RNAi agent for inhibiting MARC1 expression as well as preparation method and application of RNAi agent

Abstract

The invention belongs to the technical field of biological medicine, relates to an RNAi agent for inhibiting MARC1 expression and a preparation method and application of the RNAi agent, and further relates to application of the RNAi agent and a pharmaceutical composition containing the RNAi agent to prevention or treatment of MARC1 related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to an RNAi agent for inhibiting MARC1 expression, a preparation method thereof, and uses thereof. Background Art

[0002] The liver plays a crucial role in lipid metabolism. Abnormalities in normal liver lipid metabolism are associated with the development of various liver diseases or disorders, such as non-alcoholic fatty liver disease (NAFLD), its subsequent progression to non-alcoholic steatohepatitis (NASH), and potential other advanced liver abnormalities.

[0003] Non-alcoholic fatty liver disease (NAFLD) constitutes a spectrum of liver lesions and is the most common chronic liver disease in the world. Its prevalence has doubled in the past 20 years and now is estimated to affect approximately 20%-30% of the world's population. In some individuals, the accumulation of ectopic fat in the liver (termed steatosis) triggers inflammation and hepatocyte damage, leading to a more advanced disease, called non-alcoholic steatohepatitis (NASH). NASH is defined as lipid accumulation with signs of cell damage, inflammation, and varying degrees of scarring or fibrosis. As of 2015, it was estimated that 75 million to 100 million Americans had NAFLD, and NASH accounted for approximately 10%-30% of NAFLD diagnoses.

[0004] MARC1 (Mitochondrial amidoxime reducing component 1), also known as MTARC1, is a mammalian molybdenum-containing enzyme. It is located in the outer mitochondrial membrane and consists of an N-terminal mitochondrial signal domain facing the intermembrane space, a transmembrane domain, and a C-terminal catalytic domain facing the cytosol. It is encoded by the MOSC1 gene in humans. MARC1 contains two domains composed of discontinuous secondary structure elements. Most of the protein is dominated by β-strands, forming three-stranded and four-stranded antiparallel β-sheets. Two domains can be distinguished: one composed of β-strands 7 to 12 and helices α1 and α2, and the other surrounding a large β-barrel, with a four-stranded β-sheet forming the "lid" of the barrel and helices α4 to α9. Moco is buried within a crevice between the two domains and is accompanied by helix α3. MARC1 is mainly expressed in adipose tissue, followed by the thyroid, prostate, and liver. Recently, it has been reported that predicted loss-of-function variants of the MARC1 gene are associated with reduced blood cholesterol and liver enzyme levels, reduced liver fat, and protection against cirrhosis. Therefore, therapeutic agents targeting mARC1 function represent a new approach for reducing cholesterol levels (such as non-HDL cholesterol or LDL-cholesterol levels) and liver fibrosis, as well as treating or preventing liver diseases (especially NAFLD and NASH).

[0005] For example, Patent CN116194119A reported RNAi constructs and methods for inhibiting MARC1 expression. By using siRNA molecules to silence mARC1 mRNA in the liver, the results showed that there was still a certain silencing effect after 44 days.

[0006] Although the prior art has reported the use of siRNA to inhibit the expression of mARC1 protein in the liver, there are currently no marketed drugs. Therefore, there is still a need to provide more RNAi agents for inhibiting MARC1 expression, as well as their preparation methods and uses. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present application aims to provide an RNAi agent for inhibiting MARC1 expression, as well as its preparation method and use.

[0008] Specifically, the present invention provides an RNAi agent for inhibiting MARC1 expression. The RNAi agent comprises a sense strand and an antisense strand, and the sense strand and the antisense strand form a duplex region, wherein the sense strand and the antisense strand are selected from any one group of the sequences shown in Table 1.

[0009] Furthermore, in the duplex region, the sense strand and the antisense strand may be partially, substantially or completely complementary to each other. For example, the sense strand and the antisense strand may be 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary.

[0010] Furthermore, the sense strand and / or the antisense strand independently comprise one or more modified nucleotides.

