Inhibin subunit βe inhibitor and use thereof

Abstract

Disclosed in the present application are an siRNA targeting inhibin subunit βE (INHBE) gene expression and the use thereof. The siRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises any one of nucleotide sequences from SEQ ID NO: 1-SEQ ID NO: 2444, and the sense strand and the antisense strand are at least partially reversely complementary to form a double-stranded region. The siRNA agent of the present application can significantly inhibit the expression of INHBE gene mRNA in in-vitro experiments.

Description

Inhibitors of β-inhibitory subunits and their uses

[0001] Cross-references

[0002] This application claims priority to Chinese patent application filed on November 18, 2024, application number 202411647591.1, entitled "Inhibitor subunit βE inhibitor and its use thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of biomedical technology, specifically to an inhibitory subunit βE inhibitor and its uses. Background Technology

[0004] Inhibin subunit βE (INHBE) is a member of the transforming growth factor-β (TGF-β) superfamily and is primarily expressed in the liver. The secreted peptide inhibin E, formed by homodimerization of the βE subunit, participates in the regulation of various cellular processes, including cell proliferation, apoptosis, immune responses, and hormone secretion. Studies have found that INHBE, as a hepatic secretory factor, can alter systemic metabolic status by regulating adipose tissue responses.

[0005] In recent years, studies have shown that body fat distribution, rather than overall obesity, is a significant risk factor for cardiovascular and metabolic diseases. Body fat distribution characterized by a high waist-to-hip ratio (WHR), rather than body mass index (BMI), is considered associated with a higher cardiovascular and metabolic risk. For diseases caused by lipid metabolism disorders, such as type 2 diabetes, insulin resistance, obesity, dyslipidemia, non-alcoholic steatohepatitis, hypertension, coronary artery disease, and other atherosclerotic conditions, current treatments include lifestyle modifications, diet, exercise, and medication, including lipid-lowering agents (such as statins) and other antihypertensive and hypoglycemic drugs. However, existing therapies are often ineffective due to side effects or adherence limitations. To effectively control obesity and reduce the incidence of cardiovascular disease, new therapies are needed to meet patient needs. Small interfering RNA (siRNA), as an effective way to silence gene expression, can specifically degrade target gene mRNAs such as INHBE, effectively inhibiting the expression of secretory inhibin E, thereby regulating lipid metabolism and effectively treating diseases. Summary of the Invention

[0006] In view of the deficiencies of the prior art, one of the objectives of this invention is to provide an siRNA agent and method capable of regulating (e.g., reducing) the level of inhibin subunit βE (INHBE) in cells and subjects such as mammals (e.g., humans).

[0007] Another object of the present invention is to provide a pharmaceutical composition comprising the siRNA agent.

[0008] Another object of the present invention is to provide the use of the siRNA agent in the prevention or treatment of diseases or conditions or in the preparation of medicaments for the prevention or treatment of diseases or conditions.

[0009] The first aspect of this invention provides an siRNA agent for regulating the level of repressor subunit βE (INHBE), comprising a sense strand and an antisense strand.

[0010] Preferably, the antisense strand comprises any one of the nucleotide sequences in SEQ ID NO 1 to SEQ ID NO 2444 in Table 1, or a sequence that differs from it by no more than 3 nucleotides (e.g., 1, 2, 3).

[0011] Preferably, the justice chain and the antisense chain are at least partially inversely complementary to form a dual-chain region.

[0012] Preferably, the sense strand and / or antisense strand independently comprise one or more modified nucleotides.

[0013] Preferably, the modified nucleotide is at least one selected from 2'-fluorine modified nucleotide, 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, bicyclic nucleic acid, deoxyribonucleotide, EVP, UNA, and GNA.

[0014] Preferably, the sense strand and / or antisense strand of the siRNA each independently contain up to three unpaired nucleotides.

[0015] Preferably, the sense and / or antisense strands of the siRNA contain 20-25 nucleotides.

[0016] Preferably, the sense and / or antisense strands of the siRNA are of the same or different lengths.

[0017] Preferably, the siRNA has a nucleotide sequence as shown in any pair of sense and antisense strands in Table 1, Table 2, Table 3 or Table 4, i.e. SEQ ID NO 1-SEQ ID NO 2770.

[0018] Preferably, the siRNA agent comprises any one of the sense strands from SEQ ID NO 2540 to SEQ ID NO 2634, and / or any one of the antisense strands from SEQ ID NO 2635 to SEQ ID NO 2729.

