Oligonucleotide targeting mtarc1 gene and use thereof

Abstract

The present invention relates to an oligonucleotide targeting an MTARC1 gene and the use thereof. The oligonucleotide can effectively reduce the content of MTARC1 in the body, and is an effective MTARC1 inhibitor.

Description

Oligonucleotides targeting the MTARC1 gene and uses thereof TECHNICAL FIELD

[0001] The present disclosure relates to an oligonucleotide, in particular for the inhibition of mitochondrial amidoxime reducing component 1 (MTARC1) gene expression and for the treatment or prevention of MTARC1 related disorders, conditions and / or pathologies by inhibition of MTARC1. BACKGROUND

[0002] Mitochondrial amidoxime reducing component 1 (MTARC1, other names MARC1 or MOSC1) has molybdenum ion binding activity, molybdopterin cofactor binding activity and oxidoreductase activity, acting as a donor on other nitrogen-containing compounds, which contributes to nitrite reductase (NO formation) activity; it is involved in cellular detoxification of nitrogen compounds, in the nitrate metabolism process and in the nitric oxide biosynthesis process. MARC1 is located in the mitochondria and is part of the nitric oxide synthase complex. Genome-wide association studies (GWAS) identified and validated a common missense variant of mitochondrial amidoxime reducing component 1 (MTARC1) (p.A165T, rs2642438 G>A) that protects from all-cause cirrhosis. The minor A allele of rs2642438 is associated with lower liver fat, liver enzymes, blood cholesterol and reduced risk of NAFLD. A low-frequency coding variant of MTARC1 (p.M187K, rs17850677 T>A) and a rare stop-codon variant (p.R200Ter, rs139321832 C>T) are associated with lower blood cholesterol levels and protection from cirrhosis, suggesting that inhibition of MTARC1 can have therapeutic potential for liver disease. Carriers of the minor A allele have a mild increase in plasma triglycerides (TG) (M. K. Janik, W. Smyk, et al. Scientific Reports 2021 Vol. 11 Issue 1; P. Kalinowski, et al. Int J Mol Sci 2022 Vol. 23 Issue 24).

[0003] The molecular function of MTARC1 in the pathophysiology of NAFLD is not known. MTARC1 is a molybdenum-containing enzyme anchored to the outer mitochondrial membrane, where it associates with cytochrome b5 type B and NADH cytochrome b5 reductase to catalyze the reduction of N-oxo substrates, including nitric oxide and exogenous substances. While hepatic MTARC1 expression is regulated by nutritional stimuli such as glucose deprivation, fasting, and high-fat diet, little is known about the effects of altering MTARC1 levels on metabolism. Deletion of MTARC2, a paralog of MTARC1, reduces glycerolipid formation in adipocytes, and MTARC2 knockout mice are protected from diet-induced obesity and related metabolic disorders. It was found that MTARC1 knockdown showed reduced lipid accumulation in a primary human hepatocyte (PHH) system. In a mouse model of NASH, it was found that hepatocyte-specific MTARC1 knockout reduced steatosis and fibrosis markers. This suggested that inhibition of hepatocyte MTARC1 was sufficient to reduce liver steatosis (L.C. Lewis, et al. JHEP Reports 2023 Vol. 5 Issue 5; Y. Guo, et al. Hepatology Communications 2024 Vol. 8 Issue 5).

[0004] Nonalcoholic fatty liver disease (NAFLD) is a growing health concern, affecting an estimated 25% of the world’s population. Nonalcoholic fatty liver (NAFL) is the first stage of NAFLD, referring to a liver with a fat fraction of more than 5% without identifiable causes such as excessive alcohol consumption. NAFL can progress to nonalcoholic steatohepatitis (NASH), which can further progress to cirrhosis and hepatocellular carcinoma (HCC) (Zhang, Zhang, Wang, et al. Journal of Molecular Diagnostics and Therapeutics, 2024, 16(1)).

[0005] Researchers at DeCODE discovered DNA sequence variants associated with nonalcoholic fatty liver disease (NAFLD), including rare, protective loss-of-function variants pointing to potential drug targets. Plasma proteomic analysis further revealed the pathogenesis of NAFLD. Among the sequence variants discovered in the Icelandic population included rare, protective, predictable loss-of-function variants in MTARC1 and GPAM, suggesting that inhibition of MTARC1 or GPAM or can treat NAFL or NASH (G. Sveinbjornsson, et al. Nature Genetics 2022 Vol. 54).

[0006] Non-alcoholic fatty liver disease is a chronic liver disease associated with obesity, insulin resistance, type 2 diabetes and other metabolic syndromes. MTARC1 can affect lipid metabolism by inactivating GLP-1, neuropeptides, etc. The expression level of MTARC1 in the liver and serum of non-alcoholic fatty liver disease patients is higher than that of healthy people, and the expression of MTARC1 is related to liver damage markers such as γ-glutamyl transpeptidase (γ-GT) and alanine transaminase (ALT), indicating that the liver MTARC1 level of non-alcoholic fatty liver disease patients may be directly related to lipid metabolism and liver damage. MTARC1 inhibitors can repair liver damage and impaired glucose tolerance in non-alcoholic fatty liver disease patients (D.E. Kleiner, et al, Hepatology, 2005, 41; A. Waxler, et al, Gastroenterology, 2007, 132; A. Caligiuri, et al, Int J Mol Sci, 2016, 17).

[0007] In 1998, two American scientists Andrew Fire and Craig Mello discovered a biological mechanism that small interfering RNA molecules (Small interfering RNA; siRNA) can mediate the degradation of specific mRNA (Fire, Andrew, et al. Nature 391.6669 (1998): 806-811). When RNA molecules exist in double-stranded form in cells, this mechanism will be induced to activate, that is, the phenomenon of RNA interference occurs, which indicates the beginning of a new research field. The two scientists also won the Nobel Prize in Physiology or Medicine in 2006. When double-stranded RNA binds to the protein complex Dicer, Dicer will cut dsRNA into fragments, and then another protein complex RISC binds to these fragments. One of the two strands of siRNA double-strand is removed, but the other strand is still combined with the RISC complex. RISC recognizes and degrades the mRNA of the target gene through the guidance of single-stranded RNA, inhibits the expression of specific proteins, and then specifically causes gene silencing.

[0008] RNA interference opens a new field for the application of gene technology. Double-stranded RNA molecules have been artificially designed to silence specific genes in humans, animals or plants. This artificially designed double-stranded small interfering RNA molecule (siRNA) for gene silencing is introduced into cells and activates the RNA interference mechanism to degrade the corresponding mRNA. At present, this method is an important research tool in biology and biomedicine. In addition, a large number of siRNA drugs have been developed to treat viral infections, cardiovascular diseases, cancer, endocrine disorders and other diseases, most of which are in the research and development stage or have been approved for siRNA therapy. Since the first siRNA drug was launched in 2018, at least six siRNAs have been approved for marketing in the European Union or the United States. Therefore, using RNA interference technology to inhibit the expression of specific target genes has become an effective way to treat diseases.

[0009] The asialoglycoprotein receptor (ASGPR) in the liver is a receptor specifically expressed in hepatocytes and is a highly efficient endocytosis receptor. Because various glycoproteins in the body under physiological conditions expose galactose residues after enzymatic or acid hydrolysis of sialic acid, the sugar to which ASGPR specifically binds is galactosyl, so it is also called galactose-specific receptor. Monosaccharides and polysaccharides such as galactose, galactosamine, and N-acetylgalactosamine have high affinity for ASGPR. The main physiological function of ASGPR is to mediate the clearance of asialoglycoproteins, lipoproteins and other substances in the blood, and it is closely related to the occurrence and development of viral hepatitis, cirrhosis, liver cancer and other liver diseases. The discovery of the characteristics of ASGPR plays an important role in the diagnosis and treatment of liver diseases (Ashwell G, Harford J, Carbohydrate specific Receptors of the Liver, Ann Rev Biochem 1982 51:531-554). Liver disease treatment drugs containing galactose or galactosamine and their derivatives in the structure can specifically bind to ASGPR, thereby having active liver targeting without the need for other carrier systems for delivery. SUMMARY

[0010] The present disclosure aims to provide an inhibitor for inhibiting the expression of MTARC1, which has good efficacy, high safety, and long-lasting drug efficacy.

[0011] This disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof and a method for inhibiting MTARC1 gene expression in cells or mammals using the oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide targets the MTARC1 gene. The oligonucleotide is a double-stranded RNA (dsRNA). This document also provides compositions and methods for treating pathological conditions and diseases in mammals caused by MTARC1 gene expression. dsRNA directs the sequence-specific degradation of mRNA through a process called RNA interference (RNAi).

[0012] In one aspect, this disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting MTARC1 expression, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand having at least 80% sequence identity with any sequence or fragment thereof or modified sequence shown in SEQ ID NO. 1-94, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity; the antisense strand having at least 80% sequence identity with any sequence or fragment thereof or modified sequence shown in SEQ ID NO. 95-188, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0013] In another aspect, this disclosure provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting MTARC1 expression, comprising: (i) the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a targeting ligand and / or a lipophilic moiety, wherein at least one of the sense and antisense strands of the oligonucleotide is conjugated to the targeting ligand and / or at least one of the sense and antisense strands of the oligonucleotide is conjugated to one or more of the lipophilic moieties.

[0014] In another aspect, this disclosure provides a composition comprising the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, or the aforementioned conjugate or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0015] On the other hand, this disclosure provides the use of the aforementioned oligonucleotides or pharmaceutically acceptable salts or conjugates thereof in the preparation of medicaments for the treatment and / or prevention of MTARC1-related conditions, symptoms and / or ailments.

[0016] In another aspect, this disclosure provides a method for treating and / or preventing MTARC1-related conditions, symptoms, and / or ailments in a subject by administering a therapeutic agent (e.g., the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, or the aforementioned conjugate or a pharmaceutically acceptable salt thereof, or the aforementioned composition, or a vector or transgene encoding an oligonucleotide) to the subject.

[0017] On the other hand, this disclosure provides methods for treating and / or preventing MTARC1-related conditions, symptoms, and / or ailments in subjects in combination with the aforementioned oligonucleotides or pharmaceutically acceptable salts or conjugates or pharmaceutically acceptable salts or compositions thereof, in combination with other drugs and / or other treatments.

[0018] Inhibiting MTARC1 expression can treat or prevent metabolic conditions associated with body fat distribution, including but not limited to: type II diabetes, hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B or other lipid fractions), hepatic steatosis or fatty liver disease and its complications (such as, for example, cirrhosis, fibrosis or liver inflammation), non-alcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver damage, higher or altered levels or other markers of inflammation or steatosis in the liver, and complications of each of the above conditions.

[0019] Experiments have shown that the oligonucleotides disclosed herein can effectively reduce the MTARC1 content in hepatocytes and are effective inhibitors of MTARC1. Attached Figure Description

[0020] Figure 1 shows the solid-phase synthesis map of siRNA. Detailed Implementation

[0021] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0022] As used herein, the term “about” or “approximately” when applied to one or more target values ​​refers to a value similar to a reference value. In some embodiments, unless otherwise stated or otherwise apparent from the context, the term “approximately” or “about” refers to a range of values ​​falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in any direction (unless such a number would exceed 100% of the possible value).

[0023] As used herein, the term "complementary" refers to a structural relationship between nucleotides (e.g., two nucleotides on opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows the nucleotides to form base pairs with each other. For example, a purine nucleotide complementary to a pyrimidine nucleotide of an opposing nucleic acid can be base-paired together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides may be base-paired in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. In some embodiments, the two nucleic acids may have nucleotide sequences that are complementary to each other to form complementary regions, as described herein.

[0024] As used herein, the term "chain" refers to a single, continuous sequence of nucleotides linked together by internucleotide bonds (e.g., phosphodiester bonds, thiophosphate bonds). In some embodiments, the chain has two free ends, such as a 5'-end and a 3'-end.

[0025] As used herein, the term "deoxyribonucleotide" refers to a nucleotide that has a hydrogen atom at the 2' position of its pentose sugar compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide that has one or more modifications or substitutions (including modifications or substitutions in the sugar, phosphate group, or base) other than at the 2' position.

[0026] As used herein, the term "oligonucleotide" refers to a short nucleic acid, such as a short nucleic acid less than 100 nucleotides in length. Oligonucleotides may comprise ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, for example, modified ribonucleotides. Oligonucleotides may be single-stranded or double-stranded. Oligonucleotides may or may not have a double-stranded region. As a set of non-limiting examples, oligonucleotides may be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (dsiRNA), antisense oligonucleotides, short siRNA, or single-stranded siRNA. In some embodiments, the double-stranded oligonucleotide is an RNAi oligonucleotide.

