Rnai agent targeting inhbe

Abstract

Provided is an RNAi agent targeting INHBE. Specifically, provided are an RNAi agent targeting INHBE, a pharmaceutical composition, and a medical use thereof.

Description

RNAi agents targeting INHBE

[0001] This disclosure claims priority to Chinese patent application CN202410263661.7 filed on March 8, 2024 and Chinese patent application CN202410594262.9 filed on May 14, 2024. Technical Field

[0002] The present disclosure belongs to the field of biomedicine, and specifically relates to siRNA, conjugates, compositions and medical uses thereof targeting the inhibin βE gene (INHBE). Background Art

[0003] The inhibin beta E gene (INHBE) is a member of the TGF-β (transforming growth factor-β) superfamily of proteins and is involved in regulating numerous cellular processes, including cell proliferation, apoptosis, immune responses, and hormone secretion. Inhibin is responsible for inhibiting follicle-stimulating hormone secretion from the pituitary gland and can regulate a wide range of functions, including hypothalamic and pituitary hormone secretion, gonadal hormone secretion, germ cell development and maturation, erythrocyte differentiation, insulin secretion, neuronal survival, embryonic axial development, and skeletal growth. Furthermore, INHBE may be upregulated under conditions of endoplasmic reticulum stress, potentially inhibiting cell proliferation and growth in the pancreas and liver.

[0004] Body fat distribution is an important indicator of cardiovascular and metabolic diseases. Among various body fat distributions, a particular type is particularly susceptible to serious cardiovascular or metabolic diseases. This is characterized by a higher distribution of fat around the waist and / or a lower accumulation of fat around the hips, resulting in a larger waist-to-hip ratio (WHR). Metabolic diseases caused by abnormal body fat distribution include type 2 diabetes, hyperlipidemia, dyslipidemia (elevated or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B, or other lipid components), obesity (particularly abdominal obesity), lipodystrophy, insulin resistance, non-alcoholic steatohepatitis, hypertension, hyperglycemia, metabolic syndrome, coronary artery disease, and other atherosclerotic conditions.

[0005] Currently, the main treatments for metabolic diseases include lifestyle changes, dietary adjustments, exercise, and lipid-lowering medications (such as statins). However, these methods are less effective, and lipid-lowering medications can also cause side effects. Interfering RNAi (siRNA) is an effective way to silence gene expression. Through post-transcriptional regulatory mechanisms, it can specifically degrade the mRNA of target genes associated with metabolic diseases, such as INHBE, thereby effectively inhibiting their expression and effectively treating these diseases. Summary of the Invention

[0006] The present disclosure provides an RNAi agent targeting INHBE.

[0007] In some embodiments, the present disclosure provides an RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 9; the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence shown in any one of SEQ ID NO: 10 to SEQ ID NO: 18, wherein the sense strand comprises at least one inverted abasic modified nucleoside and / or the antisense strand comprises at least one thermally labile modified nucleoside.

[0008] Unless otherwise specified, in the context of the present disclosure, the nucleoside in the reverse abasic modified nucleotide has a structure as shown in formula (3-1), formula (3-2) or formula (3-3), wherein when the reverse abasic modified nucleoside is the first nucleoside at the 5' end of the sequence, it has a structure as shown in formula (3-1), when it is the first nucleoside at the 3' end, it has a structure as shown in formula (3-2), and when it is located in the middle of the sequence, it has a structure as shown in formula (3-3):

[0009] In some embodiments, the sense strand comprises two inverted abasic modified nucleosides, and the inverted abasic modified nucleosides are the first nucleoside at the 5' end and the first nucleoside at the 3' end of the sense strand.

[0010] In some embodiments, the “differences are no more than 3 nucleotides” means that the differences are 0, 1, 2, or 3 nucleotides.

[0011] In some embodiments, the "at least 15 consecutive nucleotides" refers to at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides; preferably, at least 17, 18, 19, 20 or 21 consecutive nucleotides.

[0012] In some embodiments, the antisense strand is at least partially complementary to the target sequence to mediate RNA interference. In some embodiments, there are no more than 5, 4, 3, 2, or 1 mismatches between the antisense strand and the target sequence. In some embodiments, the antisense strand is completely reverse complementary to the target sequence.

[0013] In some embodiments, the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region. In some embodiments, there are no more than 5, 4, 3, 2, or 1 mismatches between the sense strand and the antisense strand. In some embodiments, the sense strand and the antisense strand are completely reverse complementary.

[0014] In some embodiments, the RNAi agents of the present disclosure comprise one or two blunt ends.

[0015] In some embodiments, the sense strand and / or antisense strand of the RNAi agent of the present disclosure each independently comprises 1 or 2 unpaired nucleotides. In some embodiments, the 3' end of the antisense strand comprises an overhang formed by unpaired nucleotides.

[0016] In some embodiments, the sense and antisense strands each independently consist of 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides. In some embodiments, the sense and antisense strands each independently consist of 18, 19, 20, 21, 22, or 23 nucleotides.

[0017] In some embodiments, the sense strand and the antisense strand are the same or different in length, and the sense strand is 19-23 nucleotides long and the antisense strand is 19-26 nucleotides long. Therefore, the length ratio of the sense strand and the antisense strand of the RNAi agent provided herein can be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 19, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 19, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 21 / 27, 21 / 28, 21 / 29, 21 / 30, 21 / 31, 21 / 32, 21 / 33, 21 / 34, 21 / 35, 21 / 36, 21 / 37, 21 / 38, 21 / 39, 21 / 40, 21 / 41, 21 / 42, 21 / 43, 21 / 44, 21 / 45, 21 / 46, 21 / 47, 21 / 48, 21 / 49, 21 / 50, 21 / 51, 21 / 52, 21 / 53, 21 / 54, 21 / 55, 21 / 56, 21 / 57, 21 / 58, In some embodiments, the length ratio of the sense strand to the antisense strand of the RNAi agent is 19 / 19, 19 / 21, 21 / 21, 21 / 23, 23 / 23, or 23 / 25. In some specific embodiments, the length ratio of the sense strand to the antisense strand is 19 / 21, 21 / 21, or 21 / 23.

[0018] In some embodiments, the sense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 9; and / or the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 10 to SEQ ID NO: 18.

[0019] In some embodiments, the sense strand is a nucleotide sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 9; and / or the antisense strand is a nucleotide sequence as shown in any one of SEQ ID NO: 10 to SEQ ID NO: 18.

[0020] In some embodiments, at least one nucleotide in the sense strand and / or antisense strand is a modified nucleotide. In some embodiments, all nucleotides in the sense strand and / or antisense strand are modified nucleotides.