[0011] Further, the one or more modified nucleotides may independently be selected from the group consisting of: 5'-thiophosphate nucleotides, 5'-methylated cytosine nucleotides, 5'-methylphosphate nucleotides, 2'-O-methyl modified nucleotides, 2'-O-2-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, ethoxy nucleotides, alkyl nucleotides, 5'-C-methylphosphate nucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNA), 2'-fluoro modified nucleotides, 3'-nitrogen substituted modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, vinyl phosphonate deoxyribonucleotides (VP), thiophosphate nucleotides, dithiophosphate nucleotides, locked nucleic acids (LNA), abasic nucleotides, deoxythymidine, inverted deoxythymidine, 2'-deoxyadenosine nucleotides, 2'-deoxycytidine nucleotides, 2'-deoxyguanosine nucleotides, 2'-deoxythymidine nucleotides, 2'-amino modified nucleotides, morpholino oligonucleotides (PMO), inverted abasic nucleotides (invAb), peptide nucleotides, aminophosphates or unnatural base nucleotides.

[0012] Further, as a preferred technical solution of the present invention, the sense strand and / or the antisense strand independently comprise: one or more of 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-deoxyadenosine nucleotides, 2'-deoxycytidine nucleotides, 2'-deoxyguanosine nucleotides, 2'-deoxythymidine nucleotides or thiophosphate nucleotides.

[0013] As a preferred technical solution of the present invention, the sense strand and the antisense strand have the modified forms shown by the following formula:

[0014] Sense strand: NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm;

[0015] Antisense strand: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0016] Wherein: "N" represents a nucleotide; m indicates that the nucleotide adjacent to its left is a 2'-O-methyl modified nucleotide; f indicates that the nucleotide adjacent to its left is a 2'-fluoro modified nucleotide; s indicates that there is a thiophosphate linkage between the two nucleotides adjacent to the letter s on the left and right.

[0017] Further, as a preferred technical solution of the present invention, the RNAi agent is selected from double-stranded siRNA analogs prepared by preparing the double-stranded siRNA analogs shown in Table 1 into the modified forms shown by the following formula:

[0018] Sense strand: NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNm;

[0019] Antisense strand: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0020] Wherein: "N" represents a nucleotide, such as A, G, U, and C;

[0021] m indicates that the nucleotide adjacent to its left is a 2'-O-methyl modified nucleotide. For example, Am, Um, Gm, and Cm respectively represent 2'-O-methyl modified A, U, G, and C;

[0022] f indicates that the nucleotide adjacent to its left is a 2'-fluoro modified nucleotide. For example, Af, Uf, Gf, and Cf respectively represent 2'-fluoro modified A, U, G, and C;

[0023] s indicates that there is a phosphorothioate linkage between the two nucleotides adjacent to the letter s on its left and right.

[0024] Furthermore, as a preferred technical solution of the present invention, the RNAi agent is selected from any one of the sequences described in Table 2: wherein, m represents that a nucleotide adjacent to the left is a 2'-methoxy modified nucleotide, f represents that a nucleotide adjacent to the left of the letter f is a 2'-fluoro modified nucleotide, and the lowercase letter s indicates that there is a phosphorothioate linkage between the two nucleotides adjacent to the letter on its left and right.

[0025] Furthermore, as a preferred technical solution of the present invention, the RNAi agent is linked to a targeting ligand.

[0026] Furthermore, as a preferred technical solution of the present invention, the targeting ligand is linked to the 3' or 5' end of the sense strand or the antisense strand;

[0027] Furthermore, as a preferred technical solution of the present invention, the targeting ligand is linked to the 3' or 5' end of the sense strand;

[0028] Furthermore, as a preferred technical solution of the present invention, the targeting ligand is linked to the 3' or 5' end of the antisense strand;

[0029] Furthermore, as a preferred technical solution of the present invention, the targeting ligand contains an N-acetyl-galactosamine (GalNac) moiety.