[0027] As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide that is substantially in a double-stranded form. In some embodiments, complementary base pairings are formed between antiparallel sequences of nucleotides in covalently separated nucleic acid chains, forming one or more double-stranded regions of the double-stranded oligonucleotide. In some embodiments, complementary base pairings are formed between antiparallel sequences of nucleotides in covalently linked nucleic acid chains. In some embodiments, complementary base pairings of one or more double-stranded regions of the double-stranded oligonucleotide are formed from a single nucleic acid chain folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that are base-paired together. In some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are fully double-stranded with each other. However, in some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are partially double-stranded, for example, having overhangs at one or both ends. In some embodiments, the double-stranded oligonucleotide comprises antiparallel sequences of nucleotides that are partially complementary, and therefore may have one or more mismatches, which may include internal mismatches or terminal mismatches.

[0028] As used herein, the term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands with "sense" and "antisense" orientations relative to the target RNA (i.e., the MTARC1 gene). In some embodiments of this disclosure, the double-stranded RNA (dsRNA) triggers the degradation of the target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi. Generally, the majority of nucleotides in each strand of the dsRNA molecule are ribonucleotides, but each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides, as described in detail herein. Additionally, as used herein, "iRNA" may contain chemically modified ribonucleotides; iRNA may contain substantial modifications at multiple nucleotide sites.

[0029] As used herein, the terms “RNAi,” “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” are used interchangeably and refer to an RNA containing the terms defined herein and an agent that mediates targeted cleavage of RNA transcripts via the RNA-inducible silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs the specific degradation of mRNA sequences. RNAi regulates, for example, the inhibition of MTARC1 expression in cells, such as within an individual, such as a mammalian individual.

[0030] As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide bond, or a modified nucleobase, or any combination thereof. Therefore, the term "modified nucleotide" encompasses substitutions, additions, or removals of, for example, functional groups or atoms, of internucleotide bonds, sugar moieties, or nucleobases. Modifications applicable to pharmaceuticals disclosed herein include all types of modifications disclosed herein or known in the art.

[0031] As used herein, “conjugation” refers to the covalent connection between two or more chemical moieties, each with a specific function; correspondingly, “conjugated compound” refers to a compound formed by the covalent connection of these chemical moieties. Further, “siRNA conjugated compound” refers to a compound formed by the covalent attachment of one or more chemical moieties with specific functions to siRNA. In the following text, the siRNA conjugated compounds of this disclosure will sometimes be simply referred to as “conjugated compounds.” The term “siRNA conjugated compound” should be understood, depending on the context, as a general term for siRNA conjugated compounds, including first-type or second-type siRNA conjugated compounds, or siRNA sense strand conjugated compounds or siRNA antisense strand conjugated compounds.

[0032] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of a double-stranded RNAi. For example, a nucleotide overhang exists when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may include an overhang of at least one nucleotide; alternatively, an overhang may include at least two, three, four, five, or more nucleotides. A nucleotide overhang may include or consist of nucleotide / nucleoside analogs comprising deoxynucleotides / nucleosides. The overhang may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide may be present at the 5' end, 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0033] As used in this article, the term "naked sequence" refers to an unmodified nucleotide sequence.

[0034] As used herein, the term “inhibition” is used interchangeably with “knockdown,” “reduction,” “silence,” “downregulation,” “suppression,” and other similar terms, and includes any degree of inhibition.

[0035] As used herein, the term "lipophilic" or "lipophilic fraction" broadly refers to any compound or chemical fraction that has an affinity for lipids.

[0036] The phrase “inhibit MTARC1 expression” is intended to refer to the inhibition of the expression of any MTARC1 gene (e.g., mouse MTARC1 gene, rat MTARC1 gene, monkey MTARC1 gene, or human MTARC1 gene) and variants or mutants of the MTARC1 gene. Therefore, in the context of gene manipulation cells, cell populations, or organisms, the MTARC1 gene can be the wild-type MTARC1 gene, the mutant MTARC1 gene, or the transgenic MTARC1 gene.

[0037] "Suppression of MTARC1 gene expression" includes suppression of the MTARC1 gene at any level, such as at least partial suppression of MTARC1 gene expression. MTARC1 gene expression can be assessed based on the level or changes in the level of any variable associated with MTARC1 gene expression, such as mRNA level or MTARC1 protein level, or indirectly by suppressing the mRNA level of the Gluc and MTARC1 fusion protein gene, thereby suppressing the Gluc protein level.

[0038] The term "pharmaceutically acceptable salt" refers to salts that retain the bioavailability and properties of a free base or acid, and are not biologically or otherwise undesirable. These salts are formed using inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid (especially hydrochloric acid), and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethylsulfonic acid, p-toluenesulfonic acid, salicylic acid, and N-acetylcysteine. Furthermore, these salts can be prepared by adding an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, alkali metal salts (such as sodium, potassium, and lithium salts), ammonium salts, and alkaline earth metal salts (such as calcium and magnesium salts). Salts derived from organic bases include, but are not limited to, salts formed with organic bases such as organic amines: primary amines, secondary amines, and tertiary amines. Substituted amines include naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, and polyamine resins. The oligonucleotides of this disclosure may also exist in zwitterionic form. Particularly preferred pharmaceutical salts of this disclosure are sodium salts, lithium salts, potassium salts, and trialkylammonium salts.

[0039] As used herein, the term "subject" refers to an animal that expresses the target gene endogenously or heterologously, such as a mammal, including primates (e.g., humans, non-human primates such as monkeys and chimpanzees), non-primates (e.g., cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice), or birds. In one embodiment, the subject is a human.

[0040] As used herein, the term "treating" or "treatment" refers to a beneficial or desired outcome, such as a reduction in at least one sign or symptom of MTARC1-related disorder in a subject. Treatment also includes reducing one or more signs or symptoms associated with undesired MTARC1 expression; reducing the degree of undesired MTARC1 activation or stabilization; and improving or mitigating undesired MTARC1 activation or stabilization. Treatment also includes reducing one or more signs or symptoms associated with undesired MTARC1 expression. "Treatment" can also mean prolonged survival compared to expected survival without treatment.

[0041] As used in this article, the terms “prevention” or “preventing” when referring to a disease or condition will benefit from reduced MTARC1 gene expression or MTARC1 protein production.

[0042] As used herein, the term "therapeutic effective amount" is intended to encompass the amount of RNAi agent that, when administered to a subject with MTARC1-related conditions, is sufficient to affect the treatment of the disease (e.g., by reducing, improving, or maintaining existing disease or symptoms of one or more diseases). "Therapeutic effective amount" may vary depending on the RNAi agent, how it is administered, the disease and its severity, as well as medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the subject being treated.

[0043] As used herein, the term "preventive effective dose" is intended to encompass the amount of RNAi agent sufficient to prevent or improve the condition or one or more symptoms of the condition when administered to a subject with MTARC1-related disease. Improving the disease includes slowing its progression or reducing the severity of later-stage disease. The "preventive effective dose" may vary depending on the RNAi agent, how it is administered, the degree of disease risk, and factors such as medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0044] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium stearate, zinc stearate, or stearic acid), or solvent encapsulation material (involving the carrying or delivery of a subject compound from one organ or part of the body to another organ or part of the body). Each carrier must be "acceptable" in the sense that it is compatible with the other components of the formulation and harmless to the treated subject. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers intended for administration by injection.

[0045] This level can be assessed in individual cells or cell populations, including, for example, samples from subjects. It is understood that MTARC1 is primarily expressed in fat, but is also expressed in organs or viscera such as the liver, thyroid, and pancreas, and is present in circulation.

[0046] Inhibition can be assessed by a reduction in the absolute or relative level of one or more variables associated with MTARC1 expression compared to a control level. The control level can be any type of control level used in the art, such as baseline levels before administration, or levels determined from similar subjects who were untreated or treated with a control (e.g., a buffer-only control or an inactive agent control).

[0047] In one aspect, this disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting MTARC1 expression, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand having at least 80% sequence identity with any sequence or fragment thereof or modified sequence shown in SEQ ID NO. 1-94, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity; the antisense strand having at least 80% sequence identity with any sequence or fragment thereof or modified sequence shown in SEQ ID NO. 95-188, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0048] In some implementations, the sense strand and / or antisense strand are 15-30, 17-25, or 19-27 nucleotides in length.

[0049] In some implementations, each chain is independently 15-30, 17-25, or 19-27 nucleotides in length.

[0050] In some implementations, each chain is independently 19 to 25 nucleotides in length.

[0051] In some implementations, the antisense strand is 19 to 23 nucleotides in length.

[0052] In some implementations, the length of the positive chain is 19 to 23 nucleotides.

[0053] In some embodiments, the oligonucleotide comprises a 5' and / or 3'-overhang sequence of one or more nucleotides in length, wherein the 5' and / or 3'-overhang sequence is present on the antisense strand and / or the sense strand. In one embodiment, the antisense strand of the oligonucleotide has one to ten nucleotides at the 3' or 5' overhang, for example, one, two, three, four, five, six, seven, eight, nine, or ten nucleotides. In one embodiment, the sense strand of the dsRNA has one to ten nucleotides at the 3' or 5' overhang, for example, one, two, three, four, five, six, seven, eight, nine, or ten nucleotides. In another embodiment, one or more nucleotides in the overhang are replaced by a nucleoside thiophosphate.

[0054] In some implementations, the antisense chain has one or two protruding ends.

[0055] In some implementations, the justice chain has one or two protruding ends.

[0056] In some implementations, the oligonucleotide comprises a 3'-overhang sequence of one or two nucleotides in length.

[0057] In some implementations, the oligonucleotide comprises a 5'-protrusion sequence of one or two nucleotides in length.

[0058] In some implementations, the 3'-protruding sequence is present on the antisense strand. In some implementations, the protruding sequence is selected from: AA, AG, AU, C, CA, CC, CG, CU, G, GA, GC, GG, GU, U, UA, UC, UG, UU.

[0059] In some implementations, the 5'-protrusion sequence is present on the antisense strand. In some implementations, the protrusion sequence is selected from A and G.

[0060] In some implementations, the oligonucleotide comprises an antisense strand and a sense strand, each ranging in length from 19 to 23 nucleotides.

[0061] In some implementations, the justice chain and the antisense chain form a dual-chain region.

[0062] In some implementations, the justice chain and the antisense chain are bichain structures with 19 / 21 pairing, 21 / 21 pairing, 21 / 23 pairing, or 23 / 23 pairing, respectively.

[0063] In some embodiments, the oligonucleotide comprises a 5' overhang and a 3' overhang sequence of length 1 nucleotide, wherein the 5' overhang and the 3' overhang sequence are present on the antisense strand, and wherein the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long, such that the sense strand and the antisense strand form a double helix of length 19 nucleotides.

[0064] In some embodiments, the oligonucleotide includes a 3'-overhang sequence of 2 nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a double helix of 19 nucleotides in length.

[0065] In some embodiments, the oligonucleotide includes a 3'-overhang sequence of 2 nucleotides in length, wherein the 3'-overhang sequence is present on both the antisense and sense strands, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense and antisense strands form a doublet of 19 nucleotides in length.

[0066] In some embodiments, the oligonucleotide includes a 3'-overhang sequence of 2 nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a double helix of 21 nucleotides in length.

[0067] In some embodiments, the oligonucleotide includes a 3'-overhang sequence of 2 nucleotides in length, wherein the 3'-overhang sequence is present on both the antisense and sense strands, and wherein the sense strand is 23 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense and antisense strands form a doublet of 21 nucleotides in length.

[0068] In some embodiments, pharmaceutically acceptable salts of oligonucleotides can be prepared by adding an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, alkali metal salts (such as sodium, potassium, and lithium salts), ammonium salts, and alkaline earth metal salts (such as calcium and magnesium salts). Salts derived from organic bases (e.g., organic amines) include, but are not limited to, salts formed with the following organic bases: primary amines, secondary amines, and tertiary amines; substituted amines include naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, and polyamine resins.

[0069] In some embodiments, pharmaceutically acceptable salts of oligonucleotides include, but are not limited to, ammonium salts, such as salts of tertiary alkylamine compounds (e.g., triethylamine salts), metal salts such as sodium, potassium, and magnesium salts, etc.