[0021] In some embodiments, the sense strand contains three consecutive 2'-fluoro-modified nucleotides. In some embodiments, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 are 2'-fluoro-modified nucleotides. When one or more nucleosides at the 5' end of the sense strand are reverse-non-basic modified nucleosides, the nucleosides at positions 7, 8, and 9 refer to the 7th, 8th, and 9th positions counted from the first nucleoside that is not reverse-non-basic modified as the first position. For example, for the sense strand as shown in SEQ ID NO: 35: IBsUmsCmAmUmCmAmGfCfUfUmUmGmCmUmAmCmUmGmsUmsIB, counting begins with the second nucleoside Um as the first position. The nucleotides at the remaining positions are non-2'-fluoro-modified nucleotides. Preferably, the non-2'-fluoro-modified nucleotides are 2'-methoxy-modified nucleotides.

[0022] In some embodiments, the antisense strand contains at least 5 2'-fluoro-modified nucleotides, for example, it may contain 5, 6, 7, 8, 9 or 10 2'-fluoro-modified nucleotides. In some embodiments, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 12, 14 and 16 are each independently 2'-fluoro-modified nucleotides. In some embodiments, the antisense strand contains 5 2'-fluoro-modified nucleotides, the nucleotides at positions 2, 6, 12, 14 and 16 in the antisense strand are each independently 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-fluoro-modified nucleotides. In some embodiments, the antisense strand contains 8 2'-fluoro-modified nucleotides, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 in the antisense strand are each independently 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are non-2'-fluoro-modified nucleotides. In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, and 18 in the antisense strand are 2'-fluoro modified nucleotides, the nucleoside at position 7 is a heat-labile modified nucleoside, and the nucleotides at the remaining positions are 2'-methoxy modified nucleotides.

[0023] In some embodiments, the non-fluorinated modified nucleotide is a 2'-methoxy modified nucleotide or a thermolabile modified nucleotide. The thermolabile modified nucleotide is a modification that can lower the dissociation temperature of the nucleic acid double strand compared to the unmodified nucleic acid double strand. In some embodiments, the thermolabile modified nucleotide has a structure as shown in WO2022028462A1. In some embodiments, the nucleoside in the thermolabile modified nucleotide has a structure as shown in formula (1):

[0024] Wherein Base represents a base, and in the direction from the 5' end to the 3' end in the sequence, "*" represents the site where the nucleoside is connected to the nucleotide adjacent to its 5' end, and "#" represents the site where the nucleoside is connected to the nucleotide adjacent to its 3' end.

[0025] In some embodiments, except for the 2'-fluoro modified nucleotide, the nucleotides at the remaining positions in the antisense strand are all 2'-methoxy modified nucleotides; or the nucleoside in the 7th nucleotide in the antisense strand is a heat-labile modified nucleoside, and the nucleotides at the remaining positions are 2'-methoxy modified nucleotides.

[0026] In some embodiments, at least one phosphodiester group in the sense strand and / or antisense strand is a phosphodiester group with a modifying group. The modifying group enables the RNAi agent to have increased stability in a biological sample or environment. In some embodiments, the sense strand and / or antisense strand include a plurality of phosphodiester groups with a modifying group. In some embodiments, the sense strand and the antisense strand both contain a plurality of phosphodiester groups with a modifying group, preferably, the sense strand and the antisense strand each independently contain at least 2 phosphodiester groups with a modifying group. In some embodiments, the sense strand contains 3 phosphodiester groups with a modifying group, and the antisense strand contains 4 phosphodiester groups with a modifying group. In some embodiments, the phosphodiester group having a modifying group is present at one or more positions selected from the following: between any two adjacent nucleotides from the first to the fourth nucleotide at the 5' end and / or the 3' end of the sense strand, and / or between any two adjacent nucleotides from the first to the fourth nucleotide at the 5' end and / or the 3' end of the antisense strand. For example, it can be located between the first and second nucleotides at the 5' end of the sense strand;

[0027] between the second and third nucleotides at the 5' end of the sense strand;

[0028] between the first and second nucleotides at the 3' end of the sense strand;

[0029] between the second and third nucleotides at the 3' end of the sense strand;

[0030] between the first and second nucleotides at the 5' end of the antisense strand;

[0031] between the second and third nucleotides at the 5' end of the antisense strand;

[0032] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0033] between the second and third nucleotides at the 3' end of the antisense strand.

[0034] In some embodiments, the phosphodiester group having a modifying group is a phosphorothioate diester group.

[0035] In some embodiments, the sense strand is selected from or comprises the nucleotide sequence shown in any one of SEQ ID NO: 19 to SEQ ID NO: 45.

[0036] In some embodiments, the antisense strand is selected from or comprises the nucleotide sequence shown in any one of SEQ ID NO: 74 to SEQ ID NO: 100.

[0037] In some embodiments, the RNAi agent of the present disclosure further comprises one or more delivery groups. In some embodiments, the one or more delivery groups are connected to the sense strand. Preferably, the RNAi agent of the present disclosure contains one delivery group.

[0038] In some embodiments, the delivery group contains at least one targeting ligand targeting the liver. In some embodiments, the targeting ligand binds to asialoglycoprotein receptor (ASGPR). In some embodiments, the targeting ligand includes a galactose cluster or a galactose derivative cluster, and the galactose derivative is selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine, preferably, the targeting ligand is N-acetylgalactosamine (GalNAc) ligand. In some embodiments, the delivery group can specifically be a nucleic acid ligand as shown in formula (I) in PCT application WO2023274395A.

[0039] In some embodiments, the delivery group has a structure as shown in formula (2):

[0040] In some embodiments, the delivery group is directly linked to the nucleoside of the first nucleotide at the 3' end of the sense strand via a phosphodiester group or a phosphorothioate diester group.

[0041] In some embodiments, in the RNAi agent of the present disclosure, the sense strand comprises or is selected from the nucleotide sequence shown in any one of SEQ ID NO:46 to SEQ ID NO:73; the antisense strand comprises or is selected from the nucleotide sequence shown in any one of SEQ ID NO:101 to SEQ ID NO:128.

[0042] In some embodiments, the RNAi agent is selected from any one of the RNAi agents shown in Table 2b. In some embodiments, the RNAi agent is selected from TJR102314, TJR102316, TJR102317, TJR102318, TJR102323, TJR102526, TJR102153, TJR102705, TJR102701, TJR102702, TJR102703, TJR102704, TJR102706, TJR102707, TJR102938, TJR102939, TJR102940, TJR102942, TJR102943, TJR102944, TJR102969, TJR102970, and TJR102971.

[0043] In some embodiments, the RNAi agent is the following structure or a pharmaceutically acceptable salt thereof:

[0044] In some embodiments, the RNAi agent comprises the structure:

[0045] In some embodiments, the pharmaceutically acceptable salt of the RNAi agent has the following structure:

[0046] In the above structural formula, the direction from the 5' end to the 3' end in the sense chain is from left to right, and the direction from the 3' end to the 5' end in the antisense chain is from left to right. IB represents a reverse non-base modified nucleoside, m represents that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside, and f represents that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleoside; NAG0052' represents the delivery group NAG0052'.