[0030] Furthermore, as a preferred technical solution of the present invention, the targeting ligand can be selected from the following structures:

[0031]

[0032] Furthermore, the present invention also provides a pharmaceutical composition, which comprises the RNAi agent described in any one of the foregoing, and one or more pharmaceutically acceptable excipients and / or carriers.

[0033] Furthermore, the present invention also provides the use of the RNAi agent or pharmaceutical composition described in any one of the foregoing in the preparation of a medicament for preventing or treating a disease related to MARC1; the MARC1-related disease is preferably a liver disease; the liver disease is preferably non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0034] In some embodiments, the pharmaceutical composition of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is required and the area to be treated. Administration can be prepared by any means known in the art, such as but not limited to topical (e.g., by transdermal patch), pulmonary (e.g., by inhalation or insufflation of powder or aerosol, including by nebulizer, intratracheal, intranasal), percutaneous, transdermal, oral or parenteral routes. Parenteral administration includes but is not limited to intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous (via an implanted device), intracranial, intrasternal, intrathecal and intraventricular administration. In some embodiments, the pharmaceutical composition described herein is administered by subcutaneous injection.

[0035] In some embodiments, the gene expression level and / or mRNA level of MARC1 in a subject administered the MARC1 RNAi agent is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% relative to the subject before administration of the MARC1 RNAi agent or a subject not receiving the MARC1 RNAi agent. The gene expression level and / or mRNA level in the subject can be reduced in the cells, cell populations and / or tissues of the subject. In some embodiments, the protein level of MARC1 in a subject administered the MARC1 RNAi agent is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% relative to the subject before administration of the MARC1 RNAi agent or a subject not receiving the MARC1 RNAi agent. The protein level in the subject can be reduced in the cells, cell populations, tissues, blood and / or other fluids of the subject. Reduction of gene expression, mRNA or protein levels can be evaluated by any method known in the art. Reduction or decrease of the MARC1 mRNA level and / or protein level is collectively referred to herein as reduction or decrease of MARC1, or inhibition or reduction of MARC1 expression.

[0036] The present invention also provides a method for preparing an RNAi agent according to any one of the foregoing, wherein the sense strand and the antisense strand comprised by the RNAi agent can be conveniently and conventionally prepared by known techniques of solid-phase synthesis. Additionally or alternatively, any other method known in the art for such synthesis can be used, such as liquid-phase synthesis or fermentation. It is also known to use similar techniques to prepare other oligonucleotides (such as phosphorothioates and alkylated derivatives).

[0037] As used herein, the term "comprising" is used to mean the phrase "comprising (but not limited to)" and can be used interchangeably with that phrase, unless the context clearly dictates otherwise.

[0038] The term "antisense strand" generally refers to the strand of an RNAi agent that includes a region substantially complementary to the target sequence. As used herein, the term "complementary region" generally refers to the region on the antisense strand that is substantially complementary to a sequence defined in the present application (such as the target sequence). When the complementary region is not completely complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, for example, within 5, 4, 3 or 2 nucleotides at the 5' end and / or 3' end.

[0039] The term "sense strand" generally refers to the strand of an RNAi agent that includes a region substantially complementary to the region that is defined herein as the antisense strand. The "sense" strand is sometimes referred to as the "sense" strand, the "passenger" strand, or the "anti-guide" strand. By virtue of their sequences, the antisense strand targets the desired mRNA, while the sense strand targets a different target. Thus, if the antisense strand is incorporated into RISC, the correct target is targeted. Incorporation of the sense strand can result in off-target effects. These off-target effects can be limited by using modifications on the sense strand or by using a 5' end cap.

