Rnai agent used for inhibiting 17β-HSD13 gene expression, preparation method therefor, and use

Abstract

The present invention relates to an RNAi agent used for inhibiting 17β-HSD13 gene expression, a preparation method therefor, and a use. The RNAi agent is used for treating HSD17B13-related diseases and conditions, comprising NAFLD, NASH, liver fibrosis, or alcoholic liver disease or non-alcoholic liver disease such as cirrhosis.

Description

RNAi agents for inhibiting 17β-HSD13 gene expression and methods of making and uses thereof TECHNICAL FIELD

[0001] The present invention relates to the field of medicine, and more specifically, the present invention relates to RNAi agents for inhibiting 17β-HSD13 gene expression and methods of making and uses thereof. BACKGROUND

[0002] Liver lipid droplet protein 17β-hydroxysteroid dehydrogenase type 13 (commonly referred to as HSD17B13, 17β-HSD13, HSD17β13, 17β-HSD13, 17β-HSD 13 type, or 17-HSD13) is a member of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The 17β-HSD family consists of 14 enzymes that are involved in the reduction or oxidation of sex hormones, fatty acids, and bile acids. Tissue distribution, subcellular localization, and catalytic preference vary among the various family members. The 17β-HSD family shows different substrate specificity, including steroids, lipids, and retinoids.

[0003] 17β-HSD13 protein is distributed in a wide range of tissues in the body and is encoded by the HSD17B13 gene (alternatively referred to as the 17β-HSD13 gene). Highest expression levels are known to be found in hepatocytes of the liver, while lower levels can be detected in the ovary, bone marrow, kidney, brain, lung, skeletal muscle, urinary bladder, and testis. The function of 17β-HSD13 is not fully understood, however, some 17β-HSD family members, including 17β-HSD-4, -7, -10, and -12, have been shown to be involved in carbohydrate and fatty acid metabolism. This suggests that 17β-HSD13 can also play a role in lipid metabolic pathways. It has been reported that hepatic upregulation of 17β-HSD13 has been observed in patients with fatty liver, which supports a role for this enzyme in the pathogenesis of nonalcoholic fatty liver disease (NAFLD).

[0004] Wen Su et al. have previously identified 17β-HSD13 as a lipid droplet (LD)-associated protein in patients with NAFLD and reported that 17β-HSD13 is one of the most abundantly expressed LD proteins that specifically localize on the surface of LDs. (Wen Su et al., Comparative proteomic study reveals 17β-HSD13 as a pathogenic protein in nonalcoholic fatty live disease, 111 PNAS 11437-11442 (2014)).

[0005] Studies have shown that HSD17B13 gene expression plays an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Although the patents with international patent application publication numbers WO2019183329, WO2020061177, WO2020132564, WO2021113820, etc. disclose RNA compounds and can inhibit the expression of HSD17B13 gene, no drug has been approved for NASH or listed for other diseases and conditions of NAFLD or ARLD. SUMMARY

[0006] In view of the problems existing in the prior art, the present application provides an RNAi agent for inhibiting the expression of 17β-HSD13 gene and a preparation method and use thereof, which has a good inhibitory effect on the gene expression of 17β-HSD13.

[0007] In a first aspect, the present application provides an RNAi agent for inhibiting the expression of 17β-HSD13 gene, or a pharmaceutically acceptable salt thereof,

[0008] The RNAi agent comprises a sense strand and an antisense strand, the sense strand is at least partially complementary to the antisense strand, and the sense strand and the antisense strand are selected from the RNAi agent shown in formula (I),

[0009] Sense strand: X1UAAGAAGUCUGAUAGAUX2X3

[0010] Antisense strand: Y1Y2AUCUAUCAGACUUCUUAY3Y4Y5

[0011] (I)

[0012] Wherein, X1 is selected from nucleotide G or is absent; X2 is selected from nucleotide G, U; X3 is selected from nucleotide A, U, G or is absent;

[0013] Y1 is selected from nucleotide A, C, U or is absent; Y2 is selected from nucleotide A, C; Y3 is selected from nucleotide C, I;

[0014] Y4 is selected from nucleotide A, C, G, I; Y5 is selected from nucleotide A, C, G or is absent;

[0015] Nucleotide I represents inosine nucleotide.

[0016] Wherein, the sense strand and the antisense strand have at least 15 consecutive and complementary nucleotides, preferably, the sense strand and the antisense strand have 17, 19, 21 consecutive and complementary nucleotides, more preferably 19.

[0017] As a preferred technical solution of the present application, X1 is selected from nucleotide G, X2 is selected from nucleotide G, U, and X3 is absent;

[0018] Y1 is absent, Y2 is selected from nucleotide A, C; Y3 is selected from nucleotide C, Y4 is selected from nucleotide C, G, and Y5 is selected from A, C, G.

[0019] As a preferred technical solution of the present application, the RNAi agent is selected from:

[0020] the antisense strand comprises the nucleotide sequence of SEQ ID NO: 46 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 45;

[0021] the antisense strand comprises the nucleotide sequence of SEQ ID NO: 48 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 47;

[0022] the antisense strand comprises the nucleotide sequence of SEQ ID NO: 50 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 49;

[0023] the antisense strand comprises the nucleotide sequence of SEQ ID NO: 52 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 51;

[0024] the antisense strand comprises the nucleotide sequence of SEQ ID NO: 54 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 53;

[0025] the antisense strand comprises the nucleotide sequence of SEQ ID NO: 56 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 55;

[0026] the antisense strand comprises the nucleotide sequence of SEQ ID NO: 58 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 57;

[0027] the antisense strand comprises the nucleotide sequence of SEQ ID NO: 60 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 59;

[0028] the antisense strand comprises the nucleotide sequence of SEQ ID NO: 62 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 61;

[0029] the antisense strand comprises the nucleotide sequence of SEQ ID NO: 64 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 63;

[0030] the antisense strand comprises the nucleotide sequence of SEQ ID NO: 256 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 255;

[0031] the antisense strand comprises the nucleotide sequence of SEQ ID NO: 258 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 257.

[0032] In a second aspect, the present application further provides an RNAi agent, or a pharmaceutically acceptable salt thereof, for inhibiting the expression of HSD17B13 gene,

[0033] wherein the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is at least partially complementary to the antisense strand, and the sense strand and / or the antisense strand is selected from the sequences shown in Table 1.

[0034] wherein the sense strand and the antisense strand can be partially, substantially or completely complementary to each other, for example, the sense strand and the antisense strand can be 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary.

[0035] wherein the length of the nucleotides of the sense strand and the antisense strand can be the same or different, for example, the sense strand comprises 19 nucleotides, the antisense strand comprises 21 nucleotides, or the sense strand comprises 19 nucleotides, the antisense strand also comprises 19 nucleotides, or the sense strand comprises 21 nucleotides, the antisense strand comprises 19 nucleotides, or the sense strand comprises 21 nucleotides, the antisense strand comprises 21 nucleotides.

[0036] As a preferred technical solution of the present application, the sense strand and / or the antisense strand comprises at least one modified nucleotide, preferably each nucleotide in the sense strand and / or the antisense strand is a modified nucleotide.