[0047] The structures of the reverse abasic modified nucleoside, 2'-methoxy modified nucleoside, 2'-fluoro modified nucleoside and delivery group NAG0052' are shown in Table 1 below.

[0048] represents the anionic form of the phosphorothioate diester group, represents the anionic form of the phosphodiester group, represents the ionic form of the phosphorothioate diester group, represents the ionic form of the phosphodiester group, represents a phosphorothioate diester group, The ionic form of the phosphorothioate diester group, the phosphorothioate diester group and the structure of the phosphorothioate diester group are shown in Table 1 below.

[0049] In another aspect, the present disclosure provides a pharmaceutical composition comprising the RNAi agent of the present disclosure and one or more pharmaceutically acceptable carriers thereof; for example, but not limited to, excipients, vehicles, carriers, diluents, and / or drug delivery systems (such as delivery polymers). Various drug delivery systems are known and can be used for the RNAi agents of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing RNAi agents, receptor-mediated endocytosis, constructing nucleic acids as part of retroviruses or other vectors.

[0050] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient and / or adjuvant, which may be one or more of various preparations or compounds conventionally used in the art. For example, the pharmaceutically acceptable excipient may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0051] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

[0052] In certain embodiments, the pharmaceutical composition contains 0.01-99.99% of the aforementioned RNAi agent or its pharmaceutically acceptable salt or its isotopic substitution, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned RNAi agent or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned RNAi agent or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned RNAi agent or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned RNAi agent or its pharmaceutically acceptable salt or its isotopic substitution.

[0053] In certain embodiments, the pharmaceutical composition comprises 0.01% to 99.99% of a pharmaceutically acceptable carrier, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition comprises 0.1% to 99.9% of a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises 0.5% to 99.5% of a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises 1% to 99% of a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises 2% to 98% of a pharmaceutically acceptable carrier.

[0054] In some embodiments, when the RNAi agent or pharmaceutical composition of the present disclosure is contacted with a cell expressing the target gene, the RNAi agent or pharmaceutical composition of the present disclosure inhibits the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot or flow cytometry.

[0055] In some embodiments, when the RNAi agent or pharmaceutical composition of the present disclosure is contacted with a cell expressing the target gene, the percentage of remaining target gene mRNA expression caused by the RNAi agent or pharmaceutical composition of the present disclosure is no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot or flow cytometry.

[0056] In some embodiments, when the RNAi agent or pharmaceutical composition of the present disclosure is contacted with a cell expressing a target gene, the RNAi agent reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75% while maintaining target activity, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot, or flow cytometry.

[0057] In some embodiments, when the RNAi agent or pharmaceutical composition of the present disclosure is contacted with a cell expressing a target gene, the RNAi agent reduces on-target activity by no more than 20%, at most 19%, at most 15%, at most 10%, at most 5%, or more than 1%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot, or flow cytometry; and reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0058] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, when contacted with a cell expressing a target gene, increases on-target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%; and reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot, or flow cytometry.

[0059] In another aspect, the present disclosure provides a method for reducing INHBE expression, comprising administering to a subject an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition of the present disclosure.

[0060] In another aspect, the present disclosure provides a method for treating and / or preventing a disease associated with INHBE gene expression in a subject, comprising administering to the subject an effective amount or effective dose of an RNAi agent and / or pharmaceutical composition of the present disclosure. In some embodiments, the associated disease is a metabolic disorder. In some embodiments, the metabolic disorder is selected from one or more of metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.

[0061] On the other hand, the present disclosure provides the use of the RNAi agent and / or pharmaceutical composition described in the present disclosure in the preparation of a medicament for treating and / or preventing related diseases. In some embodiments, the related disease is a disease associated with INHBE gene expression. In some embodiments, wherein the related disease is a metabolic disorder. In some embodiments, the metabolic disorder is selected from one or more of metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.

[0062] On the other hand, the present disclosure provides the use of the RNAi agent and / or pharmaceutical composition of the present disclosure in the preparation of a drug for inhibiting the expression of INHBE

[0063] In another aspect, the present disclosure discloses a method for in vivo delivery of an RNAi agent that inhibits INHBE expression and / or replication, comprising administering to a subject an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition of the present disclosure.

[0064] The RNAi agents or pharmaceutical compositions and methods disclosed herein can reduce the level of target mRNA in a cell, a cell population, a cell population, a tissue or a subject, comprising: administering to the subject a therapeutically effective amount of the RNAi agent or pharmaceutical composition of the present disclosure, wherein the RNAi agent is linked to the delivery group, thereby inhibiting the expression of the target mRNA in the subject.

[0065] In some embodiments, prior to administration of the RNAi agents and / or pharmaceutical compositions of the present disclosure, the subject has been identified as having pathological upregulation of a target gene in the targeted cells or tissues.

[0066] The subject described in the present disclosure refers to a subject diagnosed with (or suspected of having, or susceptible to) a disease or disorder that would benefit from reduction or inhibition of target mRNA expression.

[0067] Delivery of the RNAi agents and / or pharmaceutical compositions disclosed herein can be by local administration (e.g., direct injection, implantation, or local administration), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration, and any other suitable mode of administration commonly used in the art.

[0068] In alternative embodiments, the pharmaceutical compositions provided herein can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally, or intraperitoneally.

[0069] In an alternative embodiment, after the targeting ligand is linked to the RNAi agent to form a conjugate, the RNAi agent in the form of a conjugate can be packaged in a kit.

[0070] In another aspect, the present disclosure provides a cell comprising the RNAi agent of the present disclosure, wherein the cell is incapable of developing into an animal or plant individual.

[0071] In another aspect, the present disclosure provides a kit comprising the RNAi agent and / or pharmaceutical composition of the present disclosure. In an optional embodiment, after the delivery group is linked to the RNAi agent to form a conjugate, the conjugate can be packaged in a kit.

[0072] The present disclosure also provides a method for silencing mRNA of a target gene in a cell, the method comprising the step of introducing the RNAi agent and / or pharmaceutical composition of the present disclosure into the cell.

[0073] The present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell in vivo or in vitro, the method comprising the step of introducing the RNAi agent and / or pharmaceutical composition according to the present disclosure into the cell.

[0074] The present disclosure also provides a method for inhibiting the expression of a target gene or target gene mRNA, comprising administering an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition according to the present disclosure to a subject in need thereof.

[0075] In some embodiments, the effective amount or effective dose of the RNAi agent and / or pharmaceutical composition is about 0.001 mg / kg body weight to about 200 mg / kg body weight, about 0.01 mg / kg body weight to about 100 mg / kg body weight, or about 0.5 mg / kg body weight to about 50 mg / kg body weight.

[0076] In some embodiments, the target gene is the INHBE gene and the target mRNA is the mRNA expressed by the target gene.