[0040] In the present invention, "one or more modified nucleotides" are preferably 1 - 20, 1 - 15, 1 - 10, 1 - 8, 1 - 6, 1 - 5, 1 - 4, 1 - 3 modified nucleotides, specifically including: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 modified nucleotides;

[0041] In the present invention, the modified nucleotides include but are not limited to: nucleotides with 5'-thiophosphate groups, 5'-methylated cytosine nucleotides, 5'-methylphosphate nucleotides, 2'-O-methyl modified nucleotides, reverse 2'-O-methyl nucleotides, 3'-O-methyl nucleotides, 2'-O-2-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, alkyl nucleotides, 5'-C-methylphosphate group nucleotides, 2'-F-arabinonucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNA), 2'-fluoro modified nucleotides, 3'-nitrogen-substituted modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 5'-methyl-2'-fluoro nucleotides, 2'-deoxy modified nucleotides, vinyl phosphonate deoxyribonucleotides, thiophosphate nucleotides, dithiophosphate nucleotides, locked nucleic acids (LNA), abasic nucleotides, abasic ribose (Ab), reverse deoxyribonucleotides (3'-3'-linked nucleotides or 5'-5'-linked nucleotides), phosphoethylene deoxyribonucleotides, reverse abasic nucleotides (invAb), deoxythymidine, reverse deoxythymidine, 2'-amino modified nucleotides, morpholino oligonucleotides (PMO), peptide nucleotides, aminophosphates or unnatural base nucleotides.

[0042] The term "pharmaceutically acceptable carrier" refers to any preparation carrier or medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and is non-toxic and has no side effects on the host or patient. Representative carriers include water, oils, vegetables and minerals, paste bases, lotion bases, ointment bases, etc. These bases include suspending agents, thickening agents, transdermal promoters, etc. Their preparations are well-known to those skilled in the art of the cosmetics field or the topical drug field. For other information on carriers, reference can be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the content of which is incorporated herein by reference.

[0043] The term "excipient" generally refers to the carrier, diluent, and / or medium required to formulate an effective pharmaceutical composition. Among them, excipients include (but are not limited to): absorption enhancers, anti-adhesives, defoamers, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, detergents, dextrans, dextroses, diluents, disintegrants, emulsifiers, swelling agents, fillers, flavoring agents, glidants, wetting agents, oils, polymers, preservatives, saline solutions, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, waterproofing agents, wetting agents, lubricants such as sodium lauryl sulfate and magnesium stearate, flavor correctors, and fragrances.

[0044] For a drug or a pharmacological active agent, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of the drug or agent that is non-toxic but can achieve the desired effect. For the oral dosage form in the present invention, the "effective amount" of an active substance in the composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount varies from person to person, depending on the age and general condition of the recipient, and also depends on the specific active substance. The appropriate effective amount in a particular case can be determined by those skilled in the art through routine tests.

[0045] The terms "active ingredient", "therapeutic agent", "active substance" or "active agent" refer to a chemical entity that can effectively treat a target disorder, disease or condition.

[0046] "Optionally" or "optionally" means that the subsequent described event or condition may but does not necessarily occur, and this description includes the situation where the described event or condition occurs and the situation where the described event or condition does not occur.

[0047] The compounds of the present invention can be prepared by various synthetic methods well-known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by their combination with other chemical synthesis methods, and the equivalent substitution methods well-known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention. Specific embodiments

[0048] The following further describes the present application in detail with reference to examples, but the embodiments of the present application are not limited thereto.

[0049] Example 1

[0050] Synthesis of the targeting ligand

[0051]

[0052] Among them, the synthesis route of the targeting ligand L96 refers to the document with the international patent publication number WO2009073809. The targeting ligand L96 can be linked to siRNA through a phosphate group, a phosphorothioate group, or another linking group, indicating the siRNA linking site.

[0053]

[0054] Among them, the synthesis route of NAG37 can refer to the document with the patent number CN20178�042047.6, where indicating the siRNA linking site.

[0055]

[0056] Among them, the synthesis route of M10 (conjugates 9 and 10) can refer to the document with the patent number CN202210705962.1, where R 2 represents siRNA.

[0057]

[0058] Among them, the synthesis route of L10 can refer to the synthesis route of the patent with the patent publication number WO2019105414A1, where Nu represents siRNA.

[0059] <(

[0060] Among them, the synthesis route of GLS-15 can refer to the synthesis route of the patent with the patent publication number CN202280016262.X, where indicating the siRNA linking site.