[0037] As a preferred technical solution of the present application, the modified nucleotide is selected from:

[0038] alkyl nucleotide, methoxy nucleotide, ethoxy nucleotide, methoxyethyl nucleotide, amino nucleotide, fluorinated nucleotide, deoxy nucleotide, 5'-methyl phosphate nucleotide, 5'-C-methyl phosphonate nucleotide, 2'-deoxy-2'-fluoro nucleotide, vinyl phosphonate nucleotide (VP), phosphorothioate nucleotide, phosphorodithioate nucleotide, locked nucleic acid (LNA), morpholino oligonucleotide (PMO), glycol nucleic acid (GNA); wherein the alkyl nucleotide is selected from methyl nucleotide and ethyl nucleotide; the glycol nucleic acid comprises (S)-glycol nucleic acid ((S)-GNA) and (R)-glycol nucleic acid ((R)-GNA).

[0039] wherein the sense strand and the antisense strand of the RNAi agent each comprise at least one modified nucleotide, in a particular embodiment, the sense strand comprises at least one modified nucleotide, the nucleotides of the antisense strand are unmodified, or, the nucleotides of the sense strand are unmodified, the antisense strand comprises at least one modified nucleotide. In a particular embodiment, each nucleotide of the sense strand is modified, each nucleotide of the antisense strand is also modified.

[0040] As a preferred technical solution of the present application, the RNAi agent is selected from the sequences shown in Table 2.

[0041] As a preferred technical solution of the present application, the RNAi agent is linked with a targeting ligand.

[0042] As a preferred technical solution of the present application, the targeting ligand comprises an N-acetyl-galactosamine (GalNAc) moiety.

[0043] As a preferred technical solution of the present application, the targeting ligand is linked to the 3' or 5' end of the sense strand and / or the antisense strand.

[0044] As a preferred technical solution of the present application, the RNAi agent is selected from the RNAi agent compounds shown in Table 3.

[0045] In some particular embodiments, the targeting ligand is linked to the 5' end of the sense strand. In some particular embodiments, the targeting ligand is linked to the 3' end of the sense strand. In some particular embodiments, the targeting ligand can also be linked internally to the nucleotides on the sense strand and / or the antisense strand of the RNAi agent. In some particular embodiments, the targeting ligand can also be linked to the RNAi agent via a linker, for example, the targeting ligand can also be linked to the 3' or 5' end of the sense strand via a linker, or, the targeting ligand can also be linked to the 3' or 5' end of the antisense strand via a linker, or, the targeting ligand can also be linked internally to the nucleotides on the sense strand and / or the antisense strand of the RNAi agent via a linker.

[0046] As a preferred technical solution of the present application, the targeting ligand is selected from:

[0047] further selected from

[0048] As a preferred technical solution of the present application, the pharmaceutically acceptable salt is selected from sodium salt, potassium salt, calcium salt, magnesium salt, iron salt, ammonium salt, lithium salt, hydrochloride, trifluoroacetate, oxalate, maleate, benzoate, salicylate, succinate, citrate. Among them, the ammonium salt is selected from triethylamine salt. Preferably, the pharmaceutically acceptable salt is selected from sodium salt or potassium salt, more preferably, the pharmaceutically acceptable salt is selected from sodium salt.

[0049] In a third aspect, the present application provides a pharmaceutical composition for inhibiting the expression of HSD17B13 gene, comprising the RNAi agent or a pharmaceutically acceptable salt thereof as described above.

[0050] As a preferred technical solution of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0051] Among them, the excipient includes (but is not limited to): absorption enhancers, anti-adhesion agents, antifoaming agents, antioxidants, binders, buffers, carriers, coating agents, coloring agents, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, flavoring agents, glidants, humectants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water repellents, wetting agents, lubricants such as sodium lauryl sulfate and magnesium stearate, flavorings and fragrances.

[0052] In a fourth aspect, the present application also provides a use of the RNAi agent or a pharmaceutically acceptable salt thereof as described above, or the pharmaceutical composition as described above in the preparation of a medicament for treating a disease, disorder or symptom mediated at least in part by the expression of HSD17B13 gene.

[0053] As a preferred technical solution of the present application, the disease is selected from NAFLD, NASH, liver fibrosis, or alcoholic liver disease or non-alcoholic liver disease, for example, liver cirrhosis.

[0054] Among them, the Chinese name of NAFLD is non-alcoholic fatty liver disease, also known as metabolic dysfunction-associated fatty liver disease (MAFLD); the Chinese name of NASH is non-alcoholic steatohepatitis, also known as metabolic dysfunction-associated steatohepatitis (MASH).

[0055] The RNAi agent for inhibiting the expression of HSD17B13 gene or a pharmaceutically acceptable salt thereof provided by the present application has good inhibitory activity on HSD17B13 and can be used for preventing and / or treating related diseases mediated by the expression of HSD17B13 gene.

[0056] In some embodiments, the pharmaceutical compositions of the present application can be administered in a variety of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be made by any of the 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 powders or aerosols, including by nebulizers, intratracheal, intranasal), dermal, transdermal, oral or parenteral. Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous (via an implanted device), intracranial, intrahepatic, intrathecal and intraventricular administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection.

[0057] In some embodiments, the level of gene expression and / or mRNA level of HSD17B13 in a subject administered the HSD17B13 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 prior to administration of the HSD17B13 RNAi agent or a subject not receiving the HSD17B13 RNAi agent. The level of gene expression and / or mRNA level in the subject can be reduced in a cell, cell population, and / or tissue of the subject. In some embodiments, the level of protein of HSD17B13 in a subject administered the HSD17B13 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 prior to administration of the HSD17B13 RNAi agent or a subject not receiving the HSD17B13 RNAi agent. The level of protein in the subject can be reduced in a cell, cell population, tissue, blood, and / or other fluid of the subject. Reduction in the level of gene expression, mRNA, or protein can be assessed by any method known in the art. Reduction or decrease in the level of HSD17B13 mRNA and / or protein is collectively referred to herein as reduction or decrease of HSD17B13, or inhibition or reduction of expression of HSD17B13.

[0058] The present application also provides a method of making the nucleic acid of any one of the preceding claims, wherein the sense strand and the antisense strand comprised by the nucleic acid can be conveniently and routinely made by the known techniques of solid phase synthesis. Any other method known in the art for such synthesis, such as liquid phase synthesis or fermentation, can additionally or alternatively be used. The use of similar techniques to make other oligonucleotides, such as phosphorothioates and alkylated derivatives, is also known.

[0059] The term "comprising" as used herein is used in the sense of "including", and the

[0060] The term "silencing," "reducing," "inhibiting," "down-regulating," or "knocking down gene expression" when referring to the HSD17B13 gene means that the expression of the gene (as measured by the level of RNA transcribed from the gene in a cell, cell population, or tissue in which the HSD17B13 gene is transcribed or the level of a polypeptide, protein, or protein subunit translated from the mRNA) is reduced when a cell, cell population, or tissue is treated with the HSD17B13 RNAi agent compared to the same cell, cell population, or tissue prior to administration of the HSD17B13 RNAi agent.

