine, c represents 2'-O-methylcytidine, g represents 2'-O-methylguanosine, i represents 2'-O-methylinosine; and u represents 2'-O-methyluridine; Af represents 2'-fluoroadenosine, Cf represents 2'-fluorocytidine, Gf represents 2'-fluoroguanosine, and Uf represents 2'-fluorouridine; cPrpu represents 5'-cyclopropylphosphonate-2'-O-methyluridine; and s represents a phosphorothioate linkage} It includes, consists of, or is essentially composed of, an antisense strand containing one of the modified nucleotide sequences and zero or one nucleotide that differs from them; and, The RNAi agent according to claim 1, wherein all or substantially all of the nucleotides of the sense strand are modified nucleotides.

26. The sense strand has the following nucleotide sequence (5'→3'): gauuauggAfAfUfaguucuuuca(Sequence ID 1319); aacgacaaGfAfCfaggauucugu(Sequence ID 1376); cuacugaaAfAfCfcuuuaaaiga (SEQ ID NO: 1361); or cggugucuCfAfAfugcuucaaua (Sequence ID 1377), {In the formula, a represents 2'-O-methyladenosine, c represents 2'-O-methylcytidine, g represents 2'-O-methylguanosine, u represents 2'-O-methyluridine, and i represents 2'-O-methylinosine; Af represents 2'-fluoroadenosine, Cf represents 2'-fluorocytidine, Gf represents 2'-fluoroguanosine, and Uf represents 2'-fluorouridine; s represents a phosphorothioate linkage} A modified nucleotide sequence containing, consisting of, or essentially consisting of, one of the following and zero or one nucleotide differently; and, The RNAi agent according to claim 1, wherein all or substantially all of the nucleotides of the antisense strand are modified nucleotides.

27. The RNAi agent according to any one of claims 20 to 26, wherein the sense strand further comprises a reverse debase residue at the 3' end of the nucleotide sequence, at the 5' end of the nucleotide sequence, or both.

28. The RNAi agent according to any one of claims 1 to 27, wherein the RNAi agent is linked to a targeted ligand.

29. The targeted ligand is as follows: 【Chemistry 1】 An RNAi agent according to any one of claims 1 to 28, comprising:

30. The RNAi agent according to any one of claims 1 to 29, wherein the targeted ligand is linked to the sense strand.

31. The RNAi agent according to claim 30, wherein the targeted ligand is ligated to the 5' end of the sense strand.

32. A composition comprising an RNAi agent according to any one of claims 1 to 31, wherein the composition further comprises a pharmaceutically acceptable excipient.

33. The composition according to claim 32, further comprising a second RNAi agent capable of inhibiting the expression of the MARC1 gene.

34. The composition according to any one of claims 32 to 33, further comprising one or more additional therapeutic agents.

35. The composition according to any one of claims 32 to 34, wherein the composition is formulated for administration.

36. The composition according to claim 35, wherein the composition is delivered by subcutaneous injection.

37. The composition according to any one of claims 32 to 36, wherein the pharmaceutically acceptable excipient is sodium phosphate buffer.

38. The composition according to any one of claims 32 to 36, wherein the pharmaceutically acceptable excipient is isotonic saline or water for injection.

39. A method for inhibiting the expression of the MARC1 gene in hepatocytes, comprising introducing an effective amount of an RNAi agent according to any one of claims 1 to 31 or a composition according to any one of claims 32 to 38 into the target cells.

40. The method according to claim 39, wherein the subject is a human subject.

41. The method according to any one of claims 39 to 40, wherein the MARC1 mRNA level is reduced by at least about 50% in hepatocytes or subjects.

42. The method according to any one of claims 39 to 41, wherein the MARC1 protein level is reduced by at least about 50% in hepatocytes or subjects.

43. A method for treating a MARC1-related disease, disorder, or symptom, comprising administering a therapeutically effective amount of the composition described in any one of claims 32 to 38 to a human subject in need thereof.

44. The method according to claim 43, wherein the disease is non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related diseases.

45. The method according to any one of claims 39 to 44, wherein the level of the serum MARC1 protein is reduced in the subject.

46. The method according to any one of claims 39 to 45, wherein the RNAi agent is administered to a human subject at a dose of approximately 0.05 mg / kg to approximately 5.0 mg / kg per body weight of the human subject.

47. Use of an RNAi agent according to any one of claims 1 to 31 or a composition according to any one of claims 32 to 38 for the treatment of a disease, disorder, or symptom that is at least partially mediated by a reduction in MARC1 gene expression.

48. The use according to claim 47, wherein the disease is non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, autoimmune hepatitis, hepatic fibrosis, cirrhosis, high blood cholesterol levels, hypertriglyceridemia, liver disease, and / or other MARC1-related diseases.

49. Use of an RNAi agent according to any one of claims 1 to 31 or a composition according to any one of claims 32 to 38 for the preparation of a pharmaceutical composition for treating a disease, disorder, or symptom that is at least partially mediated by a reduction in MARC1 gene expression.

50. The use according to any one of claims 47 to 49, wherein the RNAi agent is administered to a human subject at a dose of approximately 0.05 mg / kg to approximately 5.0 mg / kg per body weight of the human subject.

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