[0019] The second aspect of this application also provides the use of the aforementioned siRNA agent in the preparation of a medicament for reducing the expression of inhibin subunit βE in mammals, or for preventing and / or treating related diseases or conditions, or reducing the risk of diseases or conditions.

[0020] Preferably, the disease or condition includes diseases or conditions caused by various lipid metabolism abnormalities.

[0021] The drug comprises the aforementioned siRNA agent and one or more pharmaceutically acceptable excipients.

[0022] A third aspect of this application provides a method for preventing or treating a disease or condition, the method comprising administering an effective amount of the aforementioned siRNA agent or the aforementioned pharmaceutical composition to a target subject.

[0023] The beneficial effects of the present invention are as follows: Experiments have shown that the siRNA agent of this application can significantly inhibit the expression of the repressor subunit βE and the corresponding mRNA in in vitro experiments, and can achieve up to 99% knockdown inhibition at high doses. Detailed Implementation

[0024] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The present invention will be specifically described below with reference to specific embodiments.

[0025] The first aspect of this invention provides an siRNA agent for regulating the level of repressor subunit βE (INHBE), comprising a sense strand and an antisense strand.

[0026] The antisense strand includes any nucleotide sequence from SEQ ID NO 1 to SEQ ID NO 2444 in Table 1, or a sequence that differs from it by no more than 3 nucleotides (e.g., 1, 2, 3).

[0027] The justice chain and the antisense chain are at least partially inversely complementary, forming a dual-chain region.

[0028] Specifically, there are no more than 5, 4, 3, 2, or 1 mismatches between the justice chain and the antisense chain.

[0029] Specifically, the justice chain and the antisense chain can be completely opposite and complementary.

[0030] The sense strand and / or antisense strand independently contain one or more modified nucleotides.

[0031] Specifically, the modified nucleotide is at least one of 2'-fluorine modified nucleotide, 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, bicyclic nucleic acid, deoxyribonucleotide, EVP, UNA, and GNA.

[0032] The sense and / or antisense strands of the siRNA each independently contain up to three unpaired nucleotides.

[0033] The sense and / or antisense strands of the siRNA contain 20-25 nucleotides.

[0034] The sense and / or antisense strands of the siRNA may be the same or different in length.

[0035] Specifically, the length ratio of the justice chain to the antisense chain can be 21 / 23.

[0036] Specifically, the siRNA has a nucleotide sequence as shown in any pair of sense and antisense strands in Table 1, Table 2, Table 3 or Table 4.

[0037] Specifically, the siRNA agent includes a targeting delivery ligand conjugated to the siRNA, wherein the targeting delivery ligand is a GalNAc ligand. "GalNAc ligand" refers to a ligand containing an N-acetylgalactosamine (GalNAc) or derivative structure of the desialylate glycoprotein receptor (ASGPR).

[0038] In some embodiments, the ligand comprises Gal-6, which has the following structure:

[0039] In some embodiments, the ligand comprises three GalNAc ligand molecules or derivatives thereof. In some embodiments, the ligand comprises three Gal-6 molecules sequentially conjugated to the siRNA molecule. In some embodiments, Gal-6 molecules are sequentially conjugated to the 3' end of the sense or antisense strand, and in some embodiments, Gal-6 molecules are sequentially conjugated to the 5' end of the sense or antisense strand. In some embodiments, the ligand comprises a [Gal-6]s[Gal-6]s[Gal-6] molecule with the following structure:

[0040] In some embodiments, the ligand comprises a [Gal-6][Gal-6][Gal-6] molecule with the following structure:

[0041] The second aspect of this application also provides the use of the aforementioned siRNA agent in the preparation of a medicament for reducing the expression of inhibin subunit βE in mammals, or for preventing and / or treating related diseases or conditions, or reducing the risk of diseases or conditions.

[0042] The diseases or conditions mentioned include those caused by various lipid metabolism abnormalities.

[0043] Specifically, the diseases or conditions caused by lipid metabolism abnormalities include type II diabetes, insulin resistance, obesity, dyslipidemia, non-alcoholic steatohepatitis, hypertension, coronary artery disease, and other atherosclerotic conditions.

[0044] The drug includes pharmaceutically acceptable excipients.

[0045] Specifically, pharmaceutically acceptable excipients include liposomes, microcapsules, and microspheres.