[0061] The term "targeting ligand" can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HAS), low density lipoprotein (LDL), or globulin); a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include the following polyamino acids: polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene acid-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimeric polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine, or alpha helical peptide.

[0062] The targeting ligand can also be a cell or tissue targeting agent that binds to a specified cell type, such as a lectin, glycoprotein, lipid, or protein, e.g., an antibody. The targeting group can be a thyroid stimulating hormone, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acid, multivalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, cholic acid, folate, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic.

[0063] The targeting ligand can also be a protein, e.g., a glycoprotein, or a peptide, e.g., a molecule having a specific affinity for a co-ligand, or an antibody, e.g., an antibody that binds to a specified cell type, e.g., a hepatocyte. The ligand can also include a hormone and a hormone receptor. They can also include non-peptide species, e.g., a lipid, a lectin, a sugar, a vitamin, a co-factor, a multivalent lactose, a multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, e.g., a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.

[0064] The targeting ligand can be a substance, e.g., a drug, that can increase uptake of the RNAi agent into a cell, e.g., by perturbing the cytoskeleton of the cell, e.g., by perturbing the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, iaplakinolide, halichondrin A, phalloidin, swinholide A, indanocine, or myoservin.

[0065] The term "antisense oligonucleotide" refers to a single-stranded oligonucleotide molecule having a nucleobase sequence complementary to a corresponding segment of a target nucleic acid, e.g., a genomic sequence of interest, an mRNA precursor, or an mRNA molecule. In certain embodiments, the antisense oligonucleotide is 12 to 30 nucleobases in length. In certain embodiments, the antisense oligonucleotide is an unmodified or modified nucleic acid having a nucleotide sequence complementary to a sequence of a target nucleic acid, such as a HSD17B13 polynucleotide.

[0066] The term "antisense strand" generally refers to a strand of an RNAi agent that includes a region of substantial complementarity to a target sequence. As used herein, the term "region of complementarity" generally refers to a region on an antisense strand that is substantially complementary to a sequence defined herein, e.g., a target sequence. When the region of complementarity is not perfectly complementary to the target sequence, mismatches can be internal or at the terminal regions of the molecule. Generally, the most tolerated mismatches are at the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' terminus.

[0067] The term "sense strand" generally refers to a strand of an RNAi agent that includes a region of substantial complementarity to a region of an antisense strand as defined herein. The "sense" strand is sometimes referred to as the "plus" 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 using a 5' cap.

[0068] In the present application, "complementary" has the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases on the other strand in a complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair comprises one purine and one pyrimidine. When the adenine on one strand always pairs with the uracil on the other strand, and the guanine always pairs with the cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand, and, correspondingly, "mismatch" in the art means that the bases at the corresponding positions in a double-stranded nucleic acid are not paired in a complementary fashion. In the present specification, in some embodiments, a "mismatch" can be a nucleotide substitution of adenine (A) and / or cytosine (C) in an oligonucleotide with guanine (G) and / or uracil (U), respectively, in some embodiments, such a mismatch allows the oligonucleotide (e.g., siRNA) to form a G-U wobble base pair with the target mRNA.

[0069] The term "complementary" when used in reference to a first nucleotide sequence (e.g., a sense strand of an RNAi agent or an HSD17B13 mRNA) in relation to a second nucleotide sequence (e.g., an antisense strand of an RNAi agent) refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base-pair hydrogen bonds) and form a duplex or double helix structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimetics, as long as the above requirements regarding their hybridization ability are met. "Complementary" does not necessarily have to have nucleobase complementarity at every nucleoside. Rather, some mismatches can be tolerated.

[0070] The term "fully complementary" generally means that all (100%) of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The contiguous sequence can comprise all or a portion of the first or second nucleotide sequence. As used herein, "partially complementary" generally means that in a pair of hybridized nucleobase sequences, at least about 70% of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. As used herein, "substantially complementary" generally means that in a pair of hybridized nucleobase sequences, at least about 90% of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The terms "complementary," "fully complementary," and "substantially complementary" as used herein can be used in terms of base pairing between the sense strand and the antisense strand of an RNAi agent or between the antisense strand of an RNAi agent and the sequence of an HSD17B13 mRNA. Sequence identity or complementarity is independent of modification. For purposes of determining identity or complementarity, for example, a and Af are complementary to U (or T) and identical to A.

[0071] In the present application, "one or more modified nucleotides" 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;

[0072] In the present invention, the modified nucleotides include, but are not limited to, 5'-phosphorothioate nucleotides, 5'-methylated cytosine nucleotides, 5'-methylphosphate nucleotides, 2'-O-methyl-modified nucleotides, inverted 2'-O-methyl nucleotides, 3'-O-methyl nucleotides, 2'-O-2-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, alkyl nucleotides, 5'-C-methylphosphonate 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, vinylphosphonate nucleotides, phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), abasic nucleotides, abasic ribose (Ab), inverted deoxyribonucleotides (3'-3' linked nucleotides or 5'-5' linked nucleotides), phospho(vinyl)deoxyribonucleotides, inverted abasic nucleotides (invAb), deoxythymidine, inverted deoxythymidine, 2'-amino-modified nucleotides, morpholino oligonucleotides (PMO), polypeptide nucleotides, phosphoramidate, or non-natural base nucleotides.

[0073] wherein alkyl-modified nucleotides, such as 2'-methyl nucleotides, 2'-ethyl nucleotides, 2'-methoxy-modified nucleotides, have the structure: 2'-methoxyethyl nucleotides, have the structure: 2'-fluoro nucleotides, have the structure: vinylphosphonate ribonucleotides (VP), have the structure: phosphorothioate nucleotides (s), have the structure: phosphonate nucleotides (p), have the structure: 2'-deoxyribonucleotides, have the structure: inverted abasic nucleotides (invAb), have the structure: glycerol nucleic acids (GNA), including (S)-glycerol nucleic acids ((S)-GNA), have the structure: and (R)-glycerol nucleic acids ((R)-GNA), have the structure: inosine nucleotides (I), such as:

[0074] wherein Base represents a base, R represents an alkyl group, Me represents a methyl group, and Et represents an ethyl group.

[0075] The term "locked nucleic acid" is a nucleotide having a modified ribose moiety, wherein the ribose moiety includes an extra bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in a 3 '-endo conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and to reduce off-target effects (Elmen, J. et al. (2005) Nucleic Acids Research 33(1): 439-447; Mook, OR. et al. (2007) Mol Cancer Ther 6(3): 833-843; Grunweller, A. et al. (2003) Nucleic Acids Research 31(12): 3185-3193).

[0076] Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are incorporated herein by reference. Locked nucleic acid structures are as follows:

[0077] In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than 1 heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0078] The term "morpholino" means a sugar surrogate having the following formula:

[0079] In certain embodiments, morpholino groups can be modified, e.g., by adding or altering various substituents from the above morpholino structure. Such sugar surrogates are referred to herein as "modified morpholino groups."