[0070] In some implementations, the oligonucleotide or its salt may be in the form of a hydrate or a solvate.

[0071] In some implementations, the oligonucleotide contains at least one modified nucleotide.

[0072] In some implementations, the oligonucleotide contains at least one 2'-modified nucleotide.

[0073] In some embodiments, the 2'-modified nucleotide is selected from one or more of the following: 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-amide-modified nucleotides, 2'-substituted amide-modified nucleotides, and 2'-deoxynucleotides.

[0074] In some embodiments, the sense strand comprises an unmodified oligonucleotide selected from any of SEQ ID NO. 2, 6, 17, 23, 24, 30, 34, 35, 37, 42, 70, 71, 74, 84, 85, 86, 87, 90, 91, and 93, or a modified oligonucleotide selected from any of SEQ ID NO. 190, 194, 205, 211, 212, 218, 222, 223, 225, 230, 258, 259, 262, 272, 273, 274, 275, 278, 279, and 281; the antisense strand comprises a oligonucleotide selected from SEQ ID NO. The unmodified oligonucleotides described in any of SEQ ID NOs 96, 100, 111, 117, 118, 124, 128, 129, 131, 136, 164, 165, 168, 178, 179, 180, 181, 184, 185, and 187, or the modified oligonucleotides described in any of SEQ ID NOs 284, 288, 299, 305, 306, 312, 316, 317, 319, 324, 352, 353, 356, 366, 367, 368, 369, 372, 373, and 375.

[0075] In some implementations, the oligonucleotide comprises any of the following combinations of sense and antisense strands:

[0076] (1) The positive chain comprises the sequence shown in SEQ ID NO.2, and the negative chain comprises the sequence shown in SEQ ID NO.96;

[0077] (2) The sense chain comprises the sequence shown in SEQ ID NO.6, and the antisense chain comprises the sequence shown in SEQ ID NO.100;

[0078] (3) The positive chain contains the sequence shown in SEQ ID NO.17, and the negative chain contains the sequence shown in SEQ ID NO.111;

[0079] (4) The sense chain comprises the sequence shown in SEQ ID NO.23, and the antisense chain comprises the sequence shown in SEQ ID NO.117;

[0080] (5) The sense chain comprises the sequence shown in SEQ ID NO.24, and the antisense chain comprises the sequence shown in SEQ ID NO.118;

[0081] (6) The sense chain comprises the sequence shown in SEQ ID NO.30, and the antisense chain comprises the sequence shown in SEQ ID NO.124;

[0082] (7) The sense chain comprises the sequence shown in SEQ ID NO.34, and the antisense chain comprises the sequence shown in SEQ ID NO.128;

[0083] (8) The sense chain comprises the sequence shown in SEQ ID NO.35, and the antisense chain comprises the sequence shown in SEQ ID NO.129;

[0084] (9) The positive chain comprises the sequence shown in SEQ ID NO.37, and the negative chain comprises the sequence shown in SEQ ID NO.131;

[0085] (10) The sense chain comprises the sequence shown in SEQ ID NO.42, and the antisense chain comprises the sequence shown in SEQ ID NO.136;

[0086] (11) The sense chain comprises the sequence shown in SEQ ID NO.70, and the antisense chain comprises the sequence shown in SEQ ID NO.164;

[0087] (12) The sense chain comprises the sequence shown in SEQ ID NO.71, and the antisense chain comprises the sequence shown in SEQ ID NO.165;

[0088] (13) The positive chain comprises the sequence shown in SEQ ID NO.74, and the negative chain comprises the sequence shown in SEQ ID NO.168;

[0089] (14) The positive chain contains the sequence shown in SEQ ID NO.84, and the negative chain contains the sequence shown in SEQ ID NO.178;

[0090] (15) The positive chain comprises the sequence shown in SEQ ID NO.85, and the negative chain comprises the sequence shown in SEQ ID NO.179;

[0091] (16) The sense chain comprises the sequence shown in SEQ ID NO.86, and the antisense chain comprises the sequence shown in SEQ ID NO.180;

[0092] (17) The sense chain comprises the sequence shown in SEQ ID NO.87, and the antisense chain comprises the sequence shown in SEQ ID NO.181;

[0093] (18) The sense chain comprises the sequence shown in SEQ ID NO.90, and the antisense chain comprises the sequence shown in SEQ ID NO.184;

[0094] (19) The sense chain comprises the sequence shown in SEQ ID NO. 91, and the antisense chain comprises the sequence shown in SEQ ID NO. 185; and

[0095] (20) The positive chain contains the sequence shown in SEQ ID NO.93, and the negative chain contains the sequence shown in SEQ ID NO.187.

[0096] In some implementations, each chain is independently 19 to 30 nucleotides in length.

[0097] In some preferred embodiments, the oligonucleotide comprises any of the following combinations of sense and antisense strands:

[0098] (1) The positive chain comprises the sequence shown in SEQ ID NO.2, and the negative chain comprises the sequence shown in SEQ ID NO.96;

[0099] (2) The sense chain comprises the sequence shown in SEQ ID NO.6, and the antisense chain comprises the sequence shown in SEQ ID NO.100;

[0100] (3) The positive chain contains the sequence shown in SEQ ID NO.17, and the negative chain contains the sequence shown in SEQ ID NO.111;

[0101] (4) The sense chain comprises the sequence shown in SEQ ID NO.23, and the antisense chain comprises the sequence shown in SEQ ID NO.117;

[0102] (5) The sense chain comprises the sequence shown in SEQ ID NO.24, and the antisense chain comprises the sequence shown in SEQ ID NO.118;

[0103] (6) The sense chain comprises the sequence shown in SEQ ID NO.30, and the antisense chain comprises the sequence shown in SEQ ID NO.124;

[0104] (7) The sense chain comprises the sequence shown in SEQ ID NO.34, and the antisense chain comprises the sequence shown in SEQ ID NO.128;

[0105] (8) The sense chain comprises the sequence shown in SEQ ID NO.35, and the antisense chain comprises the sequence shown in SEQ ID NO.129;

[0106] (9) The positive chain comprises the sequence shown in SEQ ID NO.37, and the negative chain comprises the sequence shown in SEQ ID NO.131;

[0107] (10) The sense chain comprises the sequence shown in SEQ ID NO.74, and the antisense chain comprises the sequence shown in SEQ ID NO.168;

[0108] (11) The positive chain comprises the sequence shown in SEQ ID NO.84, and the negative chain comprises the sequence shown in SEQ ID NO.178;

[0109] (12) The sense chain comprises the sequence shown in SEQ ID NO.85, and the antisense chain comprises the sequence shown in SEQ ID NO.179;

[0110] (13) The sense chain comprises the sequence shown in SEQ ID NO. 90, and the antisense chain comprises the sequence shown in SEQ ID NO. 184; and

[0111] (14) The positive chain contains the sequence shown in SEQ ID NO.91, and the negative chain contains the sequence shown in SEQ ID NO.185.

[0112] In some implementations, each chain is independently 19 to 25 nucleotides in length.

[0113] In some implementations, the oligonucleotide contains at least one modified nucleotide.

[0114] In some embodiments, at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, 2'-5'-linked ribonucleotides (3'-RNA), unlocked nucleotides, conformation-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, and 2'-O-alkyl modified nucleotides. Nucleotides modified with 2'-hydroxyl, 2'-methoxyethyl, 2'-O-alkyl, morpholinonucleotides, aminophosphates, nucleotides including non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides including thiophosphate groups, nucleotides including methylphosphonate groups, nucleotides including 5'-phosphates, nucleotides including 5'-phosphate mimics, vinyl-phosphonate nucleotides, heat-labile nucleotides, glycol-modified nucleotides, nucleotides including 2'-phosphates and 2-O-(N-methylacetamide)-modified nucleotides; and combinations thereof.

[0115] In some embodiments, at least one of the modified nucleotides is selected from the group consisting of: LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxy and ethylene glycol; and combinations thereof.

[0116] In some embodiments, at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, ethylene glycol modified nucleotides (GNA), nucleotides comprising 2'-phosphate esters, and nucleotides comprising thiophosphate ester groups; and combinations thereof.

[0117] In some implementations, the oligonucleotide contains at least one 2'-modified nucleotide.

[0118] In some embodiments, the 2'-modified nucleotide is selected from the group consisting of: 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-fluorine-modified nucleotides, 2'-acylamino-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-deoxynucleotides, nucleotides including 2'-phosphate esters, and nucleotides modified with 2'-O-(N-methylacetamide); and combinations thereof. Examples include C1-C3 alkoxy groups (e.g., methoxy); substituted alkoxy groups (e.g., C1-C3 alkoxy-substituted C1-C3 alkoxy groups, such as methoxyethoxy); alkyl groups (e.g., C1-C3 alkyl groups, such as methyl); substituted alkyl groups (e.g., C1-C3 alkoxy-substituted C1-C3 alkyl groups, such as methoxymethyl, methoxyethyl); amino groups (-NH2); substituted amino groups (e.g., C1-C3 alkyl mono- or di-substituted amino groups, such as methylamino, ethylamino), but not limited thereto.

[0119] In some embodiments, the 2'-modified nucleotide is selected from the group consisting of: 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-amide-modified nucleotides, 2'-substituted amide-modified nucleotides, 2'-deoxynucleotides; and combinations thereof.

[0120] In some embodiments, the 2'-modification is selected from the group consisting of 2'-methoxy, 2'-acetamido, 2'-aminoethyl, 2'-fluorine, 2'-O-methyl, and 2'-O-methoxyethyl; and combinations thereof.

[0121] In some implementations, the 2'-modification is a 2'-methoxy modification.

[0122] In some implementations, the 2'-modification is a 2'-acetamide group modification.

[0123] In some implementations, all nucleotides of the oligonucleotide are modified.

[0124] In some implementations, the oligonucleotide may contain glycol nucleic acid (GNA) modifications.

[0125] In some implementations, the diol nucleic acid (GNA) modification is selected from adenosine-diol nucleic acid, cytidine-diol nucleic acid, thymidine-diol nucleic acid, and guanosine-diol nucleic acid.

[0126] In some implementations, the GNA modification is selected from thymidine-diol nucleic acid S-isomer (Tgn), cytidine-diol nucleic acid S-isomer (Cgn), adenosine-diol nucleic acid S-isomer (Agn), and guanosine-diol nucleic acid S-isomer (Ggn);

[0127] The structural formulas for Tgn, Cgn, Agn, and Ggn are as follows:

[0128] In some implementations, the aforementioned oligonucleotide may contain a 2'-5'-phosphodiester bond.

[0129] In some implementations, the oligonucleotide has a 5'-phosphate analog modified nucleotide at its 5' end.

[0130] In some embodiments, the 5'-phosphate analog modified nucleotide has a vinylphosphonate modified nucleotide as shown in formula (I), wherein R is selected from H, fluorine, 2'-methoxy, 2'-acetamido, 2'-aminoethyl and 2'-O-methoxyethyl, and Base represents a natural or modified nucleic acid base, preferably selected from A, G, C, T and U.

[0131] In some embodiments, the 5'-phosphate analog modified nucleotide has a vinyl phosphate modified nucleotide as shown in formula (II), wherein R is selected from H, OH, fluorine, 2'-methoxy, 2'-acetamido, 2'-aminoethyl and 2'-O-methoxyethyl.

[0132] In some embodiments, the 5'-phosphonate analog-modified nucleotide is APU as shown in formula (III) or VPUm as shown in formula (IV);

[0133] In some embodiments, the modification is selected from the following: 5'-phosphate analog modification, 2'-methoxy (CH3O-, m), 2'-fluorine (f), 2'-acetamido (CH3CO-NH-), and thiophosphate (s).

[0134] In some embodiments, the oligonucleotide comprises formula M, which is 2'-O-methyl-6-(3-(2-carboxyethyl)phenyl)-purine nucleotide as shown in formula (V);

[0135] In some embodiments, the oligonucleotide comprises uridine-2'-phosphate (U-2'5') as shown in formula (VI), guanosine-2'-phosphate (G-2'5') as shown in formula (VII), cytidine-2'-phosphate (C-2'5') as shown in formula (VIII), adenosine-2'-phosphate (A-2'5') as shown in formula (IX), and thymidine-2'-phosphate (T-2'5') as shown in formula (X).

[0136] In some implementations, the oligonucleotide contains at least one modified nucleotide inter-bond.