[0046] A third aspect of this application provides a method for preventing or treating a disease or condition, the method comprising administering an effective amount of the aforementioned siRNA agent or the aforementioned pharmaceutical composition to a target subject.

[0047] Specifically, the application includes subcutaneous injection, intramuscular injection, transdermal absorption, oral administration, etc.

[0048] In this application, the terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides or their analogues) of any length, which can be double-stranded or single-stranded molecules. Specifically, it can include: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, siRNA, miRNA, shRNA, RNAi reagents, and primers. The nucleotide sequence may contain modified nucleotides, such as methylated nucleotides and nucleotide analogues. Furthermore, the nucleotide structure can be modified before or after polymer assembly. Polynucleotides can be modified post-polymerization, for example, by coupling with a labeled component.

[0049] In this application, the term "modified nucleotide" generally means a nucleotide that contains at least one chemical modification compared to naturally occurring RNA or DNA nucleotides. A modified nucleotide comprises a modified sugar moiety and / or a modified nucleobase.

[0050] In this application, the term "antisense strand" generally refers to a strand in an RNAi agent (e.g., dsRNA) that includes a region substantially complementary to the target sequence. Herein, the term "complementary region" generally refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., the target sequence) as defined in this application. When the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the molecule or in terminal regions. Typically, the most permissible mismatches are in terminal regions, for example, within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

[0051] In this application, the term "sense strand" generally refers to a region in an RNAi agent that is substantially complementary to the region of the antisense strand. The "sense" strand is sometimes referred to as the "sense" strand. Using their sequences, the antisense strand targets the target mRNA, while the sense strand targets different targets.

[0052] In this application, the term "complementarity" refers to the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize (form base pair hydrogen bonds) with an oligonucleotide or polynucleotide containing a second nucleotide sequence under certain conditions and form a double-stranded or double-helix structure. Complementary sequences include Watson-Crickbase pairs or non-Watson-Crickbase pairs, and include native or modified nucleotides or nucleotide analogs, provided that the above requirements regarding their hybridization ability are met. "Complementarity" does not necessarily require nucleobase complementarity on every nucleotide. Instead, some mismatches are tolerable.

[0053] In this application, the term "fully complementary" generally means that all (100%) bases in the sequential sequence of the first polynucleotide will hybridize with the same number of bases in the sequential sequence of the second polynucleotide. The sequential sequence may comprise all or part of the first or second nucleotide sequence. As used herein, "partially complementary" generally means that in the hybridized base sequence pair, at least about 70% of the bases in the sequential sequence of the first polynucleotide will hybridize with the same number of bases in the sequential sequence of the second polynucleotide. As used herein, "substantially complementary" generally means that in the hybridized base sequence pair, at least about 90% of the bases in the sequential sequence of the first polynucleotide will hybridize with the same number of bases in the sequential sequence of the second polynucleotide. The terms "complementary," "fully complementary," and "substantially complementary" as used herein may be used in relation to base matching between the sense and antisense strands of the RNAi agent or between the antisense strand of the RNAi agent and the sequence of the LPAm RNA. Sequence identity or complementarity is independent of modification. For the purpose of determining identity or complementarity, for example, A and Af are complementary to U (or T) and are identical to A.

[0054] In this application, the term "expression" generally refers to the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, post-transcriptional modifications (e.g., splicing, polyadenylation, addition of a 5'-cap), and translation.

[0055] In this application, the term "pharmaceutically acceptable" generally refers to one or more non-toxic substances that do not interfere with the effectiveness of the biological activity of the active ingredient. Such formulations may typically contain salts, excipients, buffers, preservatives, compatibility carriers, and optionally other therapeutic agents. When used in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to prepare pharmaceutically acceptable salts, and these should not be excluded from the scope of this application. Such pharmacologically and pharmaceutically acceptable salts include salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium, potassium, or calcium salts.

[0056] In this application, the term "prevention and / or treatment" includes not only the prevention and / or treatment of disease, but also generally includes preventing the onset of disease, slowing or reversing the progression of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated with it and / or preventing a further increase in the severity of the disease and / or any symptoms associated with it, preventing, reducing or reversing any physiological damage caused by the disease, and any pharmacological effects that are generally beneficial to the patient being treated. The RNAi agents or pharmaceutical compositions of this application that constitute feasible therapeutic agents do not need to achieve a complete cure or eradication of any symptom or manifestation of the disease. As recognized in the relevant art, a medicine used as a therapeutic agent may reduce the severity of a given disease state, but does not need to eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, a treatment administered prophylactically constitutes a feasible preventive agent that does not need to completely and effectively prevent the onset of the condition.