[0077] The present disclosure also provides a method for preparing an RNAi agent, which comprises: synthesizing the RNAi agent described in the present disclosure.

[0078] The pharmaceutically acceptable salts of the compounds described in this disclosure are selected from inorganic salts or organic salts, and the compounds described in this disclosure can react with acidic or basic substances to form corresponding salts. In the context of this disclosure, the compounds include the RNAi agents of this disclosure. In some embodiments, the pharmaceutically acceptable salt is a sodium salt.

[0079] On the other hand, without specifying a configuration, the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present disclosure.

[0080] In addition, without specifying the configuration, the compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier.

[0081] The compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure forms or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0082] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is typically accomplished using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate formation from an amine).

[0083] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H.3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0084] In the chemical structures disclosed herein, without specifying the configuration, the bond Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or include both Although all the structural formulas described in this disclosure are drawn as certain isomers for the sake of simplicity, the present disclosure may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers. In the chemical structures of the compounds described in this disclosure, the bond No configuration is specified, i.e., the bond The configuration can be E-type or Z-type, or include both E and Z configurations.

[0085] The present disclosure incorporates the entire texts of WO2023138663A1, WO2022028462A1, WO2023274395A1, WO2023274395A and WO2024179573A1.

[0086] Explanation of terms

[0087] In order to make the present disclosure more easily understood, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0088] As used herein, "RNAi agent" (also referred to as "RNAi trigger") means an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule containing a messenger RNA (mRNA) transcript that is capable of degrading or inhibiting (e.g., degrading or inhibiting under appropriate conditions) a target mRNA in a sequence-specific manner. The RNAi agent used herein may act through an RNA interference mechanism (i.e., by inducing RNA interference through interaction with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC)) or through any alternative mechanism or pathway. Although it is believed that the term RNAi agent used herein acts primarily through an RNA interference mechanism, the disclosed RNAi agents are not bound or limited by any particular pathway or mechanism of action. The RNAi agents disclosed herein consist of a sense strand and an antisense strand, and include, but are not limited to, short (or small) interfering RNA (siRNA). The antisense strand of the RNAi agent described herein is at least partially complementary to the targeted mRNA. The RNAi agent may comprise one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0089] Unless otherwise specified, the terms "inhibin βE" and "INHBE" are used interchangeably in this disclosure. INHBEs include, but are not limited to, human INHBE, cynomolgus monkey INHBE, mouse INHBE, and rat INHBE. Their amino acid and complete coding sequences and mRNA sequences are readily available using publicly available databases, such as GenBank, UniProt, OMIM, and the Macaca mulatta genome project website.

[0090] The term "INHBE" also refers to naturally occurring DNA sequence variations of the INHBE gene, such as single nucleotide polymorphisms (SNPs) in the INHBE gene. Exemplary SNPs can be found in the dbSNP database.

[0091] The term "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of an INHBE, including mRNAs that are RNA processing products of the primary transcription product. The targeted portion of the target sequence should be long enough to serve as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is within the protein coding region of an INHBE. As used herein, in the context of RNA-mediated gene silencing, the positive strand (also known as SS, SS strand, or sense strand) refers to a strand comprising a sequence identical or substantially identical to the target mRNA sequence; the antisense strand (also known as AS or AS strand) refers to a strand having a sequence complementary or substantially complementary to the target mRNA sequence.

[0092] In the context of describing the sense strand of the RNAi agent described herein, the term "at least 15 consecutive nucleotide sequences that differ by no more than 3 nucleotides from the nucleotide sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 9" is intended to indicate that the sense strand of the RNAi agent described herein comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 9, or differs by no more than 3 nucleotide sequences (optionally, differs by no more than 2 nucleotide sequences; optionally, differs by 1 nucleotide sequence) from at least 15 consecutive nucleotide sequences of the nucleotide sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 9. Other similar descriptions in the context of this disclosure should also be understood similarly. The "difference" in this disclosure does not include the situation where nucleotides contain different modifications, that is, nucleotides containing the same base but different modifications do not belong to the nucleotides that differ in this disclosure. Other similar descriptions of the sense strand and / or antisense strand in the context of this disclosure should also be understood similarly.

[0093] In this disclosure, the "5' region," also known as the "5' end," or "5' terminus" of the sense or antisense strand may be used interchangeably. For example, nucleotides 2 through 8 in the 5' region of the antisense strand may be replaced with nucleotides 2 through 8 at the 5' terminus of the antisense strand. Similarly, the "3' region," "3' terminus," and "3' terminus" of the sense or antisense strand may be used interchangeably.

[0094] Unless otherwise specified, in the context of the present disclosure, "G", "C", "A", "T" and "U" represent nucleotides, respectively, which include the bases guanine, cytosine, adenine, thymidine and uracil, respectively. It is well known to those skilled in the art that the replacement of bases T and U will not significantly affect the properties of the RNAi agent sequence. In the sequence of the present disclosure, U can be arbitrarily replaced by T, and the sequence obtained after the replacement is also within the scope of protection of the present disclosure. In the sequence of the present disclosure, for the same nucleic acid chain, with the direction from the 5' end to the 3' end as the left to right direction, the lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside; the lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleoside; the lowercase letter s indicates that the two nucleosides adjacent to the letter s are connected by a thiophosphate diester group, and unless otherwise specified, the two nucleosides are connected by a phosphodiester group; hmpNA indicates that a nucleoside adjacent to its right in the bracket is a nucleoside modified with hmpNA; IB indicates a nucleoside without base modification in the reverse direction. Unless otherwise specified, the "RNAi agent," "nucleotide," "compound," "chemical modification," "oligonucleotide," "double-stranded RNAi inhibitor molecule," "siRNA," "dsRNA," "nucleic acid," and "RNAi" of the present disclosure may independently exist in the form of a salt, a mixed salt, or a non-salt (e.g., a free acid or a free base). When present in the form of a salt or a mixed salt, it may be a pharmaceutically acceptable salt.

[0095] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. When present in salt form, some groups may ionize to form anions or cations. For example, phosphodiester and thiophosphonate diester groups can exist as anions. Unless otherwise specified, the salt forms corresponding to the following structures are also within the scope of protection of this disclosure. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.

[0096] The above-mentioned modification and linking groups respectively have the structures shown in Table 1 below, wherein Base represents the base at the corresponding position:

[0097] Table 1

[0098] As used herein, the terms "complementary" and "reverse complement" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with bases on the other strand in a complementary manner. In DNA, the purine base adenine is always paired with the pyrimidine base thymine (or uracil in RNA); the purine base guanine is always paired with the pyrimidine base cytosine. Each base pair consists of a purine and a pyrimidine. When adenine on one strand is always paired with thymine (or uracil) on the other strand, and guanine is always paired with cytosine, the two strands are considered to be complementary to each other, and the sequence of the strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" is used in the art to mean that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary manner.