[0080] In the present invention, capital letters C, G, U, A, T represent the base composition of nucleotides, unless otherwise specified. In addition, the bases of the present invention, in addition to the above-mentioned 5 common bases, also include base analogs: for example: inosine (Inosine, I). Inosine nucleotide is also called inosine acid, which can base pair with nucleotides containing adenine (A), cytosine (C), guanine (G) or uracil (U). Therefore, nucleotides containing adenine (A), cytosine (C), guanine (G) or uracil (U) can be replaced by inosine nucleotides in the nucleotide sequences described in the present invention. Lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; LNA indicates that the nucleotide adjacent to the right is a locked nucleic acid (LNA) modification; Lowercase letter s indicates that the two nucleotides between the left and right of the letter are connected by a phosphorothioate group; VP indicates that the nucleotide adjacent to the right of the letter VP is an (E)-vinyl phosphate-modified nucleotide. invAb indicates an inverted abasic nucleotide; dN indicates any deoxyribonucleotide; dA indicates a deoxyadenine nucleotide; dT indicates a deoxythymine nucleotide; dU indicates a deoxyuracil nucleotide; dC indicates a deoxycytosine nucleotide; dG indicates a deoxyguanine nucleotide. invAb is an inverted abasic nucleotide (5'-5' linked nucleotide when at the 5' end of the strand, and 3'-3' linked nucleotide when at the 3' end of the strand), and s is a phosphorothioate bond. In some of some embodiments, invAb is covalently attached to the 5' end of the sense strand via a phosphorothioate bond (s), such as: invAbs (inverted abasic nucleotide-5'-phosphorothioate).

[0081] It should be emphasized that the "modification" of the nucleotides described in the present disclosure includes but is not limited to the above examples, and the nucleotides can also be replaced by other nucleotides, for example, (S)-glycerol nucleic acid, etc.

[0082] The term "homologous" or "homology" generally refers to the number of nucleotides of a subject nucleic acid sequence that have matched with the same nucleotides of a reference nucleic acid sequence, typically determined by a sequence analysis program (e.g., Karlin and Altschul, 1990, PNAS 87:2264-2268; Karlin and Altschul, 1993, PNAS 90:5873-5877), or by visual inspection. As used herein, the term "complete homology" or "completely homologous" generally refers to complete (100%) homology or "identity" between a reference sequence and a subject nucleic acid sequence. As used herein, the term "substantially homologous" or "substantial homology" generally refers to a subject sequence sharing at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the homologous nucleotides at the same nucleotide positions in a reference sequence.

[0083] The terms "induce," "inhibit," "enhance," "elevate," "increase," "decrease," "reduce," and the like generally indicate a quantitative difference between two states. For example, "an amount effective to inhibit the activity or expression of HSD17B13" means that the level of HSD17B13 activity or expression in a treated sample will be lower than the level of HSD17B13 activity or expression in an untreated sample. The terms apply, for example, to expression levels and activity levels. The terms "decrease" and "reduce" are used interchangeably and generally mean any change that is less than the original. "Decrease" and "reduce" are relative terms, requiring a comparison between before and after measurements. "Decrease" and "reduce" include complete depletion.

[0084] The term "at least" includes the number next to the term "at least" and all subsequent numbers or integers logically included therein, as is clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 of the 21 nucleotides in a nucleic acid molecule" means that 19, 20, or 21 nucleotides have the indicated property. When "at least" precedes a series of numbers or a range, it is understood that "at least" modifies each of the numbers in that series or range.

[0085] The term "decrease" can be an overall decrease of about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% in the expression level / amount of a gene, gene product, e.g., protein, or biomarker in a first sample as compared to the expression level / amount of the corresponding gene, gene product, e.g., protein, or biomarker in a second sample, as detected by standard methods known in the art, such as those described herein. In certain embodiments, the term "decrease" refers to a decrease in the expression level / amount of a gene or biomarker in a first sample, wherein the decrease is at least about 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold, or 0.01-fold of the expression level / amount of the corresponding gene or biomarker in a second sample. In certain embodiments, the first sample is a sample obtained from a subject, and the second sample is a reference sample.

[0086] The term "expression" generally means the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenylation, addition of 5'-cap), and translation.

[0087] The term "pharmaceutically acceptable salt" refers to a salt of a compound (RNAi agent) of the present application, prepared from a compound of the present application having specific substituents with a pharmaceutically acceptable acid or base, wherein the above-mentioned salt includes, but is not limited to, sodium salt, potassium salt, calcium salt, magnesium salt, iron salt, ammonium salt, lithium salt, hydrochloride, trifluoroacetate, oxalate, maleate, benzoate, salicylate, succinate, citrate, and the like.

[0088] The pharmaceutical compositions of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal, and / or parenteral administration. The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of a compound that produces a therapeutic effect. In general, this amount will range from about 1% to about 99% of active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30% by weight. For further

[0089] The term "preventing and / or treating" includes not only the prevention and / or treatment of a disease, but also generally includes preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or slowing the onset of one or more symptoms associated with a disease, reducing and / or alleviating one or more symptoms associated with a disease, reducing the severity and / or duration of a disease and / or any symptoms associated therewith and / or preventing further increases in the severity of a disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by a disease, and generally any pharmacological effect that is beneficial to the patient being treated. The nucleic acid or pharmaceutical composition of the present application forms a viable therapeutic agent without requiring that complete cure or eradication of any symptom or manifestation of a disease be achieved. As is recognized in the relevant art, a drug used as a therapeutic agent can reduce the severity of a given disease state, but need not eliminate every manifestation of a disease to be considered a useful therapeutic agent. Similarly, a therapeutic agent prophylactically administered constitutes a viable prophylactic agent without being completely effective in preventing the onset of a disorder. It is sufficient to simply reduce the impact of a disease in a subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another therapy, or by producing another beneficial effect), or to reduce the likelihood of a disease from occurring or worsening.

[0090] The terms "disease" or "disorder" are used interchangeably and generally refer to any deviation from the normal state of a subject, for example, any change in the state of the body or of some of its organs, which impedes or deranges the performance of the functions, and / or which causes symptoms such as malaise, malfunction, distress, or even death in a person affected or exposed to it. A disease or disorder can also be referred to as a distemper, an ailing, an ailment, a malady, a disorder, a sickness, an illness, a complaint, an inderdisposion, or an affectation.

[0091] The term "effective amount" or "therapeutically effective amount" with respect to a drug or pharmacologically active agent means a nontoxic but sufficient amount of the drug or agent to achieve the desired effect. For oral dosage forms in the present application, an "effective amount" of one active substance in a composition means the amount needed to achieve the desired effect in conjunction with another active substance in the composition. The determination of an effective amount is dependent on the age and general condition of the recipient, on the particular active substance, and an appropriate effective amount in a given case can be determined by one of ordinary skill in the art based on routine testing.

[0092] The term "active ingredient", "therapeutic agent", "active substance" or "active agent" refers to a chemical entity that is effective in treating a target disorder, disease or condition.

[0093] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0094] The compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by a combination of the specific embodiments set forth below with other chemical synthetic methods well known to those skilled in the art, and equivalents thereof as appreciated by those skilled in the art, preferred embodiments including but not limited to the examples of the present application. DETAILED DESCRIPTION

[0095] The present application will be further described in conjunction with the examples below, but the embodiments of the application are not limited thereto.