[0057] In this application, the term "administration" generally refers to the introduction of the pharmaceutical preparation of this application into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs, or tissues with the drug may be employed. Administration may include, but is not limited to, intravenous, intra-arterial, intranasal, intraperitoneal, intramuscular, subcutaneous, or oral administration. A daily dose may be divided into one, two, or more doses in suitable forms to be administered at one, two, or more times during a certain time period.

[0058] In this application, the terms "effective amount" or "effective dose" generally refer to an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutic effective amount" or "therapeutic effective dose" of a drug or therapeutic agent generally refers to any amount of drug that, when used alone or in combination with another therapeutic agent, promotes disease remission (proven by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of damage or disability due to the disease). A "preventive effective amount" or "preventive effective dose" of a drug generally refers to an amount of drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of disease development or relapse, inhibits the development or relapse of the disease. In some embodiments, "effective amount" refers to the amount of RNAi agent that produces the expected pharmacological, therapeutic, or preventive outcome.

[0059] In this application, the term "subject" generally refers to a human or non-human animal (including mammals) requiring diagnosis, prognosis, improvement, prevention, and / or treatment of a disease, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents, and adult subjects. Subjects include animal disease models.

[0060] The present application will be further explained below with reference to specific embodiments:

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available, and techniques not described in detail were performed according to standard methods well known to those skilled in the art.

[0062] Example 1: siRNA antisense strand sequence design

[0063] Computer-generated antisense siRNA sequences complementary to the human INHBE DNA sequence (NM_031479.5) were used to generate a total of 2444 sequences, which were then further screened.

[0064] Table 1. siRNA antisense strand sequences

[0065] Example 2: Design of Potential siRNA Agents

[0066] To screen for highly efficient siRNA agents, the computer-fitted siRNA antisense strands were further identified, resulting in 95 potential siRNA antisense strands. These were then paired with the sense strands to obtain double-stranded siRNA agents capable of targeting the repressor subunit βE, and their sequences are shown in Table 2. Furthermore, the inventors modified these 95 double-stranded siRNA agents, and the modified sequences are shown in Table 3.

[0067] Table 2. Sequences of unmodified double-stranded siRNA agents targeting the repressor subunit βE.

[0068] Table 3. Sequences of modified double-stranded siRNA agents targeting the repressor subunit βE.

[0069] In the table, A = adenosine-3'-phosphate.

[0070] C = Cytidine-3'-phosphate

[0071] G = Guanosine-3'-phosphate

[0072] U = uridine-3'-phosphate

[0073] Am = 2'-O-methyladenosine-3'-phosphate

[0074] Ams = 2'-O-methyladenosine-3'-thiophosphate

[0075] Cm = 2'-O-methylcytidine-3'-phosphate

[0076] Cms = 2'-O-methylcytidine-3'-thiophosphate

[0077] Gm = 2'-O-methylguanosine-3'-phosphate

[0078] Gms = 2'-O-methylguanosine-3'-thiophosphate

[0079] Um = 2'-O-methyluridine-3'-phosphate

[0080] Ums = 2'-O-methyluridine-3'-thiophosphate

[0081] Af = 2'-Fluoroadenosine-3'-phosphate

[0082] Afs = 2'-Fluoroadenosine-3'-Thiophosphate

[0083] Cf = 2'-Fluorocytidine-3'-phosphate

[0084] Cfs = 2'-Fluorocytidine-3'-Thiophosphate

[0085] Gf = 2'-Fluoroguanosine-3'-phosphate

[0086] Gfs = 2'-Fluoroguanosine-3'-Thiophosphate

[0087] Uf = 2'-fluorouridine-3'-phosphate

[0088] Ufs = 2'-fluorouridine-3'-thiophosphate

[0089] Tgn = thymine-glycolic acid-s-isomer

[0090] To further verify the effectiveness of the above nucleic acid sequences, they were conjugated with target ligands for further screening and verification of the final effect in primary liver cells and in vivo experiments. The siRNA agents conjugated with target ligands are shown in Table 4.