[0099] As used herein, the term "inhibit" can be used interchangeably with "reduce", "silence", "downregulate", "suppress" and other similar terms, and includes any level of inhibition. Inhibition can be assessed by the reduction of the absolute or relative level of one or more of these variables compared to the control level. The control level can be any type of control level used in the art, such as a baseline level before administration or a level determined from an untreated or control-treated subject, cell, or sample (e.g., only a buffer control or an inert agent control). For example, the remaining expression of mRNA can be used to characterize the degree of inhibition of RNAi agent expression of the target gene, such as the remaining expression of mRNA being no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%. The inhibition rate of target gene expression can be measured using The Luciferase Assay System was used to measure the firefly chemiluminescence value (Fir) and the Renilla chemiluminescence value (Ren), and the relative value Ratio = Ren / Fir was calculated. In the present disclosure, the ratio of remaining mRNA expression (or remaining activity %) = Ratio (RNAi-treated group) / Ratio (no-RNAi control group), and the inhibition rate (%) = 100% - remaining mRNA expression (%).

[0100] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0101] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, octanoates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamate, pyroglutamate, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbate, salicylates, 4-aminosalicylates, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0102] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. In some embodiments, the inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including natural substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Some embodiments of the organic base include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0103] "Effective amount" or "effective dose" refers to the amount of RNAi agent, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, reducing the severity, or delaying the onset of a condition, including the biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes that present during the progression of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various conditions associated with the disclosed target genes, target mRNAs, or target proteins, or ameliorating one or more symptoms of the condition, reducing the dose of another agent required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of a condition associated with the disclosed target genes, target mRNAs, or target proteins in a patient.

[0104] As used herein, "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, eg, humans or monkeys.

[0105] The RNAi agents provided herein can be obtained by conventional preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial customization services. Modified nucleotide groups can be introduced into the RNAi agents described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into RNAi agents are also well known to those skilled in the art.

[0106] The term "chemically modified" or "modification" includes all changes in a nucleotide by chemical or enzymatic means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0107] The term "base" encompasses any known DNA and RNA base, base analogues such as purines or pyrimidines, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues.

[0108] The terms "blunt end" or "blunt end" are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of an RNAi agent, i.e., no nucleotide overhangs. In most cases, an RNAi agent having both ends blunt-ended will be double-stranded throughout its entire length.

[0109] The terms "about" and "approximately" refer to values ​​that are within an acceptable error range for a specific value as determined by one of ordinary skill in the art, which depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean a standard deviation within or exceeding 1. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, or between 0.5% and 1%. In this disclosure, each instance in which a number or numerical range is preceded by the term "about" also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0110] Unless otherwise indicated, "optionally," "optional," "optional," or "optional" means that the subsequently described event or circumstance may but need not occur, and the description includes situations in which the event or circumstance occurs or does not occur. For example, "optionally, R1 and R2 are directly linked to form a ring" means that R1 and R2 may but need not be directly linked to form a ring, and the description includes situations in which R1 and R2 are directly linked to form a ring and situations in which R1 and R2 do not form a ring.

[0111] In the chemical structural formula disclosed herein, It may be linked to one or more of any groups in accordance with the scope of the invention described herein.

[0112] The term "linked" when referring to a connection between two molecules means that the two molecules are connected by a covalent bond or the two molecules are associated via a non-covalent bond (eg, hydrogen bond or ionic bond), including direct connection and indirect connection.

[0113] The term "directly linked" refers to a first compound or group being linked to a second compound or group without any intervening atoms or groups of atoms.

[0114] The term "indirectly linked" means that a first compound or group is linked to a second compound or group through an intermediate group, compound or molecule (eg, a linking group).

[0115] The term "substituted" means that any one or more hydrogen atoms on a designated atom (typically a carbon, oxygen, and nitrogen atom) are replaced by any group as defined herein, provided that the normal valence of the designated atom is not exceeded and the substitution produces a stable compound. Non-limiting examples of substituents include C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, aryl, ketone, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogen (e.g., F, Cl, Br, I). When a substituent is ketone or oxo (i.e., =O), then two (2) hydrogen atoms on the atom are replaced.

[0116] "Substituted by one or more..." means that the compound may be substituted by a single or multiple substituents. When substituted by multiple substituents, the substituents may be multiple identical substituents or a combination of one or more different substituents. DETAILED DESCRIPTION

[0117] The present disclosure is further described below with reference to the examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods in the examples of the present disclosure that do not specify specific conditions are generally performed under conventional conditions, such as the Cold Spring Harbor Laboratory Manual of Antibody Technology and the Molecular Cloning Manual; or according to the conditions recommended by the raw material or product manufacturer. Reagents whose specific sources are not specified can be obtained from any supplier of molecular biology reagents with a quality / purity suitable for molecular biology applications. Unless otherwise specified, the reagents used in the following examples are all commercially available products.

[0118] Example 1. Design of INHEBE RNAi Agents

[0119] The RNAi agents of the present disclosure were designed using the human INHBE gene (NM_031479.5) as the target gene to meet the general rules for active siRNA. The unmodified sense and antisense strand sequences are shown in Table 2a, and the sense and antisense strand sequences modified with 2'-fluoro, 2'-methoxy, etc. are shown in Table 2b.

[0120] Table 2a. Unmodified sense and antisense strands

[0121] Table 2b. RNAi agent sequences targeting the INHBE gene

[0122] Table 3. RNAi agent sequences targeting the INHBE gene

[0123] In Tables 2a, 2b, and 3 above, in the same nucleic acid sequence, G, C, A, and U represent nucleosides having guanine, cytosine, adenine, and uracil, respectively. From left to right, in the direction from the 5' end to the 3' end, the lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside; the lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleoside; the uppercase letter IB indicates a nucleoside with no base modification in the reverse direction; and hmpNA indicates the nucleoside adjacent to it in parentheses. One nucleoside is a nucleoside with hmpNA modification; IB represents a nucleoside with no base modification in reverse; the lowercase letter s indicates that the two nucleosides adjacent to the letter s or the connected nucleosides are connected to the delivery group NAG0052' or L96' via a phosphodiester group. Unless otherwise specified, the two adjacent nucleosides or the connected nucleosides are connected to the delivery group via a phosphodiester group; unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.

[0124] The structures of the 2'-methoxy-modified nucleoside, 2'-fluoro-modified nucleoside, hmpNA-modified nucleoside, phosphorothioate diester group, phosphodiester group, NAG0052', and reverse abasic-modified nucleoside are shown in Table 1 above. When the RNAi agent of the present disclosure exists in salt form, for example, in sodium salt form, the structure of the salt form corresponding to the structure in Table 1 is also within the scope of protection of the present disclosure. In the table, base represents a base.