[0096] Example 1 Synthesis of targeting ligand

[0097] The synthesis route of the targeting ligand L96 can refer to the document with international patent publication number WO2009073809, and the targeting ligand L96 can be connected to the RNA through a phosphate group, a thiophosphate group or another linking group, represents the RNA connection site.

[0098] The synthesis route of NAG37 can refer to the document with patent number CN201780042047.6, wherein, represents the RNA connection site.

[0099] The synthesis route of M10 can refer to the document with patent number CN202210705962.1, and R 2 represents the RNA.

[0100] The synthesis route of L10 can refer to the synthesis route of the patent with patent publication number WO2019105414A1, wherein Nu represents the RNA.

[0101] The synthesis route of GLS-15 can refer to the synthesis route of the patent with patent publication number CN202280016262.X, wherein, represents the RNA connection site.

[0102] The synthesis route of GL6 can refer to the synthesis method of compound 6 in the patent document WO2023241591, represents the RNA connection site.

[0103] Synthesis of RNAi agents of Example 2

[0104] The specific synthesis route is as follows:

[0105] Synthesis of sense strand and antisense strand:

[0106] By solid-phase phosphoramidite synthesis method, with blank CPG solid-phase carrier or GalNAc ligand connected CPG carrier as the starting cycle, according to the nucleotide arrangement sequence shown in Table 1, Table 2 and Table 3, nucleotide monomers and / or GalNAc ligands are connected one by one from 3'-5' direction. Each nucleotide monomer includes four-step reactions of deprotection, coupling, capping, oxidation or sulfuration.

[0107] Among them, the reagents used for the synthesis of siRNA conjugates are as follows:

[0108] The specific synthesis conditions are as follows:

[0109] The nucleotide monomers are dissolved in super-dry anhydrous acetonitrile to prepare a 0.1M solution, and 4A molecular sieves are added. The deprotection reaction conditions of each step are the same, i.e. the temperature is 25°C, the reaction time is 70 seconds, the deprotection reagent is TCA, and the volume is 300μL.

[0110] The coupling reaction conditions of each step are the same, including temperature of 25°C, reaction time of 10 minutes, nucleotide monomer injection volume of 200μL, and activated agent Act injection volume of 220μL.

[0111] The capping conditions of each step are the same, including temperature of 25°C, reaction time of 20 seconds. The capping reagent solution is a mixed solution of CapA and CapB with a molar ratio of 1:1, and the capping reagent injection volume is 300μL.

[0112] The oxidation reaction conditions of each step are the same, including temperature of 25°C, reaction time of 30 seconds. The oxidation reagent injection volume is 300μL.

[0113] The sulfuration reaction conditions of each step are the same, including temperature of 25°C, reaction time of 4 minutes, and sulfuration reagent injection volume of 300μL.

[0114] After the connection of the last nucleotide monomer is completed, the nucleic acid sequence on the solid-phase carrier is sequentially cut, deprotected, purified, and salted, and then freeze-dried to obtain the sense strand and the antisense strand. The cutting and deprotection conditions are as follows: the synthesized solid-phase carrier is added into a 2mL cryovial, 1mL concentrated ammonia is added, and the reaction is carried out in a 35°C constant temperature water bath for 16 hours. After filtration, the solid-phase carrier is washed with 50% ethanol aqueous solution three times, 1mL each time, and the filtrate is concentrated after centrifugation. The crude product is purified.

[0115] The purification and salt exchange conditions are as follows: the purification of nucleic acid samples is achieved by gradient elution of a reverse phase chromatography system using a preparative liquid chromatograph (Agilent 1260 Infinity III Bio-inert liquid chromatograph system). Specifically: the mobile phase A is 100 mM TEAA aqueous solution, the mobile phase B is 100 mM TEAA acetonitrile solution, the elution gradient is A:B = 90:10-70:30 (volume ratio), the chromatographic column is Pursuit C18, Agilent, 10x250mm, 5um, and the column temperature is 55°C. The qualified elution fractions are combined, concentrated and dried using a centrifugal concentrator, 5uL of 3M sodium acetate aqueous solution and 60uL of ethanol solution are added per OD sample for salt exchange, and the precipitated sample is washed with ethanol for several times, and then dried to obtain the sample powder.

[0116] Annealing:

[0117] The sense strand and the antisense strand are mixed at a molar ratio of 1:1, heated to 80°C in a water bath pot for 5 minutes, naturally cooled to room temperature, and freeze-dried to obtain the corresponding product.

[0118] (I) The RNAi agent is prepared by using OligoMaker ApS192 RNA synthesizer (Denmark) or Biolytic Dr.Oligo 48 synthesizer according to the above method, wherein the synthesis route can also refer to the patent document CN201980061674.3, and the sequence of the RNAi agent is shown in Table 1.

[0119] Table 1 is the sequence list of the RNAi agent

[0120] Among them, Comparative Example 1 and Comparative Example 2 are prepared by the same preparation method as the sequence in Table 1 above, and the sequences of Comparative Example 1 and Comparative Example 2 are as follows:

[0121] (II) The modified RNAi agent is prepared by using OligoMaker ApS192 RNA synthesizer (Denmark) or Biolytic Dr.Oligo 48 synthesizer according to the above preparation method, wherein the synthesis route can also refer to the patent document CN201980061674.3, and the sequence of the modified RNAi agent is shown in Table 2.

[0122] Table 2 is the sequence list of the modified RNAi agent

[0123] 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'-phosphorothioate; Ams = 2'-O-methyladenosine-3'-phosphorothioate; Ums = 2'-O-methyluridine-3'-phosphorothioate; Cms = 2'-O-methylcytidine-3'-phosphorothioate; Gms = 2'-O-methylguanosine-3'-phosphorothioate; Af = 2'-fluoroadenosine-3'-phosphate; Uf = 2'-fluorouridine-3'-phosphate; Cf = 2'-fluorocytidine-3'-phosphate; Gf = 2'-fluoroguanosine-3'-phosphate; Afs = 2'-fluoroadenosine-3'-phosphorothioate; Ufs = 2'-fluorouridine-3'-phosphorothioate; Cfs = 2'-fluorocytidine-3'-phosphorothioate; Gfs = 2'-fluoroguanosine-3'-phosphorothioate; m = 2'-O-methyl; f = 2'-fluorine; s = phosphorothioate bond, wherein the VP structure is The gn structure is The TNA structure is:

[0124] (III) Using OligoMaker ApS 192 RNA (Denmark) or Biolytic Dr. Oligo 48 synthesizer to prepare modified RNAi agents according to the preparation method described above, then connecting the targeting ligand L96 / L10 of Example 1 to the 3' end of the sense strand of the modified RNAi agent in Table 2, or connecting the NAG37 of Example 1 to the 5' end of the sense strand of the modified RNAi agent in Table 2, see Table 3. Wherein the synthesis route can also refer to patent document CN201980061674.3, the content of which is incorporated herein by reference.