[0091] Table 4. siRNA agents conjugated with targeting ligands

[0092] Among them, XHY-1-015, XHY-1-016, and XHY-1-008 are sequences in the prior art, which serve as positive controls with known efficacy. 。

[0093] Gmoes = 2'-methoxyethylguanosine-3'-thiophosphate

[0094] Cmoes = 2'-methoxyethylcytidine-3'-thiophosphate

[0095] Amoes = 2'-methoxyethyl adenosine-3'-thiophosphate

[0096] Umoes = 2'-methoxyethyluridine-3'-thiophosphate

[0097] Gmss = 2'-O-methylguanosine-3'-dithiophosphate

[0098] VP = 5'-vinylphosphonate

[0099] dA = deoxyadenosine

[0100] dG = deoxyguanosine

[0101] dC = deoxycytidine

[0102] dU = deoxyuridine

[0103] dT = deoxythymidine

[0104] IBs = reverse basic deoxyribonucleotide-5'-thioacid

[0105] The (NAG37)s structure is:

[0106] The structural formula for [Gal-6]s[Gal-6]s[Gal-6] is:

[0107] The structural formula for [Gal-6][Gal-6][Gal-6] is:

[0108] Example 3: Preliminary in vitro analysis of INHBE RNAi agent

[0109] In this embodiment, the above-screened potential INHBE RNAi agents were synthesized, and their in vitro efficacy was further screened. The results are shown in Table 5.

[0110] For screening purposes, the human INHBE cDNA sequence (accession number NM_031479.5, synthesized by GenScript) was subcloned into the commercially available reporter-based screening plasmid psiCHECK2, generating Renalis luciferase / INHBE fusion mRNA. For efficacy against human INHBE RNAi agents, Huh7 cells (human hepatocellular carcinoma line) were plated at approximately 10,000 cells / well in a 96-well configuration. Each INHBE RNAi agent was co-transfected at two concentrations (10 nM and 1 nM) with 20 ng psiCHECK2-INHBE plasmid DNA per well and 0.2 μL LipoFectamine 2000 (purchased from Invitrogen, cat#11668-019) per well. The effect of gene knockdown was determined by measuring the level of *Rhizopus luciferase* after luciferase correction using a dual luciferase reporter assay (purchased from Promega, cat#E2940). Specifically, the effect was determined by using constitutively expressed firefly luciferase, which is also present on the psiCHECK2 plasmid, as a reference.

[0111] Table 5. Gene knockdown levels detected by Renalis luciferase assay

[0112] The sequence numbers XHY_001 to XHY_095 correspond to SEQ ID NO 2540 to SEQ ID NO 2634 in Table 3, and this correspondence also applies to Table 6.

[0113] Example 4: Effect of siRNA on INHBE mRNA expression in human Huh7 cells

[0114] Each INHBE RNAi agent was mixed at a concentration of 1 nM with 1 μL of Lipofectamine per well. TM RNAiMAX (purchased from Invitrogen, cat#13778-150) was used for incubation. Huh7 cells (human hepatocellular carcinoma cell line) were seeded at approximately 80,000 cells / well in 24-well plates and incubated with the transfection complex. 24 hours post-transfection, total RNA was extracted using the RNeasy96 kit (purchased from Qiagen). cDNA synthesis and real-time PCR were performed using a reverse transcription kit (Vazyme HiScript III RT SuperMix for qPCR) and a qPCR kit (TIANGEN). INHBE mRNA levels were determined by qRT-PCR relative to Actin mRNA expression (a housekeeping transcript) in the corresponding samples; the results are shown in Table 6.

[0115] Table 6. INHBE mRNA knockdown levels in human Huh7 cells

[0116] Example 5: Effect of INHBE mRNA expression in primary hepatocytes of cynomolgus monkeys

[0117] Each INHBE RNAi agent was mixed with 1 μL of Lipofectamine per well at a concentration of 50 pM, 1 nM, or 20 nM. TM Incubation was performed using RNAiMAX (purchased from Invitrogen). Primary cynomolgus monkey hepatocytes were seeded at approximately 80,000 cells / well in 24-well plates onto the incubated transfection complex. Forty-eight hours post-transfection, total RNA was extracted using the RNeasy96 kit (purchased from Qiagen). cDNA synthesis and real-time PCR were performed using a reverse transcription kit (purchased from Vazyme) and a qPCR kit (purchased from TIANGEN). INHBE mRNA levels were determined by qRT-PCR relative to Actin mRNA expression (a housekeeping transcript) in the corresponding samples. The results are shown in Table 7. The results indicate that incubation with 1 nM or 20 nM siRNA significantly suppressed INHBE gene mRNA expression in primary cynomolgus monkey hepatocytes, reducing the expression level to only approximately 20%. After incubation with 50 pM siRNA, the expression level of INHBE gene mRNA in primary liver cells of cynomolgus monkeys was suppressed, especially in XHY-1-002, XHY-1-005, XHY-1-009, XHY-1-013, XHY-1-018, XHY-1-021, XHY-1-022, XHY-1-025, XHY-1-026, XHY-1-029, and XHY-1-030, with the expression level remaining at only about 30%.