[0125] Example 2. Synthesis of RNAi Agents of the Present Disclosure

[0126] The synthesis of the RNAi agent disclosed in the present invention is no different from the conventional phosphoramidite solid phase synthesis method. The synthesis process is briefly described as follows: on a Dr.Oligo48 synthesizer (Biolytic), starting with a universal CPG carrier, unmodified nucleoside phosphoramidite monomers or nucleoside phosphoramidite monomers with modifications at corresponding positions in the sequence are connected one by one according to the synthesis procedure. The nucleoside phosphoramidite monomers used in the embodiments of the present invention were purchased from Shanghai Zhaowei Company and Suzhou Jima Company. 5-Ethylthio-1H-tetrazole (ETT) was used as an activator (0.6M acetonitrile solution), 0.22M PADS dissolved in a 1:1 volume ratio of acetonitrile and 3-methylpyridine (Shanghai Lingjiang) solution was used as a sulfurizing reagent, and iodine pyridine / water solution (Shanghai Lingjiang) was used as an oxidant.

[0127] After solid-phase synthesis is completed, the oligoribonucleotide is cleaved from the solid support and soaked in a 3:1 28% ammonia and ethanol solution at 50°C for 16 hours. Then centrifuge, transfer the supernatant to another centrifuge tube, concentrate and evaporate to dryness, and purify using C18 reverse chromatography with a mobile phase of 0.1M TEAA and acetonitrile. The target oligonucleotide is collected and freeze-dried, and identified as the target product by LC-MS, and then quantified by UV (260nm). The resulting single-stranded oligonucleotide is annealed according to an equimolar ratio and complementary pairing. The final double-stranded RNAi agent is dissolved in 1×PBS and adjusted to the concentration required for the experiment.

[0128] The RNAi agent of the present disclosure containing NAG0052' was prepared according to the method described in patent application WO2023138663A1. The RNAi agent of the present disclosure containing hmpNA-modified nucleotides was prepared according to the method described in patent application WO2022028462A1. The RNAi agent containing L96' was prepared according to the same method as described in the examples of WO2014025805A1.

[0129] Example 3. psiCHECK Validation at Target Level

[0130] The RNAi agents of the present invention were screened for on-target activity at the molecular level in vitro using 9 concentration gradients in HEK293A cells.

[0131] The psi-CHECK plasmid used in this example was purchased from Sangon Biotech (Shanghai) Co., Ltd. The on-target sequence corresponding to the RNAi agent was constructed using the INHBE gene and inserted into the psiCHECK-2 plasmid, i.e., the GSCM on-target plasmid. This plasmid contains the Renilla luciferase gene and the firefly luciferase gene. As a dual reporter gene system, the target sequence of the RNAi agent is inserted into the 3'UTR region of the Renilla luciferase gene. The activity of the RNAi agent for the target sequence can be reflected by the detection of the expression of Renilla luciferase after calibration with firefly luciferase. The detection was performed using the Dual-Luciferase Reporter Assay System (Promega, E2940).

[0132] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 8 × 10 cells per well. 3 cells, 100 μL culture medium per well.

[0133] According to the instructions, cells were co-transfected with RNAi agents and corresponding plasmids using Lipofectamine 2000 (ThermoFisher, 11668019). 0.2 μL of Lipofectamine 2000 was used per well. The plasmid transfection amount was 20 ng per well. For the target sequence plasmid, the RNAi agent was set at a total of 9 concentration points, with the highest concentration point being 20 nM. The final concentration was 4-fold serial dilution: 20 nM, 5 nM, 1.25 nM, 0.3125 nM, 0.0781 nM, 0.0195 nM, 0.0049 nM, 0.0012 nM, and 0.0003 nM. 24 hours after transfection, the Dual-Luciferase Reporter Assay System (Promega, E2940) was used to detect target levels.

[0134] The experimental materials and instruments used in this example are detailed in Tables 4 and 5. The dilution scheme used in this example is shown in Table 6. The experimental results are shown in Table 7, which shows that the RNAi agent disclosed herein has excellent on-target activity in the pis-Check system.

[0135] Table 4. Psi-CHECK experimental consumables and reagents

[0136] Table 5. Psi-CHECK test equipment

[0137] Table 6. Sample dilution scheme for multiple concentrations

[0138] Table 7. Psi-CHECK on-target activity screening results

[0139] Example 4: Inhibitory activity of INHBE at multiple concentrations in primary monkey hepatocytes (PCH)

[0140] 24 hours before transfection, PCH cells were seeded at approximately 30,000 cells / well in 96-well plates with 100 μL of culture medium per well. For transfection, samples were transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150) according to the product instructions. The final concentrations of the transfection gradient were 20, 4, 0.8, 0.16, 0.032, 0.0064, and 0.00128 nM. After 24 hours of treatment, total RNA was extracted using a high-throughput cell RNA extraction kit (FG0417-L / FG0418-XL, magnetic bead method). RNA reverse transcription was performed (Takara, RR037A) and real-time quantitative PCR (Thermo, 4444557) to measure monkey INHBE mRNA levels. Monkey INHBE mRNA levels were corrected for GAPDH levels. The instruments used in this experiment are shown in Table 8.

[0141] Table 8. Experimental instruments

[0142] Among them, in the real-time quantitative PCR detection, the probe Q-PCR detection experiment was used, and its primer information is shown in Table 9.

[0143] Table 9. Taqman primer information table

[0144] Result analysis method

[0145] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct values: Comparative Ct refers to calculating the difference in gene expression by the difference between the Ct value and the internal reference gene, also known as 2-△△Ct, △△Ct = [(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)].

[0146] Inhibition rate (%) = (1-remaining expression level of target gene)*100%.

[0147] The results are expressed as the remaining percentage of monkey INHBE mRNA expression relative to cells treated with the RNAi agent. The data in Table 10 show that the RNAi agent of the present disclosure has a high level of on-target inhibitory activity against the INHBE gene in PCH cells.

[0148] Table 10. INHBE inhibitory activity of RNAi agents in PCH cells

[0149] Example 5: Single-site inhibitory activity of INHBE in primary monkey hepatocytes (PCH)

[0150] 24h before transfection, PCH cells were seeded in 96-well plates at approximately 30,000 cells / well with 100 μL of culture medium per well. During transfection, the sample was transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150) with reference to the product instruction manual, and the final gradient concentration of the sample transfection was 4nM. After 24 hours of treatment, total cell RNA was extracted using a high-throughput cell RNA extraction kit (FG0417-L / FG0418-XL, magnetic bead method), RNA reverse transcription experiment (Takara, RR037A) and real-time quantitative PCR (Thermo, 4444557) were used to detect the mRNA level of monkey INHBE, and the mRNA level of monkey INHBE was corrected according to the GAPDH internal reference gene level. Its instruments, primers, calculation methods, etc. are all referred to Example 4. The data results in Table 11 show that the RNAi agent disclosed herein has a high level of on-target inhibitory activity against the INHBE gene in PCH cells.