[0061]

[0062] Among them, the synthesis route of GL6 can refer to the synthesis method of compound 6 in the patent document WO2023241591. Indicates the siRNA ligation site.

[0063] Example 2

[0064] Synthesis of RNAi agent

[0065] An OligoMaker ApS 192 RNA synthesizer (produced in Denmark) was used to prepare the RNAi agent. Among them, the specific synthesis route can refer to the patent document CN202180077184.X. The designed RNAi agent sequences are shown in Table 1 below.

[0066] Table 1 RNAi agent sequences

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] <°

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] Example 3: In vitro testing of MARC1 RNAi agent in HuH7 cells

[0085] The effect of the RNAi agents in Table 1 on the expression of MARC1 gene in HuH7 cells was detected by qPCR experiment (Taqman probe method).

[0086] To determine the inhibition efficiency at the mRNA level, after transfecting MARC1 siRNA into HuH7 cells, a qPCR experiment was performed to measure the expression level of MARC1 mRNA. Specifically, HuH7 cells in the logarithmic growth phase were seeded in 96-well plates at a density of 1.0×10 4 / well and cultured overnight in a 37°C, 5% CO2 incubator. siRNA and Lipo2000 (Invitrogen-52887) diluted to a final concentration of 10 nM, 1 nM, and 0.01 nM with Opti-MEM (Gibco-31985-062) were added, and transfection was carried out according to the protocol provided by Invitrogen. After 48 h of transfection, total cellular RNA was extracted using the Baypure magnetic bead method tissue cell total RNA extraction kit (BayBio-TIRM-48-K-D), and then the extracted cellular RNA was reverse transcribed using TransScript UniAll-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Trans-AU341-02-V2) to convert RNA into cDNA. Finally, qPCR experiments were performed on the obtained cDNA using EZ-Probe qPCR Master Mix-UDG (EZB-EZB-Probe-U2).

[0087] The expression level of the target gene mRNA in each sample was calculated by the ΔΔCT relative quantification method. The relative expression level of the target gene was expressed as 2-ΔΔCT. The specific method was to subtract the CT value of the internal reference gene (GAPDH) from the CT value of the target gene in each sample to obtain the ΔCT value, then subtract the ΔCT value of the control group containing only the transfection reagent (RNAiMAX Control) from the ΔCT value of the drug administration group (sample) to obtain the ΔΔCT, and finally perform the 2-ΔΔCT conversion on the ΔΔCT to obtain the relative expression level of CFBmRNA (value of sample).

[0088] The inhibition rate of the MARC1 gene mediated by the RNAi agent was calculated according to the following formula.

[0089] Inhibition rate of MARC1 gene % = (1 - value of sample / Average value of RNAiMAX Control) * 100.

[0090] Example 4 Synthesis of Modified Sequences of RNAi Agents

[0091] An OligoMaker ApS 192 RNA synthesizer (produced in Denmark) was used to prepare the modified sequences of RNAi agents. Specifically, the double-stranded siRNA analogs shown in Table 1 were prepared into double-stranded siRNA analogs with the modified form shown in the following formula:

[0092] Sense strand: NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm;

[0093] Antisense strand: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0094] Where: "N" represents nucleotides, such as A, G, U, and C;

[0095] m indicates that the nucleotide adjacent to its left is a 2'-O-methyl modified nucleotide. For example, Am, Um, Gm, and Cm represent 2'-O-methyl modified A, U, G, and C respectively;

[0096] f indicates that the nucleotide adjacent to its left is a 2'-fluoro modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-fluoro modified A, U, G, and C respectively;

[0097] s indicates that there is a phosphorothioate linkage between the two nucleotides adjacent to the letter s on the left and right.

[0098] Among them, the specific synthesis route can refer to the patent document CN202180077184.X. The sequences of some modified RNAi agents are shown in Table 2 below.