[0137] In some embodiments, at least one modified nucleotide inter-bond is a phosphate-thioester bond. Phospho-thioester inter-bond modification can occur at any position on any nucleotide of the sense strand, antisense strand, or both strands. For example, the inter-bond modification can occur on each nucleotide of the sense strand or antisense strand; each inter-bond modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand can contain two inter-bond modifications in an alternating pattern. The alternating pattern of the inter-bond modification on the sense strand can be the same as or different from that on the antisense strand, and the alternating pattern of the inter-bond modification on the sense strand can be offset relative to the alternating pattern of the inter-bond on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6 to 8 phosphate-thioester inter-bonds. In some embodiments, the antisense strand comprises two phosphate-thioester inter-bonds at the 5' end and two phosphate-thioester inter-bonds at the 3' end, and the sense strand comprises at least two phosphate-thioester inter-bonds at either the 5' or 3' end.

[0138] In some implementations, the oligonucleotide comprises any of the following combinations of sense and antisense strands:

[0139] (1) The sense chain comprises the sequence shown in SEQ ID NO.190, and the antisense chain comprises the sequence shown in SEQ ID NO.284;

[0140] (2) The sense chain comprises the sequence shown in SEQ ID NO.194, and the antisense chain comprises the sequence shown in SEQ ID NO.288;

[0141] (3) The sense chain comprises the sequence shown in SEQ ID NO.205, and the antisense chain comprises the sequence shown in SEQ ID NO.299;

[0142] (4) The sense chain comprises the sequence shown in SEQ ID NO.211, and the antisense chain comprises the sequence shown in SEQ ID NO.305;

[0143] (5) The sense chain comprises the sequence shown in SEQ ID NO.212, and the antisense chain comprises the sequence shown in SEQ ID NO.306;

[0144] (6) The sense chain comprises the sequence shown in SEQ ID NO.218, and the antisense chain comprises the sequence shown in SEQ ID NO.312;

[0145] (7) The sense chain comprises the sequence shown in SEQ ID NO.222, and the antisense chain comprises the sequence shown in SEQ ID NO.316;

[0146] (8) The sense chain comprises the sequence shown in SEQ ID NO.223, and the antisense chain comprises the sequence shown in SEQ ID NO.317;

[0147] (9) The sense chain comprises the sequence shown in SEQ ID NO.225, and the antisense chain comprises the sequence shown in SEQ ID NO.319;

[0148] (10) The sense chain comprises the sequence shown in SEQ ID NO.230, and the antisense chain comprises the sequence shown in SEQ ID NO.324;

[0149] (11) The sense chain comprises the sequence shown in SEQ ID NO.258, and the antisense chain comprises the sequence shown in SEQ ID NO.352;

[0150] (12) The sense chain comprises the sequence shown in SEQ ID NO.259, and the antisense chain comprises the sequence shown in SEQ ID NO.353;

[0151] (13) The sense chain comprises the sequence shown in SEQ ID NO.262, and the antisense chain comprises the sequence shown in SEQ ID NO.356;

[0152] (14) The sense chain comprises the sequence shown in SEQ ID NO.272, and the antisense chain comprises the sequence shown in SEQ ID NO.366;

[0153] (15) The sense chain comprises the sequence shown in SEQ ID NO.273, and the antisense chain comprises the sequence shown in SEQ ID NO.367;

[0154] (16) The sense chain comprises the sequence shown in SEQ ID NO.274, and the antisense chain comprises the sequence shown in SEQ ID NO.368;

[0155] (17) The sense chain comprises the sequence shown in SEQ ID NO.275, and the antisense chain comprises the sequence shown in SEQ ID NO.369;

[0156] (18) The sense chain comprises the sequence shown in SEQ ID NO.278, and the antisense chain comprises the sequence shown in SEQ ID NO.372;

[0157] (19) The sense chain comprises the sequence shown in SEQ ID NO. 279, and the antisense chain comprises the sequence shown in SEQ ID NO. 373; and

[0158] (20) The sense chain comprises the sequence shown in SEQ ID NO.281, and the antisense chain comprises the sequence shown in SEQ ID NO.375;

[0159] Each chain is independently 19 to 30 nucleotides in length.

[0160] In some preferred embodiments, the oligonucleotide comprises any of the following combinations of sense and antisense strands:

[0161] (1) The sense chain comprises the sequence shown in SEQ ID NO.190, and the antisense chain comprises the sequence shown in SEQ ID NO.284;

[0162] (2) The sense chain comprises the sequence shown in SEQ ID NO.194, and the antisense chain comprises the sequence shown in SEQ ID NO.288;

[0163] (3) The sense chain comprises the sequence shown in SEQ ID NO.205, and the antisense chain comprises the sequence shown in SEQ ID NO.299;

[0164] (4) The sense chain comprises the sequence shown in SEQ ID NO.211, and the antisense chain comprises the sequence shown in SEQ ID NO.305;

[0165] (5) The sense chain comprises the sequence shown in SEQ ID NO.212, and the antisense chain comprises the sequence shown in SEQ ID NO.306;

[0166] (6) The sense chain comprises the sequence shown in SEQ ID NO.218, and the antisense chain comprises the sequence shown in SEQ ID NO.312;

[0167] (7) The sense chain comprises the sequence shown in SEQ ID NO.222, and the antisense chain comprises the sequence shown in SEQ ID NO.316;

[0168] (8) The sense chain comprises the sequence shown in SEQ ID NO.223, and the antisense chain comprises the sequence shown in SEQ ID NO.317;

[0169] (9) The sense chain comprises the sequence shown in SEQ ID NO.225, and the antisense chain comprises the sequence shown in SEQ ID NO.319;

[0170] (10) The sense chain comprises the sequence shown in SEQ ID NO.262, and the antisense chain comprises the sequence shown in SEQ ID NO.356;

[0171] (11) The sense chain comprises the sequence shown in SEQ ID NO.272, and the antisense chain comprises the sequence shown in SEQ ID NO.366;

[0172] (12) The positive chain comprises the sequence shown in SEQ ID NO.273, and the negative chain comprises the sequence shown in SEQ ID NO.367;

[0173] (13) The sense chain comprises the sequence shown in SEQ ID NO. 278, and the antisense chain comprises the sequence shown in SEQ ID NO. 372; and

[0174] (14) The positive chain contains the sequence shown in SEQ ID NO.279, and the negative chain contains the sequence shown in SEQ ID NO.373.

[0175] In another aspect, this disclosure provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting MTARC1 expression, comprising: (i) the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a ligand conjugated to the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more target ligands. The conjugation of at least one nucleotide of the oligonucleotide to one or more target ligands forms an siRNA conjugate. The aforementioned siRNA conjugate contains the aforementioned siRNA and a conjugating group conjugated to the siRNA. The term "oligonucleotide salt" refers to an oligonucleotide compound in salt form. Oligonucleotide salts include salts of oligonucleotide conjugated compounds and salts of unconjugated oligonucleotide compounds. Oligonucleotide salts are advantageously present in solid powder form.

[0176] In some implementations, the target ligand is conjugated at the 3' or 5' end of the positive-angle strand.

[0177] Generally, the aforementioned conjugation group comprises at least one pharmaceutically acceptable targeting ligand and an optional linker, and the aforementioned siRNA, the aforementioned linker, and the aforementioned targeting ligand are sequentially linked. The targeting group can be a ligand conventionally used in the field of siRNA drug delivery, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference. In some embodiments, there are 2-4 aforementioned targeting ligands. The aforementioned siRNA molecule can be non-covalently or covalently conjugated to the aforementioned conjugation group, for example, it can be covalently conjugated to the aforementioned conjugation group. The conjugation site of the siRNA to the conjugation group can be at the 3' end and / or 5' end of the siRNA's sense strand and / or antisense strand, or it can be within the siRNA's internal sequence. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3' end or 5' end of the siRNA's sense strand. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3' end or 5' end of the siRNA's antisense strand. In some preferred embodiments, the conjugation site of the aforementioned siRNA and the conjugating group is located at the 3' end of the siRNA's positive strand.

[0178] In some embodiments, the targeting ligand comprises a desialyl glycoprotein receptor ligand. In some embodiments, the desialyl glycoprotein receptor ligand comprises or is composed of one or more galactose derivatives. As used herein, the term "galactose derivative" includes galactose and lactose derivatives with an affinity for the desialyl glycoprotein receptor equal to or greater than that for galactose. Galactose derivatives include, but are not limited to, galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-butyryl-galactosamine, and N-isobutyryl-galactosamine. Galactose derivatives and clusters of galactose derivatives that can be used to target the liver in vivo with oligonucleotides and other molecules are known in the art. Galactose derivatives have been used to target molecules to hepatocytes in vivo by binding to the desialyl glycoprotein receptor (ASGPr) expressed on the surface of hepatocytes. Binding of ASGPr ligands to ASGPr(s) facilitates cell-specific targeting of hepatocytes and the entry of endocytic molecules into hepatocytes. ASGPr ligands can be monomers (e.g., having a single galactose derivative) or polymers (e.g., having multiple galactose derivatives). Galactose derivatives or clusters of galactose derivatives can be linked to the 3' or 5' end of the siRNA using methods known in the art.

[0179] In some embodiments, the pharmaceutically acceptable targeting ligand in the aforementioned siRNA conjugate can be galactose or N-acetylgalactosamine (GalNAc), wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent meanings refer to the molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in the siRNA conjugate being 1:1, 1:2, 1:3, or 1:4, respectively, after the siRNA molecule forms a conjugate with a conjugate group containing galactose or N-acetylgalactosamine as a targeting ligand. In some embodiments, the pharmaceutically acceptable targeting ligand is N-acetylgalactosamine. In some embodiments, when the siRNA of the present invention is conjugated with a conjugate group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA of the present invention is conjugated with a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0180] In some embodiments of this disclosure, the aforementioned targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0181] In some embodiments of this disclosure, the aforementioned GalNAc portion is a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion, or a tetravalent GalNAc portion.

[0182] In some embodiments of this disclosure, the targeting ligand includes one to four 2'-O-alkyl modifications. Optionally, the 2'-O-alkyl modified nucleotide is a 2'-C16-modified nucleotide. Optionally, the RNAi agent includes a single 2'-O-C16-modified nucleotide. Optionally, the single 2'-C16-modified nucleotide is located at the sixth nucleotide position at the 5' end of the positive strand, or at the 3' or 5' terminal nucleotide position of the positive strand.

[0183] The aforementioned targeting ligand is L96.

[0184] This disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting MTARC1 expression, comprising: (i) the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) one or more lipophilic moieties conjugated to one or more internal positions on at least one chain and / or one or more lipophilic moieties conjugated to the 3' and / or 5' ends on at least one chain, optionally via a linker or vector.

[0185] In some embodiments of this disclosure, another modification of the RNAi agent chemically links one or more ligands, portions or conjugates that enhance RNAi activity, cellular distribution or cellular uptake to the RNA. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553-6556), bile acids (Manohara et al., (1994) Biorg. Med. Chem. Let., 4: 1053-1060), thioethers, such as beryl-S-triphenylmethylthiol (Manoharan et al., (1992) Ann. NY Acad. Sci., 660: 306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3: 2765-2770), sulfur cholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20: 533-538), and fatty acid chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett, 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), phospholipids, such as di-hexadecyl-racemic-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-phosphate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), palmitic moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or octadecylamine or hexylamino-carbonyl-oxocholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0186] In some embodiments, the lipophilic moiety is an aliphatic, cyclic (e.g., alicyclic) or polycyclic (e.g., polyalicyclic) compound, such as a steroid (e.g., a sterol) or a straight-chain or branched aliphatic hydrocarbon. The lipophilic moiety may typically comprise a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, such as oxygen or nitrogen atoms. Such lipophilic aliphatic moietyes include, but are not limited to, saturated or unsaturated C4-C... 30 Hydrocarbons (e.g., C6-C) 22 Hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C464 ... 10 Terpenes, C 15 Sesquiterpenes, C 20 Diterpenes, C 30 Triterpenes and C 40 Tetraterpenes and other polycyclic hydrocarbons. For example, the lipophilic moiety can contain C4 to C5. 30 Hydrocarbon chains (e.g., C4 to C5) 30 Alkyl or alkenyl). In some embodiments, the lipophilic moiety comprises saturated or unsaturated C6 to C6 groups. 18 Hydrocarbon chains (e.g., straight-chain C6 to C15) 22 (alkyl or alkenyl). In one embodiment, the lipophilic moiety comprises saturated or unsaturated C. 16 Hydrocarbon chains (e.g., straight-chain C) 16 Alkyl or alkenyl). The lipid moiety is 2'-O-alkyl, consisting of 10-30 hydrocarbon chains, including single and / or branched chains.