[0125] Table 3

[0126] Wherein, the sequence of Comparative Example 3 is prepared by the same preparation method as the sequence in Table 3, and the specific sequence of Comparative Example 3 is as follows:

[0127] 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'-phosphorothioate; Ams = 2'-O-methyladenosine-3'-phosphorothioate; Ums = 2'-O-methyluridine-3'-phosphorothioate; Cms = 2'-O-methylcytidine-3'-phosphorothioate; Gms = 2'-O-methylguanosine-3'-phosphorothioate; Af = 2'-fluoroadenosine-3'-phosphate; Uf = 2'-fluorouridine-3'-phosphate; Cf = 2'-fluorocytidine-3'-phosphate; Gf = 2'-fluoroguanosine-3'-phosphate; Afs = 2'-fluoroadenosine-3'-phosphorothioate; Ufs = 2'-fluorouridine-3'-phosphorothioate; Cfs = 2'-fluorocytidine-3'-phosphorothioate; Gfs = 2'-fluoroguanosine-3'-phosphorothioate; m = 2'-O-methyl; f = 2'-fluoro; s = phosphorothioate, wherein the VP structure is wherein Nu represents the site of attachment to the siRNA.

[0128] Example 3 In vitro testing of HSD17B13 RNAi agents in HuH7 cells

[0129] ​​​​​​The human HSD17B13 gene exon region (GENBANK Accession No. NM_178135.5) was cloned into a reporter-based screening plasmid psiCHECK2 (Promega-C8021) to generate a Renilla luciferase / HSD17B13 fusion mRNA. HuH7 cells were cultured in DMEM (Gibco-10313021) medium containing 10% fetal bovine serum (ExCell Bio-FSP500), 1% glutamine (Gibco-35050061), 1% non-essential amino acids (Gibco-11140050), 1% penicillin-streptomycin (HyClone-SV30010). The HSD17B13-psiCHECK2 plasmid, RNAi agent and Lipo2000 (Invitrogen-11668019) transfection reagent diluted with Opti-MEM (Gibico-11058021) were added to the HuH7 cell suspension, and plated in a 96-well plate at a cell density of 1×10 5 / ml, so that the final concentration of the RNAi agent was 1 nM, 0.1 nM, 0.11 nM, 0.012 nM, 0.02 nM. After 24 hours of culture, the relative level of Renilla luciferase normalized to the level of constitutively expressed firefly luciferase also present in the psiCHECK2 plasmid was measured using a dual luciferase reporter assay (Promega-E2920).

[0130] The inhibition rate of the HSD17B13 gene mediated by the RNAi agent in Tables 1 and 2 was calculated according to the following formula.

[0131] The HSD17B13 gene inhibition rate (%) = (1 - sample Renilla luciferase relative level / control group Renilla luciferase relative level) × 100. The results are shown in Tables 4 and 5.

[0132] Table 4 is the inhibition rate of the RNAi agent on the HSD17B13 gene

[0133] wherein D≥50%.

[0134] Table 5

[0135] wherein H≥80%.

[0136] As can be seen from Tables 4 and 5, the RNAi agent of the present application has good inhibitory activity on the HSD17B13 gene.

[0137] Example 4

[0138] The siRNA stock solution was diluted with opti-MEM (Gbico, 31985070) to the required concentration, and a series of gradient dilutions were prepared, and then the siRNA working solution was further prepared. The RNAiMAX (Invitrogen, 13778100) was diluted with opti-MEM in proportion, and then mixed with the siRNA working solution, and the mixed solution was taken and added to the 96-well plate pre-coated with Matrigel (Miaoshun, HCTPM-R-001). After the PCH cells were recovered and centrifuged, they were resuspended and counted using the plating medium (Miaoshun, HCTPM-R-001), and then inoculated into the above-mentioned 96-well plate at a density of 70,000 cells per well, and the final concentration of siRNA in the cell culture system was 20, 4, 0.8, 0.16, 0.032, 0.0064 nM, and the cells were cultured at 37°C, 5% CO2 for 48 hours. After the culture ended, the culture medium was discarded, and the cells were washed with PBS, and the RNA was extracted according to the instructions of the kit (Transgene, AC301) and reverse transcribed, and the qPCR analysis (Transgene, AC732) was performed for the HSD17B13 mRNA, and the expression amount of the target gene in each test sample was determined by relative quantification using the comparative CT (ΔΔC) method, which measures the difference (ΔCt) between the target gene and the internal reference gene (GAPDH), and the formula is as follows:

[0139] ΔCT = average Ct of target gene - average Ct of GAPDH;

[0140] ΔΔCT = ΔCT (sample) - ΔCT (vehicle control);

[0141] Relative amount of mRNA of target gene = 2^-ΔΔCt;

[0142] Inhibition rate = (relative amount of vehicle control - relative amount of sample) / relative amount of vehicle control x 100%.

[0143] The inhibition rate of each concentration point is the average of the inhibition rates of two duplicate wells, and the relative IC50 value is calculated by Prism-GraphPad software, and the calculation results are shown in Table 6.

[0144] Table 6

[0145] As can be seen from Table 6, compared with the prior art, the RNAi agent of the present application has better inhibitory activity on the HSD17B13 gene.

[0146] Example 5 Evaluation of the in vivo activity of the RNAi agent using the HSD17B13-SEAP system in mice

[0147] To evaluate the in vivo activity of the RNAi compounds, 6-8 week old Balb / C mice were injected with the HSD17B13-SEAP system plasmid via hydrodynamically-tail vein injection (HDI) at least 7 days in advance, which was used to achieve transient transfection in mice. The plasmid contains a SEAP (secreted human placental alkaline phosphatase) reporter gene, with the HSD17B13 cDNA sequence (GenBank NM_178135.5) inserted into the 3'UTR of the SEAP gene. A total volume of 10% of the mouse body weight of normal saline containing 20 ug of the plasmid was injected into the mice via the tail vein within 3-5 seconds to achieve the construction of the HSD17B13-SEAP model mice. Subsequent treatment of the mice with the RNAi compounds inhibits the expression of HSD17B13 and is accompanied by the inhibition of SEAP expression. Before the administration of the RNAi (day -1), the baseline expression level of SEAP in the serum of the mice was measured using the Phospha-Light TM The baseline expression level of SEAP in the serum of the mice was measured using the Phospha-Light SEAP reporter gene detection system (Invitrogen), and the mice were grouped according to the average baseline SEAP level. The serum of the mice was collected on day 4, day 5, day 7, day 8, day 14, day 15, day 21, day 22, day 28, day 29, day 35, and day 36 after administration, and the SEAP expression at each time point was measured.

[0148] The SEAP expression normalization rate of a particular mouse was calculated by dividing the SEAP expression level of the mouse at a certain time point by the baseline SEAP expression level of the mouse. The inhibition rate of HSD17B13-SEAP at each time point was calculated according to the following formula: Inhibition rate of HSD17B13-SEAP (%) = (1 - SEAP expression normalization rate of a particular mouse / average of SEAP expression normalization rates of control mice) * 100.

[0149] Using the method described above, HSD17B13-SEAP mice were constructed by HDI, and RNAi compounds shown in Table 3 were administered at a dose of 1 mg / kg, 3 mg / kg, or 10 mg / kg by subcutaneous injection. The serum SEAP level at different time points was continuously detected, and the inhibition rate at the corresponding time point was calculated, and the results are shown in Tables 7 and 8, respectively.