[0099] Table 2 Modified Sequences of RNAi Agents

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] Wherein, A = adenosine-3'-phosphate; U = uridine-3'-phosphate; C = cytidine-3'-phosphate; G = guanosine-3'-phosphate; Am = 2'-O-methyladenosine-3'-phosphate; Um = 2'-O-methyluridine-3'-phosphate; Cm = 2'-O-methylcytidine-3'-phosphate; Gm = 2'-O-methylguanosine-3'-phosphate; Gs = guanosine-3'-thiotriphosphate; Ams = 2'-O-methyladenosine-3'-thiotriphosphate; Ums = 2'-O-methyluridine-3'-thiotriphosphate; Cms = 2'-O-methylcytidine-3'-thiotriphosphate; Gms = 2'-O-methylguanosine-3'-thiotriphosphate; Af = 2'-fluoroadenosine-3'-phosphate; Uf = 2'-fluorouridine-3'-phosphate; Cf = 2'-fluorocytidine-3'-phosphate; Gf = 2'-fluoroguanosine-3'-phosphate; Afs = 2'-fluoroadenosine-3'-thiotriphosphate; Ufs = 2'-fluorouridine-3'-thiotriphosphate; Cfs = 2'-fluorocytidine-3'-thiotriphosphate; Gfs = 2'-fluoroguanosine-3'-thiotriphosphate; m = 2'-O-methyl; f = 2'-fluoro; s = thiotriphosphate bond.

[0107] Example 5

[0108] Synthesis of RNAi conjugate

[0109] An RNAi agent containing modifications was prepared using an OligoMaker ApS192 RNA synthesizer (produced in Denmark), and then the targeting ligand L96 of Example 1 was linked to the 3'-end of the sense strand of the RNAi agent including the modifications in Table 2 of Example 2. Specifically, the synthetic route can refer to the patent document CN202180077184.X, and the content of this patent document can be incorporated into this application by reference.

[0110] Example 6

[0111] In vitro test of MARC1 RNAi agent in HuH7 cells

[0112] The effect of the RNAi agent in Table 1 on the expression of MARC1 gene in HuH7 cells was detected by qPCR experiment (Taqman probe method).

[0113] To determine the inhibition efficiency at the mRNA level, after transfecting MARC1 siRNA into HuH7 cells, a qPCR experiment was performed to measure the expression level of MARC1 mRNA. Specifically, HuH7 cells in the logarithmic growth phase were taken, and according to 1.0×10 4 / The 96-well plates were seeded with cells and incubated overnight at 37°C in a 5% CO2 incubator. siRNA diluted to a final concentration of 10 nM, 1 nM, and 0.05 nM with Opti-MEM (Gibco-31985-062) and Lipo2000 (Invitrogen-52887) were added, and transfection was performed according to the protocol provided by Invitrogen. At 48 h after transfection, total cellular RNA was extracted using the Baypure magnetic bead method tissue cell total RNA extraction kit (BayBio-TIRM-48-K-D). Then, the extracted cellular RNA was reverse transcribed into cDNA using TransScript UniAll-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Trans-AU341-02-V2). Finally, qPCR experiments were performed on the resulting cDNA using EZ-Probe qPCR Master Mix-UDG (EZB-EZB-Probe-U2).

[0114] The expression level of the target gene mRNA in each sample was calculated by the ΔΔCT relative quantification method. The relative expression of the target gene was expressed as 2-ΔΔCT. The specific method was to subtract the CT value of the internal reference gene (GAPDH) from the CT value of the target gene in each sample to obtain the ΔCT value. Subsequently, the ΔΔCT was obtained by subtracting the ΔCT of the drug administration group (sample) from the ΔCT of the control group containing only the transfection reagent (RNAiMAX Control). Finally, the relative expression of CFB mRNA (value of sample) was obtained by converting ΔΔCT to 2-ΔΔCT.

[0115] The inhibition rate of the MARC1 gene mediated by the RNAi agent was calculated according to the following formula. The results are shown in Table 3.