[0187] In another embodiment, the lipophilic portion comprises lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytriphenylmethyl, or phenoxazine.

[0188] The lipophilic moiety is bound to the double-stranded RNAi agent via a linker, which may contain ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphate diester, sulfonamide bond, click reaction (e.g., triazole from azide-alkynyl cycloaddition), or carbamate.

[0189] In some embodiments of this disclosure, the aforementioned ligands that enhance RNAi activity, cell distribution, or cell uptake comprise N-acetylgalactosamine (GalNAc) and / or C... 12 Alkyl, C 18 Alkyl, C 22Alkyl or branched lipids such as DDA (cationic dimethyl dioctadecyl ammonium) and TDB (trehalose 6,6,9-disorbate).

[0190] The straight-chain lipophilic portion (C16) attached to one position on the chain has the following structure:

[0191] Base is a nucleotide base or a nucleotide base analogue, optionally, wherein Base is selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

[0192] For example, 2'-O-hexadecyluridine has the following structure:

[0193] This disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting MTARC1 expression, wherein the lipid conjugate has the following structure:

[0194] A is a modified double-stranded oligonucleotide or a modified single-stranded oligonucleotide, wherein the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide is conjugated to a lipid-containing moiety at the 3' end of one strand of the modified double-stranded oligonucleotide or the 3' end of the modified single-stranded nucleic acid.

[0195] X1 is:

[0196] L1 is -(CH2)n-, -(CH2) n L2(CH2) n - or key;

[0197] L2 is -C(=O)NH-, -C(=O)O-, -OC(=O)O-, -NHC(=O)O-, -NHC(=O)NH-, -C(=S)NH-, -C(=O)S-, -NH-, O(oxygen)

[0198] Or S (sulfur),

[0199] Each m is an integer from 10 to 18, and each n is an integer from 1 to 6.

[0200] For example, DTX-1

[0201] This disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting MTARC1 expression, comprising: (i) the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a targeting ligand and a lipophilic moiety, wherein at least one of the sense strand and antisense strand of the oligonucleotide is conjugated to the targeting ligand, and at least one of the sense strand and antisense strand of the oligonucleotide is conjugated to one or more lipophilic moieties.

[0202] In another aspect, this disclosure provides a composition comprising the aforementioned oligonucleotide or a pharmaceutically acceptable salt or conjugate thereof, and optionally a pharmaceutically acceptable carrier.

[0203] In some embodiments, the composition is in the form of an oral, intravenous, subcutaneous, or intramuscular injection, preferably a subcutaneous injection.

[0204] In some embodiments, the composition also includes other medicines for treating and / or preventing MTARC1-related conditions.

[0205] On the other hand, this disclosure provides the use of the aforementioned oligonucleotides or pharmaceutically acceptable salts, conjugates or compositions thereof in the preparation of medicaments for the treatment and / or prevention of MTARC1-related conditions.

[0206] In some implementations, MTARC1-related diseases, conditions, and / or symptoms are selected from the group consisting of: liver diseases (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDL-C, low HDL-C, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance, diabetes), cardiovascular diseases (e.g., hypertension, endothelial cell dysfunction), kidney diseases (e.g., acute kidney disease, renal tubular dysfunction, pro-inflammatory changes in the proximal tubules), metabolic syndrome, adipocyte dysfunction, visceral fat deposition, obesity, hyperuricemia, gout, eating disorders, and excessive sugar cravings; and combinations thereof.

[0207] In some embodiments of the method disclosed herein, the expression of the MTARC1 gene is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the assay detection level. In a preferred embodiment, the expression of MTARC1 is inhibited by at least 70%. It should also be understood that it may be desirable to inhibit the expression of MTARC1 in certain tissues (e.g., liver) without significantly inhibiting its expression in other tissues (e.g., brain). In a preferred embodiment, the expression level is determined in a suitable species-matched cell line using the assay method provided in Example 2 with a concentration of 0.5 nM siRNA.

[0208] In some implementations, inhibition of in vivo expression is determined by knocking down the human gene group in rodents expressing human genes, for example, in AAV-infected mice expressing the human target gene (i.e., MTARC1), for example, by confirming the inhibitory effect on the human gene when administered as a single dose, for example, by the point of lowest MTARC1 expression after a subcutaneous injection of 3 mg / kg. Such systems are useful when the nucleic acid sequences of the human gene and the model animal gene are sufficiently similar so that human RNAi provides effective knockdown of the model animal gene.

[0209] Inhibition of MTARC1 gene expression can be represented by a reduction in the amount of mRNA expressed in a cell line (such cells may be present, for example, in a sample derived from a subject), in which the MTARC1 gene has been transcribed and treated (e.g., by contacting one or more cells with the RNAi of this disclosure, or by administering the RNAi of this disclosure to a subject in which cells are present or were previously present), such that MTARC1 gene expression is inhibited compared to a substantially identical cell line that has not been treated (control cells not treated with RNAi or not treated with RNAi targeting the target gene). In a preferred embodiment, inhibition is assessed in a species-matched cell line using a 0.5 nM siRNA concentration as described in Example 2, expressed using the following formula: ΔCT = CT / 2^(-ΔΔCT) of mRNA expression level in the treated cells relative to the mRNA level in the control cells. MTARC1 -CT GAPDH △△CT=△CT 处理细胞 -△CT 对照细胞 mRNA level = 2^-△△CT

[0210] In other embodiments, inhibition of MTARC1 gene expression can be assessed based on a decrease in parameters associated with MTARC1 gene expression function, such as the MTARC1 protein level in the blood or serum of a subject. MTARC1 gene silencing can be determined in any cell expressing MTARC1, whether endogenous or heterologous from the expression construct, and by any assay known in the art.

[0211] Inhibition of MTARC1 protein expression can be demonstrated by a decrease in the level of MTARC1 protein expressed in cells or cell populations or in a subject sample (e.g., protein levels in a blood sample from a subject). As described above, to assess mRNA inhibition, inhibition of protein expression levels in treated cells or cell populations can similarly be expressed as a percentage of protein levels in control cells or cell populations, or as a change in protein levels in a subject sample (e.g., blood or serum from which it is derived).

[0212] mRNA inhibition percentage = (protein expression level) 处理细胞 - Protein expression level 对照细胞 Protein expression level 对照细胞 *100%

[0213] Control cells, cell populations, or subject samples that can be used to assess inhibition of MTARC1 gene expression include cells, cell populations, or subject samples that have not yet been exposed to the RNAi agent of this disclosure. For example, control cells, cell lines, or subject samples may be derived from individual subjects (e.g., human or animal subjects) before treating subjects or appropriately matched cohorts with the RNAi agent.

[0214] In some embodiments of the methods disclosed herein, RNAi is administered to a subject to deliver RNAi to a specific site within the subject. Inhibition of MTARC1 expression can be assessed by measuring the level or changes of MTARC1 mRNA or MTARC1 protein or fusion secreted luciferase in fluid or tissue samples from a specific site in the subject (e.g., liver or blood), or by assessing the effectiveness of RNAi by detecting biomarkers such as blood glucose, glycated hemoglobin (HbA1c), blood glucose, and total cholesterol.

[0215] This disclosure also provides methods for using the RNAi of this disclosure or compositions containing the RNAi of this disclosure to inhibit MTARC1 expression, thereby preventing or treating the following diseases, conditions and / or symptoms, including but not limited to: type II diabetes, hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B or other lipid fractions), insulin resistance or higher or altered insulin levels during fasting or metabolic challenges, hepatic steatosis or fatty liver disease and its complications (such as, for example, cirrhosis, fibrosis or liver inflammation), non-alcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver damage, higher or altered or elevated other markers of inflammation or steatosis in the liver, higher or hyperglycemia or blood glucose, chronic diseases such as hepatitis C, and complications of each of the above conditions.

[0216] Various formulations have been developed to facilitate the use of oligonucleotides. For example, oligonucleotides can be delivered to a subject or cellular environment using formulations that minimize degradation, facilitate delivery and / or uptake, or provide another beneficial property to the oligonucleotide or its salt, or its conjugate, or a salt of its conjugate in the formulation. In some embodiments, compositions comprising oligonucleotides (e.g., single-stranded or double-stranded oligonucleotides) to reduce MTARC1 expression are provided herein. Such compositions can be suitably formulated such that when administered to a subject (in the direct environment of target cells or systemically), a sufficient fraction of the oligonucleotide enters the cells to reduce MTARC1 expression. Any of a variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for reducing MTARC1 as disclosed herein. In some embodiments, the oligonucleotide or its conjugate is formulated in a buffer solution, such as an aqueous solution of phosphate-buffered saline, liposomes, micelle structures, and shells. The buffer solution may be selected from solutions of acetate, citrate, glutenin, carbonate, or phosphate, or any combination thereof. In some embodiments, the naked oligonucleotide or its conjugate is formulated in water or an aqueous solution (e.g., pH-adjusted water) without a delivery agent. In some embodiments, the naked oligonucleotide or its conjugate is formulated in an alkaline buffered aqueous solution (e.g., PBS).

[0217] Formulations of oligonucleotides containing cationic lipids can be used to promote the transfection of oligonucleotides into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used.

[0218] Therefore, in some embodiments, the formulation comprises lipid nanoparticles. In some embodiments, the excipient comprises liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticles, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need.

[0219] In some embodiments, the formulations disclosed herein include excipients. In some embodiments, the excipients impart to the composition enhanced stability, enhanced absorption, enhanced solubility, and / or therapeutic enhancement of the active ingredient. In some embodiments, the excipients are buffers (e.g., sodium citrate, sodium phosphate, tris base, or sodium hydroxide) or mediators (e.g., buffer solutions, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, oligonucleotides or their salts, or conjugates thereof, or salts of conjugates thereof are lyophilized to extend their shelf life and then formulated into solutions prior to use (e.g., administration to a subject). Therefore, the excipients in compositions comprising any of the oligonucleotides or their salts, or conjugates thereof, or salts of conjugates thereof described herein may be lyophilization protectants (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or collapse temperature modifiers (e.g., dextran, ficoll, or gelatin).

[0220] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Typically, the route of administration is intravenous or subcutaneous.

[0221] Pharmaceutical compositions suitable for injectable applications include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL.™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The aforementioned carriers can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In many cases, isotonic agents, such as sugars, and polyols such as mannitol, sorbitol, and sodium chloride, are preferably included in the composition. Sterile injectable solutions can be prepared by incorporating the desired amount of an oligonucleotide or its salt, or a conjugate thereof, or a salt of a conjugate thereof, with one or a combination of the desired ingredients listed above into a selected solvent, followed by filtration sterilization.

[0222] In some embodiments, the composition may contain at least about 0.1% or more of a therapeutic agent (e.g., an oligonucleotide or its salt, or a conjugate thereof, for reducing MTARC1 expression), although the percentage of one or more active ingredients may be between about 1% and about 80% or more of the total composition by weight or volume. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore various dosages and treatment regimens may be desired.

[0223] Cells suitable for treatment using the methods of this disclosure can be any cell expressing the MTARC1 gene, such as hepatocytes, brain cells, or kidney cells, but hepatocytes are preferred. Cells suitable for use in the methods of this disclosure can be mammalian cells, such as primate cells (e.g., human cells, including human cells in chimeric nonhuman animals, or nonhuman primate cells, such as monkey cells or chimpanzee cells) or non-primate cells. In some embodiments, the cells are human cells, such as human hepatocytes. In the methods of this disclosure, the expression of MTARC1 in the cells is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the assayed detection level.

[0224] The in vivo methods of this disclosure may include administering a composition comprising RNAi to a subject, wherein the RNAi comprises a nucleotide sequence complementary to at least a portion of the RNA transcript of the MTARC1 gene of the mammal to which the RNAi agent is administered. The composition may be administered in any manner known in the art, including but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and local (including oral and sublingual) administration. In some embodiments, the composition is administered by intravenous infusion or injection. In some embodiments, the composition is administered subcutaneously. In some embodiments, the composition is administered by intramuscular injection.