[0150] Table 7 shows the in vivo activity of the RNAi compounds administered at a dose of 3 mg / kg

[0151] Table 8 shows the in vivo activity of the RNAi compounds administered at a dose of 1 mg / kg

[0152] wherein D≥50%.

[0153] As shown in Table 7 and Table 8, the RNAi compounds of the present application have good in vivo inhibitory activity against SEAP.

[0154] Example 6 Mouse in vivo activity of RNAi compounds evaluated using an adeno-associated viral expression system

[0155] To evaluate the in vivo activity of HSD17B13 RNAi compounds, C57BL / 6J mice (4-9 per group) infected with adeno-associated virus (AAV) were used. Each mouse was injected with 2X10^11 or 5X10^11 viral particles of AAV expressing human HSD17B13 via tail vein before dosing. Fourteen days after virus infection, the mice were administered with 1 mg / kg, 3 mg / kg or 5 mg / kg dose of RNAi compounds or saline (vehicle group). Ten to fifteen days after dosing, human HSD17B13 expression in the mouse liver was detected by RT-PCR. The expression level was evaluated by normalizing the relative expression level of human HSD17B13 mRNA to Gapdh mRNA of the corresponding mouse, and the inhibition rate of human HSD17B13 was calculated by the following formula:

[0156] Human HSD17B13 inhibition rate (%) = (1 - sample human HSD17B13 mRNA relative expression level / average of vehicle group human HSD17B13 mRNA relative level) x 100.

[0157] Table 9 Inhibitory activity of 5 mg / kg dose of RNAi compounds against HSD17B13

[0158] wherein E > 50%.

[0159] Example 7 Mouse in vivo activity evaluation

[0160] To evaluate the in vivo activity of HSD17B13 RNAi compounds, human HSD17B13 transgenic mice (B6.129-Hsd17b13tm1Cgn / J) were used (4-6 per group). The mice were administered with 1 mg / kg, 3 mg / kg, 5 mg / kg or 10 mg / kg dose of RNAi compounds or saline (vehicle group). Ten to fifteen days after dosing, human HSD17B13 expression in the mouse liver was detected by RT-PCR. The expression level was evaluated by normalizing the relative expression level of human HSD17B13 mRNA to Gapdh mRNA of the corresponding mouse, and the inhibition rate of human HSD17B13 was calculated by the following formula:

[0161] Human HSD17B13 inhibition rate (%) = (1 - sample human HSD17B13 mRNA relative expression level / average of human HSD17B13 mRNA relative level of the vehicle group) x 100. The calculation results are shown in Table 10.

[0162] Table 10 is the inhibition activity of the RNAi compound administered at a dose of 3 mg / kg

[0163] Table 11 is the inhibition activity of the RNAi compound administered at a dose of 5 mg / kg

[0164] wherein the inhibition rate F is greater than or equal to 60%.

[0165] As can be seen from Tables 10 and 11, the RNAi compound of the present application has good in vivo inhibition activity on HSD17B13.

[0166] Example 8 Evaluation of in vitro activity of cynomolgus monkey primary hepatocytes

[0167] To evaluate the in vitro activity of the RNAi conjugate, we performed a free uptake experiment in cynomolgus monkey primary hepatocytes (PCH), and the specific experimental operation is as follows: PCH cells were cultured in CP medium (BIOIVT catalog number BIOIVTS03316) containing 10% fetal bovine serum (ExCell Bio FSP500). PCH cells (5 x 10 4 Cells / well) were seeded into 96-well cell plates coated with collagen in advance, and the RNAi conjugate was mixed with RNase-Free water at the same time, then added to the cell mixture, and the final concentration of the RNAi conjugate was set to 150.00, 50.00, 16.67, 5.56, 1.85, 0.62, 0.21, 0.069, 0.023 and 0.008 nM at 10 concentration points, and each concentration point had 2 replicate wells. At the same time, a no-compound cell control group containing RNase-Free water was set. After incubation for 48 hours, the culture medium was removed, total RNA extraction (QIAGEN-74182) and reverse transcription (Vazyme-R323-1) were performed. The target cDNA was detected by TaqMan method, and β-actin cDNA was detected as an internal control for parallel detection.

[0168] The expression level of each sample target gene mRNA was calculated by the ΔΔCT relative quantification method. The relative expression of the target gene was expressed by 2-ΔΔCt. The specific method was to subtract the CT value of the internal reference gene (ACTB) from the CT value of the target gene in each sample to obtain the ΔCT value, then subtract the ΔCT value of the compound-free control group (RNase-Free water Control) from the ΔCT value of the sample group to obtain the ΔΔCT value, finally convert the ΔΔCT value by 2-ΔΔCt, and finally obtain the relative expression value of HSD17B13 mRNA (value of sample).

[0169] The inhibition rate of HSD17B13 gene in each sample mediated by the RNAi conjugate was calculated according to the following formula:

[0170] HSD17B13 inhibition rate % = (1-value of sample / Average value of RNase-Free water Control)*100.

[0171] The inhibition rate of each concentration point is the average of the inhibition rates of 2 duplicate wells; the absolute EC 50 value (Absolute EC50) was calculated by Prism-GraphPad software. The results are shown in Table 12.

[0172] Table 12

[0173] From Table 12, it can be seen that the compound of the present application has better inhibition activity on HSD17B13 gene compared with the prior art.

[0174] Example 9 In vitro activity evaluation of human primary hepatocytes

[0175] In order to evaluate the in vitro activity of the RNAi conjugate, we carried out a free uptake experiment in human primary hepatocytes (PHH), and the specific experimental operation was as follows: the PHH cells were cultured in CP medium (BIOIVT item number BIOIVTS03316) containing 10% fetal bovine serum (ExCell Bio FSP500). The PHH cells were seeded (5×10 4The cells were plated into 96-well cell plates coated with collagen in advance, and the RNAi conjugate was mixed with RNase-Free water, and then added to the cell mixture, and the final concentration of the RNAi conjugate was set to 150.00, 50.00, 16.67, 5.56, 1.85, 0.62, 0.21, 0.069, 0.023 and 0.008 nM at 10 concentration points, and each concentration point had 2 duplicate wells. At the same time, a no compound cell control group containing RNase-Free water was set. After incubation for 48 hours, the culture medium was removed, total RNA extraction (QIAGEN-74182) and reverse transcription (Vazyme-R323-1) were performed. The target cDNA was detected by TaqMan method, and β-actin cDNA was detected as an internal control for parallel detection.

[0176] The expression level of the target gene mRNA of each sample was calculated by the ΔΔCT relative quantification method. The relative expression amount of the target gene was represented by 2-ΔΔCt. Specifically, the CT value of the target gene in each sample was subtracted from the CT value of the internal reference gene (ACTB) to obtain the ΔCT value, then the ΔCT of the sample was subtracted from the ΔCT value of the no compound control group (RNase-Free water Control) to obtain the ΔΔCT, finally the ΔΔCT was converted by 2-ΔΔCt, and finally the relative expression amount (value of sample) of HSD17B13 mRNA was obtained.