[0116] Inhibition rate of MARC1 gene % = (1 - value of sample / Average value of RNAiMAX Control) * 100. A indicates that the inhibition rate is greater than 80%; B indicates that the inhibition rate is 50% - 80%; C indicates that the inhibition rate is less than 50%.

[0117] Table 3 Inhibition rates of different RNAi agents on the MARC1 gene at 1 nM and 0.05 nM concentrations

[0118]

[0119]

[0120] As can be seen from the results in Table 3, the RNAi agent of the present invention has a very good inhibitory effect on the MARC1 gene at different concentrations.

[0121] Example 7

[0122] In vitro test of MARC1 RNAi agent modified sequence in HuH7 cells

[0123] The effect of siRNA on the expression of MARC1 gene in HuH7 cells was detected by q-PCR experiment (Taqman probe method).

[0124] To determine the inhibition efficiency at the mRNA level, after transfecting MARC1 siRNA into HuH7 cells, q-PCR experiment was performed to measure the expression level of MARC1 mRNA. Specifically, HuH7 cells were cultured in DMEM medium (Gibco 11965-092) containing 10% fetal bovine serum (ExCell BioFSP500), 1% glutamine (Gibco 35050061), 1% NEAA (Gibco 11140050), 1% penicillin-streptomycin (HyClone SV30010). HuH7 cells in the logarithmic growth phase were taken and seeded in a 96-well cell culture plate at a density of 2×10 4 cells / well. At the same time of plating, siRNA was mixed with Lipofectamine TM RNAiMAX (INVITROGEN 13778150) so that the final concentration of the RNAi agent mixture was 10 nM, 1 nM, 0.1 nM, 0.05 nM, and then the RANi agent mixture was transfected into the cells. The cells were cultured overnight in a 37°C 5% CO2 incubator, and 2 replicates were measured in parallel. At the same time, a cell control group without compound containing Lipofectamine TM RNAiMAX was set up. After 48 h of transfection, the culture medium was removed, and total cellular RNA was extracted (QIAGEN-74182) and reverse transcribed (Vazyme-R323-1). The target cDNA was detected by the TaqMan method, and GAPDH cDNA was detected as an internal control for parallel detection.

[0125] The expression level of the target gene mRNA in each sample was calculated by the ΔΔCT relative quantification method. The relative expression level of the target gene was expressed using 2 -ΔΔCt . The specific method was to subtract the CT value of the internal reference gene (GAPDH) from the CT value of the target gene in each sample to obtain the ΔCT value. Subsequently, the ΔCT of the dosing group (sample) was subtracted from the ΔCT value of the control group containing only the transfection reagent (RNAiMAXControl) to obtain ΔΔCT. Finally, 2 was performed on ΔΔCT-ΔΔCt After conversion, the relative expression level of MARC1 mRNA (value of sample) was finally obtained.

[0126] The inhibition rate of the MARC1 gene mediated by the siRNA analog was calculated according to the following formula, and the results are shown in Tables 4, 5, and 6.

[0127] MARC1 inhibition rate % = (1 - value of sample / Average value of RNAiMAX Control) * 100; A indicates that the inhibition rate is greater than 80%; B indicates that the inhibition rate is 50% - 80%; C indicates that the inhibition rate is less than 50%.

[0128] Table 4 MARC1 gene inhibition rates of different modified RNAi agents at a concentration of 1 nM

[0129]

[0130]

[0131] From the results in Table 4, it can be seen that the modified sequences of the RNAi agents of the present invention all have very good inhibitory effects on the MARC1 gene.