[0151] Table 11. INHBE inhibitory activity of RNAi agents in PCH cells

[0152] Example 6: Efficacy of RNAi in an AAV-constructed liver hINHBE obese (DIO) mouse model

[0153] The DIO male mice in this example (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. and its subsidiaries) were 4-5 weeks old and weighed 30-32 g.

[0154] All mice were acclimated to the animal facility for 5 days before being switched to a 60% HFD diet. At 6 weeks of age, 15 mice were selected based on body weight and injected intravenously with a blank vector (2 x 10E11 vg / mouse), while the remaining mice were injected intravenously with AAV-hINHBE (2 x 10E11 vg / mouse). Liver biopsies were performed 2 weeks after blank vector and AAV injections, and hINHBE expression was assessed by qPCR. One week after liver biopsy, 80-100 μl of blood was collected from all mice and centrifuged to obtain plasma. Plasma was diluted 2-fold and analyzed for liver function tests (ALT, AST) and four lipid profiles (TC, TG, HDL-C, LDL-C). Groups were then divided into groups 1, 2, and 3 based on qPCR, liver function test results, and body weight. Groups 1, 2, and 3 each had 12 mice, with four mice at each time point. The day of grouping was designated D-1. Group 1 contained blank vector mice, while Groups 2 and 3 contained AAV-treated mice.

[0155] Dosing started on D0. PBS was given to mice in Group 1 and Group 2, and the RNAi agent TJR102316 of the present disclosure was given to mice in Group 3. The dosage was 3 mg / kg, the volume was 5 μl / g, the administration method was subcutaneous administration, and the administration frequency was single administration.

[0156] The efficacy period is 4 weeks. Four mice were selected from each group for endpoints on days 7, 14, and 28: (1) On the endpoint day, the mice were fasted for 5-6 hours, and blood was collected. A portion was centrifuged to obtain plasma, which was diluted 2-fold and tested for liver function (ALT, AST) and four blood lipid parameters (TC, TG, HDL-C, LDL-C); (2) Liver tissue was collected and weighed. A portion of the liver tissue was quickly frozen in liquid nitrogen and then analyzed by Western blotting (Flag). Another portion of the liver tissue was stored in RNALater and subsequently analyzed by qPCR for hINHBE expression. In the context of this disclosure, "hINHBE" refers to human INHBE.

[0157] Table 12 records the expression levels of hINHBE mRNA in the liver of different groups at D7, D14 and D28. The data in Table 12 show that compared with Group 2, the hINHBE-mRNA level in mice administered with the RNAi agent TJR102316 of the present disclosure was significantly decreased, indicating that the RNAi agent of the present disclosure can effectively inhibit the expression of hINHBE-mRNA in mice.

[0158] Table 12. hINHBE-mRNA levels in mice (average hINHBE mRNA expression in liver, normalized to group 2) t-test, * indicates p < 0.05 compared with group 2, ** indicates p < 0.01 compared with group 2, *** indicates p < 0.001 compared with group 2, **** indicates p < 0.0001 compared with group 2.

[0159] Table 13 records the expression levels of the tag protein detected by WB (Flag) in the livers of different groups at D7, D14 and D28. The data in Table 13 show that compared with Group 2, the tag protein level in mice administered with the RNAi agent TJR102316 of the present invention was significantly decreased, indicating that the RNAi agent of the present invention can effectively inhibit the expression of the INHBE gene in mice.

[0160] Table 13 One-way ANOVA test, * indicates p<0.05 compared with group 2, ** indicates p<0.01 compared with group 2, *** indicates p<0.001 compared with group 2, **** indicates p<0.0001 compared with group 2.

[0161] Example 7. Pharmacodynamic evaluation of RNAi agents in a high-fat-fed hINHBE mouse obesity model

[0162] The DIO-hINHBE transgenic homozygous male mice used in this example were purchased from Saiye (Suzhou) Biotechnology Co., Ltd.

[0163] Humanized hINHBE transgenic mice were used and fed a 60% HFD (Research Diet D12492) diet to establish an obese mouse (DIO) model. Before the experiment, the mice were randomly divided into 5 groups according to body weight and body fat ratio (Lean, Fat%). Group 1 was the control group and was given normal saline as a vehicle control group. Groups 2-5 were given the RNAi agent TJR102316 of the present invention at different doses (0.3 mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg). Each group was administered subcutaneously. The frequency of administration of the RNAi agent TJR102316 of the present invention was once every 3 weeks, and the efficacy period was 6 weeks. The day of grouping was defined as D-1, and the administration period was 2 days from D0 to D42.

[0164] Results are presented as mean values. Comparisons between two groups were performed using the independent sample t-test, univariate comparisons between multiple groups were performed using one-way analysis of variance (ANOVA), and bivariate comparisons between multiple groups were performed using two-way analysis of variance (ANOVA). Data were analyzed using Graphpad Prism 9. **** indicates p < 0.0001; *** indicates p < 0.001; ** indicates p < 0.01; * indicates p < 0.05, with P < 0.05 considered statistically significant.

[0165] Tables 14 and 15 show the weight change rate and food intake changes of mice in different groups, respectively. Combining the experimental results in Tables 14 and 15, it can be seen that the RNAi agent TJR102316 of the present application not only has a good weight-reducing effect at a low dose, but also has no significant effect on the food intake of mice at all doses. This demonstrates that the RNAi agent TJR102316 of the present disclosure can achieve a good weight-reducing effect without affecting the food intake of mice.

[0166] Table 14. Changes in mouse body weight (compared to group 1)

[0167] Table 15. Changes in food intake of mice (unit: g)

[0168] Table 16 records the LDL-C levels, mesenteric fat (MAT) mass, liver triglyceride levels, and serum β-hydroxybutyrate levels in mice on day 42.

[0169] As shown in Table 16, the RNAi agent TJR102316 of the present application can effectively reduce the LDL-C level in serum, mesenteric fat mass and liver triglycerides.

[0170] At the same time, the β-hydroxybutyric acid in mice administered with the RNAi agent TJR102316 of the present application showed an upward trend, indicating that the RNAi agent of the present application can promote β-oxidation of liver lipids and use β-hydroxybutyric acid as energy for the body.

[0171] Table 16. Serum LDL-C content, mesenteric fat mass, liver triglyceride level and β-hydroxybutyrate content in D42 mice

[0172] Example 8. psiCHECK Validation at Target Level

[0173] The RNAi agents listed in Table 3 were screened for on-target activity at the in vitro molecular level using 9 concentration gradients in HEK293A cells.