[0177] The inhibition rate of HSD17B13 gene in each sample mediated by the RNAi conjugate was calculated according to the following formula:

[0178] HSD17B13 inhibition rate % = (1-value of sample / Average value of RNase-Free water Control)*100.

[0179] The inhibition rate of each concentration point was the average of the inhibition rates of 2 duplicate wells; the absolute EC 50 value (Absolute EC50) was calculated by Prism-GraphPad software. The results are shown in Table 13.

[0180] Table 13

[0181] As can be seen from Table 13, compared with the prior art, the compound of the present application has better inhibition activity on HSD17B13 gene.

[0182] Example 10 In vivo activity evaluation in cynomolgus monkeys

[0183] To evaluate the in vivo efficacy of the RNAi agent compounds of the present application, male cynomolgus monkeys were used to test the in vivo activity of the RNAi agent conjugates in Table 3, according to the following procedure: A small amount of tissue from the left and right lobes of the liver was obtained from the cynomolgus monkeys by liver puncture 7 days prior to administration of the RNAi agent compounds (Day -7), and the endogenous HSD17B13 expression in the monkey liver tissue was detected by RT-PCR and normalized to the ACTB mRNA level in the corresponding tissue to determine the baseline relative expression of HSD17B13. On Day 0, each group of cynomolgus monkeys (n = 4) was subcutaneously injected with a 3 mg / kg dose of the RNAi agent compound, and then liver puncture was performed on Day 21, 42, 70, 98 and 126 to collect liver tissue and detect the relative expression of endogenous HSD17B13 in the animals, and the change in the HSD17B13 expression level relative to the baseline in the animals was calculated according to the following formula: Inhibition rate relative to baseline (%) = (1 - HSD17B13 expression level in a particular cynomolgus monkey at a particular time point / HSD17B13 expression level in the monkey on Day -7) * 100%. The in vivo efficacy of the RNAi agent compounds was evaluated by calculating the average of the relative baseline inhibition rate of HSD17B13 in the animals in the corresponding group at different time points.

[0184] Table 14 is the inhibition activity data on Day 21 and Day 42

[0185] As can be seen from Table 14, the RNAi agent compounds of the present application have good in vivo inhibition activity on HSD17B13 compared with the prior art Comparative Example 3.

[0186] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and all shall be included in the protection scope of the present application.

Claims

1. An RNAi agent for inhibiting the expression of a 17β-HSD13 gene, or a pharmaceutically acceptable salt thereof, characterized in that, The RNAi agent comprises a sense strand and an antisense strand, the sense strand being at least partially complementary to the antisense strand, the sense strand and the antisense strand being selected from the group consisting of RNAi agents represented by formula (I), Sense strand: X1UAAGAAGUCUGAUAGAUX2X3 Antisense strand: Y1Y2AUCUAUCAGACUUCUUAY3Y4Y5 (I) wherein X1is selected from nucleotide G or absent; X2is selected from nucleotide G, U; X3is selected from nucleotide A, U, G or absent; Y1is selected from nucleotide A, C, U or absent; Y2is selected from nucleotide A, C; Y3is selected from nucleotide C, I; Y4is selected from nucleotide A, C, G, I; Y5is selected from nucleotide A, C, G or absent; Nucleotide I represents inosine nucleotide.

2. The RNAi agent, or a pharmaceutically acceptable salt thereof, of claim 1, characterized in that, The X1is selected from nucleotide G, X2is selected from nucleotide G, U, X3is absent; the Y1is absent, Y2is selected from nucleotide A, C; the Y3is selected from nucleotide C, the Y4is selected from nucleotide C, G, the Y5is selected from A, C, G.

3. The RNAi agent, or a pharmaceutically acceptable salt thereof, of claim 1, wherein The RNAi agent is selected from the group consisting of: The antisense strand comprises the nucleotide sequence of SEQ ID NO: 46 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 45; The antisense strand comprises the nucleotide sequence of SEQ ID NO: 48 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 47; The antisense strand comprises the nucleotide sequence of SEQ ID NO: 50 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 49; The antisense strand comprises the nucleotide sequence of SEQ ID NO: 52 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 51; The antisense strand comprises the nucleotide sequence of SEQ ID NO: 54 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 53; The antisense strand comprises the nucleotide sequence of SEQ ID NO: 56 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 55; The antisense strand comprises the nucleotide sequence of SEQ ID NO: 58 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 57; The antisense strand comprises the nucleotide sequence of SEQ ID NO: 60 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 59; The antisense strand comprises the nucleotide sequence of SEQ ID NO: 62 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 61; The antisense strand comprises the nucleotide sequence of SEQ ID NO: 64 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 63; The antisense strand comprises the nucleotide sequence of SEQ ID NO: 256 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 255; The antisense strand comprises the nucleotide sequence of SEQ ID NO: 258 and the sense strand comprises the nucleotide sequence of SEQ ID NO:

257.

4. An RNAi agent for inhibiting the expression of a 17β-HSD13 gene, or a pharmaceutically acceptable salt thereof, characterized in that, The RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is at least partially complementary to the antisense strand, and the sense strand and / or the antisense strand is selected from the sequences shown in Table 1.

5. The RNAi agent, or a pharmaceutically acceptable salt thereof, of any one of claims 1-4, characterized in that, The sense strand and / or the antisense strand comprises at least one modified nucleotide, preferably each nucleotide of the sense strand and / or the antisense strand is a modified nucleotide.

6. The RNAi agent, or a pharmaceutically acceptable salt thereof, of claim 5, characterized in that, The modified nucleotide is selected from the group consisting of: alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5'-methylphosphonate nucleotides, 5'-C-methylphosphonate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinyl phosphonate nucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), glycol nucleic acids (GNA).

7. The RNAi agent, or a pharmaceutically acceptable salt thereof, of claim 5, wherein The RNAi agent is selected from the sequences shown in Table 2.

8. The RNAi agent, or a pharmaceutically acceptable salt thereof, of any one of claims 1-4, characterized in that, The RNAi agent is linked to a targeting ligand.

9. The RNAi agent, or a pharmaceutically acceptable salt thereof, of claim 8, characterized in that, The targeting ligand comprises an N-acetyl-galactosamine (GalNAc) moiety.

10. The RNAi agent, or a pharmaceutically acceptable salt thereof, of claim 8, characterized in that, The targeting ligand is linked to the 3' or 5' end of the sense strand and / or the antisense strand.

11. The RNAi agent, or a pharmaceutically acceptable salt thereof, of any one of claims 1-4, characterized in that, The RNAi agent is selected from the RNAi agent compounds shown in Table 3.

12. A pharmaceutical composition for inhibiting expression of a HSD17B13 gene, characterized by, The pharmaceutical composition comprises the RNAi agent of any one of claims 1-11, or a pharmaceutically acceptable salt thereof.

13. Use of the RNAi agent of any one of claims 1-11, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 12, for the manufacture of a medicament for the treatment of a disease, disorder, or symptom mediated at least in part by expression of the HSD17B13 gene.

14. Use according to claim 13, characterized in that, The disease is selected from the group consisting of NAFLD, NASH, liver fibrosis, or alcoholic or non-alcoholic liver disease, for example cirrhosis.

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