[0225] In one aspect, this disclosure also provides a method for inhibiting the expression of the MTARC1 gene in mammals. The method comprises administering to a mammal an oligonucleotide or a pharmaceutically acceptable salt thereof, a conjugate thereof, a salt of a conjugate thereof, or a combination thereof. The oligonucleotide is a double-stranded RNA (dsRNA) that targets the MTARC1 gene in mammalian cells and sustains the mammal for a sufficient time to allow for the degradation of the mRNA transcript of the MTARC1 gene, thereby inhibiting the expression of the MTARC1 protein in the cells. The reduction in gene expression can be assessed by any method known in the art and by methods such as qRT-PCR as described herein, for example, as in Example 2. The reduction in protein product can be assessed by any method known in the art (e.g., ELISA). In other embodiments, a blood sample is used as a subject sample to monitor the reduction in MTARC1 protein expression.

[0226] This disclosure also provides methods of treatment in subjects in need, such as subjects diagnosed with MTARC1-related conditions, including but not limited to: type II diabetes, hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B or other lipid fractions), hepatic steatosis or fatty liver disease and its complications (such as, for example, cirrhosis, fibrosis or liver inflammation), non-alcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver damage, higher or elevated or altered other markers of inflammation or steatosis in the liver, and complications of each of the above conditions.

[0227] In one implementation, MTARC1-related diseases include, but are not limited to: type II diabetes, hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B or other lipid fractions), hepatic steatosis or fatty liver disease and its complications (such as, for example, cirrhosis, fibrosis or liver inflammation), non-alcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver damage, other chronic diseases such as higher or elevated or altered markers of inflammation or steatosis in the liver, and complications of each of the above conditions.

[0228] The RNAi disclosed herein can be administered as “free RNAi.” Free RNAi is administered in the absence of a pharmaceutical composition. Naked RNAi can be administered in a suitable buffer solution. The buffer solution may contain acetate, citrate, lactate, tartrate, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and osmotic pressure of the buffer solution containing RNAi can be adjusted to make it suitable for administration to a subject.

[0229] The administration of RNAi according to the methods of this disclosure can lead to the prevention or treatment of MTARC1-related conditions, including but not limited to: type II diabetes, hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B or other lipid fractions), hepatic steatosis or fatty liver disease and its complications (such as, for example, cirrhosis, fibrosis or liver inflammation), non-alcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver damage, higher or elevated or altered other markers of inflammation or steatosis in the liver, and complications of each of the above conditions.

[0230] A therapeutic dose of RNAi can be administered to the subject, such as at a dose ranging from about 0.01 mg / kg to about 200 mg / kg (e.g., about 0.1 mg / kg to about 100 mg / kg). In some embodiments, the oligonucleotide or its salt, or a conjugate thereof, or a salt of a conjugate thereof, is administered to the subject at a dose ranging from about 0.1 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 20 mg / kg, 0.3 mg / kg to about 18 mg / kg, 0.5 mg / kg to about 15 mg / kg, or 0.5 mg / kg to about 12 mg / kg, more preferably from about 1 mg / kg to about 10 mg / kg. Preferably, the dose is from 1 mg / kg to about 50 mg / kg. RNAi is preferably administered subcutaneously, i.e., by subcutaneous injection. One or more injections can be used to deliver the desired dose of RNAi to the subject. Injections can be repeated over a period of time.

[0231] In some embodiments, RNAi is administered to the subject at a fixed dose of about 50 mg to about 800 mg. In some embodiments, RNAi is administered to the subject at a fixed dose of about 50 mg to about 200 mg, about 200 mg to about 400 mg, or about 400 mg to about 800 mg. In some embodiments, RNAi is administered to the subject at a fixed dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, or about 800 mg.

[0232] It can be administered repeatedly at regular intervals. In some embodiments, treatment can be administered at a lower frequency after the initial treatment regimen. Repeated dosing regimens may include the regular administration of therapeutic doses of RNAi, such as once a month to once a year. In some embodiments, RNAi is administered approximately once a month to approximately every three months, or approximately every three months to approximately every six months, or even once a year.

[0233] In some embodiments, a fixed dose is administered to the subject at monthly intervals. In some embodiments, a fixed dose is administered to the subject at six-month intervals.

[0234] In some embodiments, the subject is administered a fixed dose of about 50 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 100 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 150 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 300 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 300 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 600 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 800 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 800 mg approximately every six months.

[0235] In some implementations, the subject to be treated is a human (e.g., a human patient) or a non-human primate or other mammal subject. Other exemplary subjects include domestic animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.

[0236] This disclosure further provides that combination therapy with RNAi agents or pharmaceutical compositions thereof, in conjunction with other drugs and / or other treatments (e.g., known drugs and / or known treatments, such as those currently used to treat these conditions), will benefit patients with liver disease, dyslipidemia, cardiovascular disease, impaired glycemic control, etc. Combination therapy may include oral medications such as metformin, alpha-glucosidase inhibitors, insulin secretagogues (sulfonylureas and meglitinides), thiazolidinediones (TZDs), MTARC1 inhibitors, and SGLT2 inhibitors. Injectable medications include GLP-1 receptor agonists and insulin. Lipid-lowering drugs include statins, fibrates, cholesterol absorption inhibitors, proprotein convertase subtilisin 9 (PCSK9) inhibitors, etc. Drugs that inhibit uric acid synthesis, such as xanthine oxidase inhibitors (XOIs): XOIs inhibit uric acid synthesis, including allopurinol and febuxostat; drugs that increase uric acid excretion, such as benzbromarone and probenecid, etc.

[0237] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described.

[0238] The present disclosure will now be described in more detail with reference to specific embodiments. However, the embodiments are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0239] Example

[0240] Unless otherwise specified in this article, such reagents can be obtained from any molecular biology reagent supplier, and their quality / purity standards are applicable to molecular biology.

[0241] Abbreviations for nucleotide monomers used in nucleic acid sequence representation. It should be understood that when a nucleotide contains a 2'-fluorine modification, the fluorine replaces the hydroxyl group at that position in the parent nucleotide (i.e., it is a 2'-deoxy-2'-fluoronucleotide).

[0242] Table A. Abbreviations for nucleotide monomers used in nucleic acid sequence representation

[0243] Example 1. Preparation of targeting ligands and siRNA

[0244] Preparation of targeted ligands

[0245] L96 was prepared according to the method described in patent CN104717982B.

[0246] C16 was purchased from Chengdu Pioneer Pharmaceuticals Co., Ltd.

[0247] The preparation of DTX was carried out in accordance with the method described in patent application CN113166191A.

[0248] Preparation of oligonucleotides

[0249] (1) Preparation of siRNA

[0250] First, a computer-based algorithm was used to generate candidate oligonucleotide sequences complementary to human MTARC1 mRNA (NM_022746.4, Table 1). Some of these sequences were also complementary to or had no more than two mismatches with cynomolgus monkey MTARC1 mRNA (XM_065542183.1, Table 1). Some were designed as double-stranded siRNAs with 19 / 21 pairings on the sense and antisense strands, with the antisense strand having two drooping ends complementary to the mRNA sequence. In some cases, the drooping ends of the antisense strand were non-complementary UU. Some sequences were designed as double-stranded siRNAs with 21 / 23 pairings on the sense and antisense strands, with the antisense strand having two drooping ends complementary to the mRNA sequence. Some sequences were designed as double-stranded siRNAs with 21 / 21 and 23 / 23 pairings. In some complementary pairing sequences, the first base at the 5' end of the antisense strand (the last base at the 3' end of the sense strand) was replaced with a base that did not match the MTARC1 mRNA.

[0251] Table 1. Human and cynomolgus monkey MTARC1 mRNA sequences

[0252] The siRNA sequence was synthesized separately on a solid support via the sense strand (SS) and antisense strand (AS), and was obtained after deprotection, cleavage, purification, annealing, purification and lyophilization.

[0253] Solid-phase synthesis (Figure 1): The sense and antisense strands were synthesized separately on a solid support using an automated oligonucleotide synthesizer, employing phosphoramide technology. The synthesizer used was, for example, the AKTA Oligopilot (Cytiva) or Dr. Oligo 192XLc (Kunshan Berlik Precision Instruments Co., Ltd.). Solid-phase synthesis began at the 3' end of the sequence, with monomers sequentially coupled into the sequence. Each coupling of a phosphoramide monomer involved four chemical steps: 1) unblocking or deprotection (de-hydroxyl protecting group); 2) coupling; 3) oxidation; and 4) end-capping. All phosphoramidite monomers, reagents, and purification consumables used were commercially available. For example, various phosphoramidite monomers (such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) were purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (such as 40wt% methylamine aqueous solution and 28wt% ammonium hydroxide aqueous solution) were purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used in this paper are as described in US20130178612A1 and US2015100197A1; the synthesis methods for VPUm and APU structural sequences are as described in J.Med.Chem.2018,61,734-744.

[0254] (2) Preparation of double-stranded RNA (dsRNA) drugs

[0255] (a) The synthesis of the chain of justice

[0256] Solid-phase phosphoramide synthesis is a mature method for synthesizing oligonucleotides. A computer-controlled synthesizer is used, and the reaction takes place in a stainless steel column. The positive chain synthesis begins with a solid support loaded with a targeting ligand (e.g., L96), or directly with the solid support. Different starting materials, reagents, and solvents are injected sequentially from sequence 3' to 5' using the solid-phase synthesizer, linking phosphoramide nucleoside monomers one by one. The reaction process involves four cyclic steps: DMT protection removal, condensation, oxidation or thiolation, and end-capping. One nucleotide unit is linked in each cycle, yielding an oligonucleotide sequence of 19 or 21 nucleotides. After synthesis, the protecting group (2-cyanoethyl) is removed on the solid-phase column, and the synthesized sequence is cleaved from the solid support via ammonolysis. The sequence is filtered, the filter cake is washed with ethanol, and the filtrate and washings are collected and concentrated to obtain the crude positive chain. The crude product is purified by chromatography (SOURCE 15Q) and lyophilized to obtain the target product, the positive chain. In the synthesizer, siRNA positive strand conjugates were synthesized starting with a solid support loaded with a targeting ligand (e.g., L96); siRNA was synthesized directly starting with a solid support.

[0257] (b) Synthesis of antisense chains

[0258] Similar to the sense strand synthesis, the antisense strand is synthesized using a solid-phase synthesizer. Different starting materials, reagents, and solvents are injected sequentially from the 3' to 5' ends of the sequence through different tubing, linking phosphoramidine nucleoside monomers one by one. The reaction process involves four cyclic steps: DMT protection removal, condensation, oxidation or thiolation, and end-capping. One nucleotide unit is linked in each cycle, yielding an oligonucleotide sequence of 21 or 23 nucleotides. After synthesis, the protecting group (2-cyanoethyl) is removed on a solid-phase column, and the synthesized sequence is cleaved from the solid support via ammonolysis. The sequence is filtered, the filter cake is washed with ethanol, and the filtrate and washings are collected and concentrated to obtain the crude antisense strand. The crude product is purified by chromatography (SOURCE 15Q), ultrafiltered, and lyophilized to obtain the target product, antisense siRNA.

[0259] (c) Preparation of double-stranded siRNA

[0260] The AS and SS strands were dissolved separately in injection water and mixed in a defined ratio (1.01:1.0-1.2:1.0). The mixture was incubated at 30-50°C for 30-90 minutes and then cooled to room temperature. The double-stranded siRNA product was obtained by freeze-drying.

[0261] The double-stranded siRNA agents listed in Tables 2, 3, and 4 were prepared using the same method.

[0262] In Tables 2, 3, and 4, “G”, “C”, “A”, “U”, and “T” typically represent nucleotides with guanine, cytosine, adenine, uracil, and thymine as bases, respectively. The naked sequences in Tables 2, 3, and 4 refer to unmodified oligonucleotide sequences.

[0263] Modifications: m represents 2'-methoxy; f represents 2'-deoxy-2'-fluorine; s represents thiophosphate; VPUm is 2'-methoxy modified uridine; L96 is N-[tris(GalNAc-alkyl)amidodecyl]-4-hydroxyprolyl (Hyp-(GalNAc-alkyl)3).

[0264] Table 2. Naked Oligonucleotide Sequences

[0265] Table 3 Oligonucleotide Modification Sequences

[0266] Example 2. In vitro activity evaluation of MTARC1-siRNA in cell lines

[0267] (1) Cell culture and transfection:

[0268] Hep3B cells (Wuhan Pronosei Life Sciences Co., Ltd., catalog number CL-0102) were incubated at 37℃ in a 5% CO2 incubator using DMEM medium (Shanghai Biotechnology Co., Ltd., catalog number iCell-0001) supplemented with 10% FBS (GIBCO, 12483020) and 1% penicillin-streptomycin (GIBCO, 15140-122). Once the cell confluence reached 90%, the cells were digested with trypsin-EDTA (Thermo, 25200-072), counted using a Countstar (IC1000), and seeded at 190 μl / well in 96-well plates. The seeding density of Hep3B cells was 2*10n. 4 Cells / wells will adhere to the culture vessel the following day for transfection.