[0174] The psi-CHECK plasmid used in this example was purchased from Sangon Biotech (Shanghai) Co., Ltd. The on-target sequence corresponding to the RNAi agent was constructed using the INHBE gene and inserted into the psiCHECK-2 plasmid, i.e., the GSCM on-target plasmid. This plasmid contains the Renilla luciferase gene and the firefly luciferase gene. As a dual reporter gene system, the target sequence of the RNAi agent is inserted into the 3'UTR region of the Renilla luciferase gene. The activity of the siRNA against the target sequence can be reflected by the detection of the expression of the Renilla luciferase after calibration with firefly luciferase. The detection was performed using the Dual-Luciferase Reporter Assay System (Promega, E2940).

[0175] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 8 × 10 cells per well. 3 cells, 100 μL culture medium per well.

[0176] Cells were co-transfected with siRNA and the corresponding plasmid using Lipofectamine 2000 (ThermoFisher, 11668019) according to the manufacturer's instructions. 0.2 μL of Lipofectamine 2000 was used per well. A total of 20 ng of plasmid was transfected per well. For the target sequence plasmid, siRNA was administered at nine concentration points, with the highest concentration being 20 nM. A four-fold serial dilution series was then performed, with the following concentrations: 20 nM, 5 nM, 1.25 nM, 0.3125 nM, 0.0781 nM, 0.0195 nM, 0.0049 nM, 0.0012 nM, and 0.0003 nM. 24 hours after transfection, on-target levels were detected using the Dual-Luciferase Reporter Assay System (Promega, E2940).

[0177] The experimental materials and instruments used in this example are detailed in Tables 17 and 18. The dilution scheme used in this example is shown in Table 19. The experimental results are shown in Tables 20, 21, 22, and 23.

[0178] Table 17. Psi-CHECK experimental consumables and reagents

[0179] Table 18. Psi-CHECK test equipment

[0180] Table 19. Sample dilution scheme for multiple concentrations

[0181] Table 20. RNAi agent on-target activity screening results

[0182] Table 21. RNAi agent on-target activity screening results

[0183] Table 22. RNAi agent on-target activity screening results

[0184] Table 23. RNAi agent on-target activity screening results

[0185] Example 9. psiCHECK Validation at Target Level

[0186] Two concentration gradients were used to screen the RNAi agents for on-target activity in vitro at the molecular level in HEK293A cells. The experimental process, experimental materials and instruments used, dilution process, and result analysis methods were the same as those in Example 8.

[0187] Table 24. RNAi agent inhibitory effect on INHBE

Claims

1. An RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, The sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 9 by no more than 3 nucleotides; The antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence shown in any one of SEQ ID NO: 10 to SEQ ID NO: 18 by no more than 3 nucleotides, in, The sense strand contains at least one reverse abasic modified nucleoside; and / or The antisense strand comprises at least one heat-labile modified nucleoside.

2. The RNAi agent of claim 1, wherein: The sense strand comprises two reverse abasic modified nucleosides, and the reverse abasic modified nucleosides are the first nucleoside at the 5' end and the first nucleoside at the 3' end of the sense strand.

3. The RNAi agent of claim 1 or 2, wherein: From the 5' end to the 3' end, the nucleotides at positions 2, 6, 12, 14, and 16 in the antisense strand are each independently a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions are non-2'-fluoro-modified nucleotides; alternatively, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, and 18 in the antisense strand are each independently a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions are non-2'-fluoro-modified nucleotides.

4. The RNAi agent of claim 3, wherein: The remaining nucleotides in the antisense strand are all 2'-methoxy-modified nucleotides; or The nucleoside in the 7th nucleotide of the antisense strand is a heat-labile modified nucleoside, and the nucleotides in the remaining positions are 2'-methoxy-modified nucleotides; Preferably, the thermally unstable modified nucleoside has a structure as shown in formula (1): Wherein Base represents a base, and in the direction from the 5' end to the 3' end in the sequence, "*" represents the site where the nucleoside is connected to the nucleotide adjacent to its 5' end, and "#" represents the site where the nucleoside is connected to the nucleotide adjacent to its 3' end.

5. The RNAi agent of any one of claims 1 to 4, wherein At least one phosphodiester group in the sense strand and / or antisense strand is a phosphodiester group having a modified group; Preferably, the phosphodiester group having a modifying group is a thiophosphate diester group; The phosphodiester group having a modifying group is present in at least one position selected from the following: between the first and second nucleotides at the 5' end of the sense strand; between the second and third nucleotides at the 5' end of the sense strand; between the first and second nucleotides at the 3' end of the sense strand; between the second and third nucleotides at the 3' end of the sense strand; between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; between the second and third nucleotides at the 3' end of the antisense strand; Preferably, the sense strand and the antisense strand each independently contain at least two phosphodiester groups having a modification group.

6. The RNAi agent according to any one of claims 1 to 5, wherein, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 in the sense strand are 2'-fluoro nucleotides, and the nucleotides at the remaining positions are non-2'-fluoro modified nucleotides.

7. The RNAi agent according to any one of claims 1 to 6, The sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO: 19 to SEQ ID NO: 45; The antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO: 74 to SEQ ID NO:

100.

8. The RNAi agent according to any one of claims 1 to 7, further comprising one or more delivery groups, wherein the delivery groups are linked to the sense strand and / or antisense strand.

9. The RNAi agent according to claim 8, wherein the delivery group has a structure as shown in formula (2):

10. The RNAi agent according to claim 9, wherein The sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO: 46 to SEQ ID NO: 73; and / or The antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 101 to SEQ ID NO:

128.

11. The RNAi agent according to any one of claims 1 to 10, wherein The RNAi agent is selected from the group consisting of TJR102314, TJR102316, TJR102317, TJR102318, TJR102323, TJR102526, TJR102153, TJR102705, TJR102701, TJR102702, TJR102703, TJR102704, TJR102706, TJR102707, TJR102938, TJR102939, TJR102940, TJR102942, TJR102943, TJR102944, TJR102969, TJR102970, and TJR102971.

12. A pharmaceutical composition comprising: The RNAi agent of any one of claims 1 to 11, and Pharmaceutically acceptable carrier.

13. A kit comprising: The RNAi agent of any one of claims 1 to 11; and / or The pharmaceutical composition of claim 12.

14. A method for reducing inhibin beta E (INHBE) expression, comprising administering to a subject an effective amount or an effective dose of the RNAi agent according to any one of claims 1 to 11 and / or the pharmaceutical composition according to claim 12.

15. A method for treating and / or preventing metabolic disorders in a subject, comprising administering to the subject an effective amount or effective dose of the RNAi agent according to any one of claims 1 to 11 and / or the pharmaceutical composition according to claim 12; The metabolic disorder is selected from the group consisting of metabolic syndrome, cardiovascular disease, obesity, hepatitis, and kidney disease.

16. A method for in vivo delivery of an RNAi agent that inhibits INHBE expression and / or replication, the method comprising administering to a subject the RNAi agent of any one of claims 1 to 11 and / or the pharmaceutical composition of claim 12.

17. A method for preparing an RNAi agent, comprising: Synthesize the RNAi agent according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 12.