[0132] Table 5 MARC1 gene inhibition rates of different modified RNAi agents at a concentration of 0.1 nM

[0133]

[0134]

[0135] Table 6 MARC1 gene inhibition rates of different modified RNAi agents at a concentration of 0.05 nM

[0136] Number Inhibition rate (%) Number Inhibition rate (%) Number Inhibition rate (%) Number Inhibition rate (%) M341 B M343 B M344 B M357 B M361 B M372 B M390 B M405 B M416 B M457 B M458 B M460 B M462 B M463 B M562 B M564 B M566 B M571 B M572 B M575 A M578 B M584 B M609 B M613 B M614 B M615 A M616 B M618 B M624 B M631 B M651 B M671 A M672 B M674 B M684 B M685 A M687 B M688 A M690 A M691 A M692 B M722 B M726 B M727 A M728 A M730 B M731 A M740 B M745 B M746 B M747 A M748 A M754 B M755 B M756 A M757 B M758 B M783 B M781 B M782 B M785 A M786 B M787 B M788 A M789 B M791 B M827 B M828 A M831 B M867 B M874 B M893 B M894 B M897 B M899 B M900 A M901 B M902 B M903 A M904 A M905 A

[0137] From the results in Tables 5 and 6, it can be seen that the modified sequences of the RNAi agents of the present invention all have very good inhibitory effects on the MARC1 gene at low concentrations (e.g., 0.1 nM or 0.05 nM).

[0138] The sequence listing was prepared in accordance with WIPO Sequence STANDARD ST.26, wherein, Table A – Conventional Nucleotide Symbols, and Definition:

[0139] Table A

[0140]

[0141] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An RNAi agent for inhibiting MARC1 expression, the RNAi agent comprising a sense strand and an antisense strand, the sense strand and the antisense strand forming a duplex region, wherein, The sense strand and the antisense strand are selected from any one group of the sequences described in Table 1.

2. The RNAi agent according to claim 1, wherein The sense strand and / or the antisense strand independently comprises one or more modified nucleotides.

3. The RNAi agent according to claim 2, wherein, The modified nucleotides are selected from: alkyl nucleotides, 2'-O-methyl modified nucleotides, 2'-O-2-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 5'-methyl phosphate nucleotides, 5'-C-methyl phosphate group nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, vinyl phosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, dithiophosphate nucleotides, locked nucleic acids (LNA), glyceryl nucleic acids (GNA), or morpholino oligonucleotides (PMO), or one or more of them.

4. The RNAi agent according to any one of claims 1-3, characterized in that, The RNAi agent is selected from double-stranded siRNA analogs prepared from the double-stranded siRNA analogs shown in Table 1 and having a modified form represented by the following formula: Sense strand: NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm; Antisense strand: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm; Wherein: "N" represents a nucleotide, such as A, G, U, and C; m means that the nucleotide adjacent to its left is a 2'-O-methyl modified nucleotide. For example, Am, Um, Gm, and Cm respectively represent 2'-O-methyl modified A, U, G, and C; f means that the nucleotide adjacent to its left is a 2'-fluoro modified nucleotide. For example, Af, Uf, Gf, and Cf respectively represent 2'-fluoro modified A, U, G, and C; s means that there is a phosphorothioate group linkage between the two nucleotides adjacent to the letter s on the left and right.

5. The RNAi agent according to claim 4, characterized in that, The RNAi agent is selected from any one group of the sequences described in Table 2, wherein m means that one nucleotide adjacent to the left is a 2'-methoxy modified nucleotide, f means that one nucleotide adjacent to the left of the letter f is a 2'-fluoro modified nucleotide, and the lowercase letter s means that there is a phosphorothioate group linkage between the two nucleotides adjacent to the letter on the left and right.

6. The RNAi agent according to any one of claims 1-5, characterized in that, The RNAi agent is linked to a targeting ligand. Preferably, the targeting ligand is linked to the 3' or 5' end of the sense strand or the antisense strand.

7. The RNAi agent according to any one of claims 1-6, characterized in that, The targeting ligand is selected from:

8. A pharmaceutical composition, characterized in that, Comprising the RNAi agent according to any one of claims 1-7 and one or more pharmaceutically acceptable excipients and / or carriers.

9. Use of the RNAi agent according to any one of claims 1-7 or the pharmaceutical composition according to claim 8 in the preparation of a drug for preventing or treating a disease related to MARC1.

10. The use according to claim 9, characterized in that, The MARC1-related diseases are selected from liver diseases. Preferably, the liver diseases are selected from non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

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