[0269] Transfection was performed using Lipofectamine™ RNAiMAX (thermofisher, 13778150). A transfection complex was prepared by mixing 2.2 μl (100 nM) of the diluted compound, 19.1 μl of Opti-MEM (thermofisher, 1105821), and 0.7 μl of RNAiMAX. After incubation for 5 minutes, the transfection complex was added to the cells (two replicates per complex), 10 μl per well, with a final siRNA concentration of 0.5 nM. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0270] (2) RNA extraction and detection

[0271] (i) Total RNA was extracted using the RNA-Quick Purification Kit (RN001, Yishan Biotechnology):

[0272] Remove the 12-well plate from the incubator, aspirate the culture medium, wash once with an appropriate amount of PBS, add 500 μl of lysis buffer to each well, and transfer the supernatant to a new 1.5 ml centrifuge tube. Add 500 μl of anhydrous ethanol to the lysed cells and mix thoroughly (if precipitation occurs, this is normal; continue the procedure). Invert the centrifuge tube several times, or use a pipette to forcefully aspirate 10 times to disperse the precipitate, then add the liquid to the centrifuge column. Place the centrifuge tube symmetrically in a centrifuge (Eppendorf, 5430) and centrifuge at 4000 × g for 1 min. Remove the centrifuge tube, add 500 μl of wash buffer to the column, and centrifuge at 12000 × g for 1 min. After centrifugation, remove the column, discard the waste liquid, and reassemble the RNA column into the collection tube. Centrifuge the empty tube once to remove any remaining wash buffer. Place the column on a clean, RNase-free 1.5 ml centrifuge tube and allow it to air dry for 2 minutes. Add 30 μl of elution buffer to the center of the RNA column membrane, incubate at room temperature for 2 minutes, centrifuge at 2000×g for 1 min to elute the RNA, and then place on ice. Measure the concentration of the eluted RNA for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80℃ for later use.

[0273] (ii) Use IIQ RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novazia, R223-01) for cDNA synthesis:

[0274] Prepare a mixture in an RNase-free centrifuge tube: 4 μl 4×g DNA wiper Mix, 1 μg template RNA, and RNase-free ddH2O to a final volume of 16 μl to remove genomic DNA. Gently pipette to mix and incubate at 42°C for 2 min. Then, directly add 4 μl 5×HiScript II qRT SuperMix II to the reaction tube and gently pipette to mix. Incubate in a PCR instrument (Applied Biosystems, 9700) at 50°C for 15 min, then at 85°C for 5 sec, and finally at 4°C. The product can be used immediately for qPCR or stored at -20°C and used within six months. For long-term storage, aliquot and store at -80°C. Avoid repeated freeze-thaw cycles for cDNA.

[0275] (iii) Quantitative analysis using ChamQ SYBR qPCR Master Mix (Novazia, Q311-02):

[0276] Prepare a 20 μl mixture by adding 10 μl of 2×ChamQ SYBR qPCR Master Mix, 0.5 μl of Forward primer (Ruiboxingke), 0.5 μl of Reverse primer (Ruiboxingke), 1 μl of Template cDNA, and 8 μl of ddH2O. Each sample was tested in triplicate. The 96-well plate was placed in a qPCR instrument (ROCGENE, Archimed). The following program was executed: pre-denaturation, 95℃, 30 sec; amplification, 95℃, 10 sec, 60℃, 30 sec, 40 cycles; melting curve, 95℃, 15 sec, 60℃, 60 sec, 95℃, 15 sec.

[0277] (3) Data statistical analysis:

[0278] Export the data to Excel format using CT. MTARC1 -CT GAPDH The control group was normalized. To calculate the fold change in relative silencing efficiency, the data were analyzed using the ΔΔCT method. The mean and standard deviation of the three parallel replicates were calculated.

[0279] The results of the two screenings of Hep3B cells are shown in Table 4.

[0280] As shown in Table 4, at a dosage of 0.5 nM, the inhibition rate of MTARC1 mRNA by some siRNAs can reach or approach 30%, such as AL0345002, AL0345006, AL0345017, AL0345023, AL0345024, AL0345030, AL0345034, AL0345035, AL0345037, AL0345042, AL0345070, AL0345071, AL0345074, AL0345084, AL0345085, AL0345086, AL0345087, AL0345090, AL0345091, and AL0345093; some sequences inhibit MTARC1. The inhibition rate of mRNA can reach or approach 50%, such as AL0345002, AL0345006, AL0345017, AL0345023, AL0345024, AL0345034, AL0345035, AL0345037, AL0345074, AL0345084, AL0345085, AL0345090 and AL0345091.

[0281] Table 4 shows the MTARC1 siRNA knockdown levels in Hep3B.

[0282] Example 3. Evaluation of the in vitro activity of MTARC1-siRNA in primary monkey hepatocytes

[0283] (1) Cell culture and transfection:

[0284] Cynomolgus monkey hepatocytes (Beijing Ruide Biotechnology Co., Ltd., cmTCSC) were used. The culture medium was preheated. The thawed culture medium (Beijing Ruide Biotechnology Co., Ltd., HEPO24) was transferred to a biosafety cabinet. 4 mL of FBS was added to 36 mL of thawed culture medium (TPCS, HEPO24) to prepare a completely thawed medium, which was then heated in a 37°C water bath for 10 minutes. The cells were then treated with coating medium (Beijing Ruide Biotechnology Co., Ltd., HEPO44) in a CO2 incubator at 37°C for 0.5 h. Cells were removed from liquid nitrogen and revived in a 37°C water bath. After approximately 2 minutes, the cells were transferred to a preheated 40 mL thawed culture medium. The cryovials were washed with 2 mL of completely thawed culture medium. The cell suspension was centrifuged at 180 × g for 1 minute, the supernatant was discarded, and 2 mL of preheated CM seeding medium (Beijing Ruide Biotechnology Co., Ltd., CMHEP054) was added. The cell suspension was gently dispersed and mixed. 20 μL of the cell suspension was used for cell counting. Based on the counting results, inoculate into 12-well plates, 3*10 5 / well, placed in an incubator at 37℃, 5% CO2. After 4-5 hours of adhesion, the CM seeding medium was aspirated and replaced with preheated medium (Beijing Ruide Biotechnology Co., Ltd., CMHEP064). Transfection was performed 6 hours after adhesion.

[0285] Use Lipofectamine TM RNAiMAX (thermofisher, 13778150) was used for transfection. 2.2 μl (100 nM) of diluted compound, 19.1 μl of Opti-MEM (thermofisher, 1105821), and 0.7 μl of RNAiMAX were mixed to prepare the transfection complex. After incubation for 5 minutes, the transfection complex was added to the cells (two technical replicates for each complex), 10 μl per well, with a final siRNA concentration of 0.5 nM. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0286] (2) RNA extraction and detection

[0287] Same as Example 2.

[0288] (3) Data statistical analysis:

[0289] Same as Example 2.

[0290] As shown in Table 5, in primary hepatocytes of cynomolgus monkeys, when the dosage was 0.5 nM, AL0345002, AL0345023, AL0345024, AL0345030, AL0345074 and AL0345090 all showed an inhibition rate of over 40% against MTARC1 mRNA. Some sequences, such as AL0345002, AL0345023 and AL0345090, showed an inhibition rate of over 60% against MTARC1 mRNA.

[0291] Table 5. MTARC1 siRNA knockdown levels in primary hepatocytes of cynomolgus monkeys.

Claims

1. An oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting MTARC1 expression, said oligonucleotide comprising a sense strand and an antisense strand, said sense strand having at least 80% sequence identity with any sequence or fragment thereof or modified sequence shown in SEQ ID NO. 1-94, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity; said antisense strand having at least 80% sequence identity with any sequence or fragment thereof or modified sequence shown in SEQ ID NO. 95-188, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity; Preferably, the modifying sequence of the justice chain comprises a sequence selected from any of SEQ ID NO:189-282; Preferably, the modified sequence of the antisense strand comprises a sequence selected from any of SEQ ID NO:283-376.

2. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, wherein, The oligonucleotide or its pharmaceutically acceptable salt is selected from carboxylates, alkali metal salts, ammonium salts, alkaline earth metal salts, salts formed with organic bases, and other pharmaceutically acceptable salts. Preferably, the oligonucleotide or its pharmaceutically acceptable salt is more preferably its sodium salt or triethylamine salt.

3. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein, The sense strand comprises an unmodified oligonucleotide selected from any of SEQ ID NO. 2, 6, 17, 23, 24, 30, 34, 35, 37, 42, 70, 71, 74, 84, 85, 86, 87, 90, 91, and 93, or a modified oligonucleotide selected from any of SEQ ID NO. 190, 194, 205, 211, 212, 218, 222, 223, 225, 230, 258, 259, 262, 272, 273, 274, 275, 278, 279, and 281; the antisense strand comprises a oligonucleotide selected from SEQ ID NO. The unmodified oligonucleotides described in any of SEQ ID NOs 96, 100, 111, 117, 118, 124, 128, 129, 131, 136, 164, 165, 168, 178, 179, 180, 181, 184, 185, and 187, or the modified oligonucleotides described in any of SEQ ID NOs 284, 288, 299, 305, 306, 312, 316, 317, 319, 324, 352, 353, 356, 366, 367, 368, 369, 372, 373, and 375.

4. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein, The oligonucleotide comprises any of the following combinations of sense and antisense strands: (1) The positive chain comprises the sequence shown in SEQ ID NO.2, and the negative chain comprises the sequence shown in SEQ ID NO.96; (2) The sense chain comprises the sequence shown in SEQ ID NO.6, and the antisense chain comprises the sequence shown in SEQ ID NO.100; (3) The positive chain contains the sequence shown in SEQ ID NO.17, and the negative chain contains the sequence shown in SEQ ID NO.111; (4) The sense chain comprises the sequence shown in SEQ ID NO.23, and the antisense chain comprises the sequence shown in SEQ ID NO.117; (5) The sense chain comprises the sequence shown in SEQ ID NO.24, and the antisense chain comprises the sequence shown in SEQ ID NO.118; (6) The sense chain comprises the sequence shown in SEQ ID NO.30, and the antisense chain comprises the sequence shown in SEQ ID NO.124; (7) The sense chain comprises the sequence shown in SEQ ID NO.34, and the antisense chain comprises the sequence shown in SEQ ID NO.128; (8) The sense chain comprises the sequence shown in SEQ ID NO.35, and the antisense chain comprises the sequence shown in SEQ ID NO.129; (9) The positive chain comprises the sequence shown in SEQ ID NO.37, and the negative chain comprises the sequence shown in SEQ ID NO.131; (10) The sense chain comprises the sequence shown in SEQ ID NO.42, and the antisense chain comprises the sequence shown in SEQ ID NO.136; (11) The sense chain comprises the sequence shown in SEQ ID NO.70, and the antisense chain comprises the sequence shown in SEQ ID NO.164; (12) The sense chain comprises the sequence shown in SEQ ID NO.71, and the antisense chain comprises the sequence shown in SEQ ID NO.165; (13) The positive chain comprises the sequence shown in SEQ ID NO.74, and the negative chain comprises the sequence shown in SEQ ID NO.168; (14) The positive chain contains the sequence shown in SEQ ID NO.84, and the negative chain contains the sequence shown in SEQ ID NO.178; (15) The sense chain comprises the sequence shown in SEQ ID NO.85, and the antisense chain comprises the sequence shown in SEQ ID NO.179; (16) The sense chain comprises the sequence shown in SEQ ID NO.86, and the antisense chain comprises the sequence shown in SEQ ID NO.180; (17) The positive chain comprises the sequence shown in SEQ ID NO.87, and the negative chain comprises the sequence shown in SEQ ID NO.181; (18) The sense chain comprises the sequence shown in SEQ ID NO.90, and the antisense chain comprises the sequence shown in SEQ ID NO.184; (19) The sense chain comprises the sequence shown in SEQ ID NO. 91, and the antisense chain comprises the sequence shown in SEQ ID NO. 185; and (20) The sense chain comprises the sequence shown in SEQ ID NO.93, and the antisense chain comprises the sequence shown in SEQ ID NO.187; Preferably, the oligonucleotide comprises any combination of the following sense and antisense strands: (1) The positive chain comprises the sequence shown in SEQ ID NO.2, and the negative chain comprises the sequence shown in SEQ ID NO.96; (2) The sense chain comprises the sequence shown in SEQ ID NO.6, and the antisense chain comprises the sequence shown in SEQ ID NO.100; (3) The positive chain contains the sequence shown in SEQ ID NO.17, and the negative chain contains the sequence shown in SEQ ID NO.111; (4) The sense chain comprises the sequence shown in SEQ ID NO.23, and the antisense chain comprises the sequence shown in SEQ ID NO.117; (5) The sense chain comprises the sequence shown in SEQ ID NO.24, and the antisense chain comprises the sequence shown in SEQ ID NO.118; (6) The sense chain comprises the sequence shown in SEQ ID NO.30, and the antisense chain comprises the sequence shown in SEQ ID NO.124; (7) The sense chain comprises the sequence shown in SEQ ID NO.34, and the antisense chain comprises the sequence shown in SEQ ID NO.128; (8) The sense chain comprises the sequence shown in SEQ ID NO.35, and the antisense chain comprises the sequence shown in SEQ ID NO.129; (9) The positive chain comprises the sequence shown in SEQ ID NO.37, and the negative chain comprises the sequence shown in SEQ ID NO.131; (10) The sense chain comprises the sequence shown in SEQ ID NO.74, and the antisense chain comprises the sequence shown in SEQ ID NO.168; (11) The positive chain comprises the sequence shown in SEQ ID NO.84, and the negative chain comprises the sequence shown in SEQ ID NO.178; (12) The sense chain comprises the sequence shown in SEQ ID NO.85, and the antisense chain comprises the sequence shown in SEQ ID NO.179; (13) The sense chain comprises the sequence shown in SEQ ID NO. 90, and the antisense chain comprises the sequence shown in SEQ ID NO. 184; and (14) The positive chain contains the sequence shown in SEQ ID NO.91, and the negative chain contains the sequence shown in SEQ ID NO.

185.

5. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein, The oligonucleotide contains at least one modified nucleotide; Preferably, at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, 2'-5'-linked ribonucleotides (3'-RNA), unlocked nucleotides, conformation-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, and 2'-O-alkyl modified nucleotides. Nucleotides modified with 2'-hydroxyl, 2'-methoxyethyl, 2'-O-alkyl, morpholinonucleotides, aminophosphates, nucleotides including non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides including thiophosphate groups, nucleotides including methylphosphonate groups, nucleotides including 5'-phosphates, nucleotides including 5'-phosphate mimics, vinyl-phosphonate nucleotides, heat-labile nucleotides, glycol-modified nucleotides, nucleotides including 2'-phosphates and 2-O-(N-methylacetamide)-modified nucleotides; and combinations thereof; Preferably, at least one of the modified nucleotides is selected from the group consisting of: LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxy and ethylene glycol; and combinations thereof; Preferably, at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, ethylene glycol modified nucleotides (GNA), nucleotides comprising 2'-phosphate esters and nucleotides comprising thiophosphate ester groups; and combinations thereof. Preferably, the oligonucleotide comprises at least one 2'-modified nucleotide; Preferably, the 2'-modified nucleotide is selected from the group consisting of: 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-fluorine-modified nucleotides, 2'-acylamino-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-amide-modified nucleotides, 2'-substituted amide-modified nucleotides, 2'-deoxynucleotides, nucleotides including 2'-phosphate esters, and nucleotides modified with 2'-O-(N-methylacetamide); and combinations thereof; Preferably, the 2'-modification is selected from the group consisting of: 2'-methoxy, 2'-acetamido, 2'-aminoethyl, 2'-fluorine, 2'-O-methyl, and 2'-O-methoxyethyl; and combinations thereof; Preferably, the oligonucleotide has a 5'-phosphate analog modified nucleotide at its 5' end; preferably, the 5'-phosphate analog modified nucleotide has a vinylphosphonate modified nucleotide as shown in formula (I), wherein R is selected from H, OH, fluorine, 2'-methoxy, 2'-acetamide, 2'-aminoethyl and 2'-O-methoxyethyl, and Base represents a natural or modified nucleic acid base, preferably selected from A, G, C, T and U; preferably, the 5'-phosphate analog modified nucleotide has a vinylphosphonate modified nucleotide as shown in formula (II), wherein R is selected from H, OH, fluorine, 2'-methoxy, 2'-acetamide, 2'-aminoethyl and 2'-O-methoxyethyl; more preferably, the 5'-phosphate analog modified nucleotide is APU as shown in formula (III) or VPUm as shown in formula (IV); Preferably, the oligonucleotide is a 6-(3-(2-carboxyethyl)phenyl)purine-modified nucleotide; preferably, the oligonucleotide comprises formula M, which is a 2'-O-methyl-6-(3-(2-carboxyethyl)phenyl)-purine nucleotide as shown in formula (V); Preferably, the oligonucleotide comprises uridine-2'-phosphate (U-2'5') of formula (VI), guanosine-2'-phosphate (G-2'5') of formula (VII), cytidine-2'-phosphate (C-2'5') of formula (VIII), adenosine-2'-phosphate (A-2'5') of formula (IX), and thymidine-2'-phosphate (T-2'5') of formula (X); 6. The oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-5, wherein, The oligonucleotide contains at least one modified internucleotide bond; Preferably, the at least one modified nucleotide inter-bond is a phosphate thioester bond.

7. The oligonucleotide or a pharmaceutically acceptable salt thereof of any one of claims 1-6, wherein, The oligonucleotide comprises any of the following combinations of sense and antisense strands: (1) The sense chain comprises the sequence shown in SEQ ID NO.190, and the antisense chain comprises the sequence shown in SEQ ID NO.284; (2) The sense chain comprises the sequence shown in SEQ ID NO.194, and the antisense chain comprises the sequence shown in SEQ ID NO.288; (3) The sense chain comprises the sequence shown in SEQ ID NO.205, and the antisense chain comprises the sequence shown in SEQ ID NO.299; (4) The sense chain comprises the sequence shown in SEQ ID NO.211, and the antisense chain comprises the sequence shown in SEQ ID NO.305; (5) The sense chain comprises the sequence shown in SEQ ID NO.212, and the antisense chain comprises the sequence shown in SEQ ID NO.306; (6) The sense chain comprises the sequence shown in SEQ ID NO.218, and the antisense chain comprises the sequence shown in SEQ ID NO.312; (7) The sense chain comprises the sequence shown in SEQ ID NO.222, and the antisense chain comprises the sequence shown in SEQ ID NO.316; (8) The sense chain comprises the sequence shown in SEQ ID NO.223, and the antisense chain comprises the sequence shown in SEQ ID NO.317; (9) The sense chain comprises the sequence shown in SEQ ID NO.225, and the antisense chain comprises the sequence shown in SEQ ID NO.319; (10) The sense chain comprises the sequence shown in SEQ ID NO.230, and the antisense chain comprises the sequence shown in SEQ ID NO.324; (11) The sense chain comprises the sequence shown in SEQ ID NO.258, and the antisense chain comprises the sequence shown in SEQ ID NO.352; (12) The sense chain comprises the sequence shown in SEQ ID NO.259, and the antisense chain comprises the sequence shown in SEQ ID NO.353; (13) The sense chain comprises the sequence shown in SEQ ID NO.262, and the antisense chain comprises the sequence shown in SEQ ID NO.356; (14) The sense chain comprises the sequence shown in SEQ ID NO.272, and the antisense chain comprises the sequence shown in SEQ ID NO.366; (15) The sense chain comprises the sequence shown in SEQ ID NO.273, and the antisense chain comprises the sequence shown in SEQ ID NO.367; (16) The sense chain comprises the sequence shown in SEQ ID NO.274, and the antisense chain comprises the sequence shown in SEQ ID NO.368; (17) The sense chain comprises the sequence shown in SEQ ID NO.275, and the antisense chain comprises the sequence shown in SEQ ID NO.369; (18) The sense chain comprises the sequence shown in SEQ ID NO.278, and the antisense chain comprises the sequence shown in SEQ ID NO.372; (19) The sense chain comprises the sequence shown in SEQ ID NO. 279, and the antisense chain comprises the sequence shown in SEQ ID NO. 373; and (20) The sense chain comprises the sequence shown in SEQ ID NO.281, and the antisense chain comprises the sequence shown in SEQ ID NO.375; Preferably, the oligonucleotide comprises any combination of the following sense and antisense strands: (1) The sense chain comprises the sequence shown in SEQ ID NO.190, and the antisense chain comprises the sequence shown in SEQ ID NO.284; (2) The sense chain comprises the sequence shown in SEQ ID NO.194, and the antisense chain comprises the sequence shown in SEQ ID NO.288; (3) The sense chain comprises the sequence shown in SEQ ID NO.205, and the antisense chain comprises the sequence shown in SEQ ID NO.299; (4) The sense chain comprises the sequence shown in SEQ ID NO.211, and the antisense chain comprises the sequence shown in SEQ ID NO.305; (5) The sense chain comprises the sequence shown in SEQ ID NO.212, and the antisense chain comprises the sequence shown in SEQ ID NO.306; (6) The sense chain comprises the sequence shown in SEQ ID NO.218, and the antisense chain comprises the sequence shown in SEQ ID NO.312; (7) The sense chain comprises the sequence shown in SEQ ID NO.222, and the antisense chain comprises the sequence shown in SEQ ID NO.316; (8) The sense chain comprises the sequence shown in SEQ ID NO.223, and the antisense chain comprises the sequence shown in SEQ ID NO.317; (9) The sense chain comprises the sequence shown in SEQ ID NO.225, and the antisense chain comprises the sequence shown in SEQ ID NO.319; (10) The sense chain comprises the sequence shown in SEQ ID NO.262, and the antisense chain comprises the sequence shown in SEQ ID NO.356; (11) The sense chain comprises the sequence shown in SEQ ID NO.272, and the antisense chain comprises the sequence shown in SEQ ID NO.366; (12) The positive chain comprises the sequence shown in SEQ ID NO.273, and the negative chain comprises the sequence shown in SEQ ID NO.367; (13) The sense chain comprises the sequence shown in SEQ ID NO. 278, and the antisense chain comprises the sequence shown in SEQ ID NO. 372; and (14) The positive chain contains the sequence shown in SEQ ID NO.279, and the negative chain contains the sequence shown in SEQ ID NO.

373.

8. Conjugates or pharmaceutically acceptable salts thereof for inhibiting MTARC1 expression, comprising: (i) the oligonucleotide of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, and (ii) a targeting ligand and / or a lipophilic moiety, wherein at least one of the sense strand and antisense strand of the oligonucleotide is conjugated to the targeting ligand and / or at least one of the sense strand and antisense strand of the oligonucleotide is conjugated to one or more of the lipophilic moieties; Preferably, the ligand comprises an N-acetylgalactosamine (GalNAc) moiety; Preferably, the GalNac portion is a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion, or a tetravalent GalNAc portion; Preferably, the lipophilic moiety is an aliphatic, alicyclic, or polycyclic alicyclic compound; preferably, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain; preferably, the lipophilic moiety comprises C16, C18, C22, or Dtx. Preferably, the lipophilic portion comprises a lipid portion; Preferably, the ligand is L96 or a lipophilic portion; Preferably, the lipophilic portion is located at the sixth position of the positive chain or at one end of the positive chain; 9. A composition comprising the oligonucleotide of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, or the conjugate of claim 8 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier; Preferably, the dosage form of the composition is an oral preparation, an intravenous injection, a subcutaneous injection, or an intramuscular injection, preferably a subcutaneous injection; Preferably, the composition further comprises other medicines for treating and / or preventing MTARC1-related conditions, symptoms, and / or ailments.

10. The oligonucleotide of any one of claims 1-8 or a pharmaceutically acceptable salt thereof, or the composition of claim 9, in the preparation for the treatment and / or prevention of MTARC1-related conditions, symptoms, and / or ailments; The MTARC1-related diseases, conditions, and / or symptoms include, but are not limited to: type II diabetes, hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B, or other lipid fractions), insulin resistance or higher or altered insulin levels during fasting or metabolic challenges, hepatic steatosis or fatty liver disease and its complications (such as cirrhosis, fibrosis, or liver inflammation), non-alcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver damage, higher or altered levels or changes in other markers of inflammation or steatosis in the liver, higher or higher blood glucose or blood glucose, chronic diseases such as hepatitis C, and complications of each of the above conditions.

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