[0118] Table 7. Effects of INHBE mRNA expression in primary hepatocytes of cynomolgus monkeys. " / " indicates that no detection was performed.

[0119] The serial numbers XHY-1-001 to XHY-1-0036 above correspond to the serial numbers in Table 4, and this correspondence also applies to Table 8-11.

[0120] Example 6: Screening INHBE siRNA in a mouse model of HDI human INHBE plasmid

[0121] Four days prior to drug administration (D-4), male NOD-SCID mice (HDI mouse model) were injected via high-pressure intravenous injection (HDI) with the human INHBE plasmid carrying the luciferase gene. On day 0 (D0), mice were divided into groups of four and received a single subcutaneous injection of 3 mg / kg of the test substance. Serum was collected two days prior to drug administration (D-2) and on days 2, 7, and 14 post-administration to separate luciferase expression levels. The percentage of luciferase expression was calculated and compared with the pre-administration (D-2) level (as shown in Table 8) to reflect the effect of the test substance on INHBE gene expression. Specific results are shown in Table 8.

[0122] Table 8 Results of luciferase expression detection

[0123] As shown in Table 7, XHY-1-003, XHY-1-006, XHY-1-007, XHY-1-008, and XHY-1-012 exhibited a certain inhibitory effect on INHBE gene expression. On day 2 after drug administration, the expression level was 50%-80% of the pre-drug expression level, and on day 7 after drug administration, the expression level was 70%-90% of the pre-drug expression level.

[0124] Example 7: Inhibition of human INHBE mRNA expression in transgenic mice by low-dose siRNA.

[0125] C57BL / 6Smoc-Inhbeem1(hINHBE)Smoc mice (male, 6-8 weeks, NM-HU-233512) had their serum collected one day before drug administration (Day 1). On Day 0, the mice were divided into groups of 6 mice each and administered the drug subcutaneously (3 mg / kg). On Day 14 post-administration, liver cells were harvested, and mRNA was extracted from the liver. The expression level of human INHBE mRNA in the liver was detected by qPCR. The results are shown in Table 9. It can be seen that, compared with the blank solvent group (PBS), XHY-1-003, XHY-1-004, XHY-1-005, XHY-1-006, and XHY-1-007 showed an inhibitory effect on INHBE mRNA expression, with expression levels ranging from 43% to 67% of those in the blank solvent group.

[0126] Table 9. Results of human INHBE mRNA expression in the liver of transgenic mice.

[0127] Example 8: Inhibition of human INHBE mRNA expression in transgenic mice by high-dose siRNA.

[0128] C57BL / 6Smoc-Inhbeem1(hINHBE)Smoc mice (male, 6-8 weeks, NM-HU-233512) had their blood collected and serum separated one day before drug administration (Day-1). On Day 0, mice were divided into groups of 5 and subcutaneously administered the drug (10 mg / kg). Day 0 was the day of drug administration. Liver and blood samples were collected and serum separated on Day 14 post-administration. mRNA was extracted from the liver, and the expression level of human INHBE mRNA in the liver was detected by qPCR. The content of INHBE protein in serum was detected by ELISA. The expression level of INHBE protein in the serum of each group was normalized to the protein expression level one day before drug administration (Day-1). Table 10 shows that, compared with the blank solvent group (PBS), XHY-1-002, XHY-1-015, and XHY-1-016 inhibited the expression of INHBE mRNA, with the expression level of INHBE mRNA being 59%–70% of that in the blank solvent group. Table 11 shows that on day 14 after drug administration, the serum INHBE protein level was significantly reduced. XHY-1-001, XHY-1-005, and XHY-1-011 reduced protein expression to 20%, while XHY-1-002 reduced protein expression to 8%.

[0129] Table 10. Results of human INHBE mRNA expression in mouse liver.

[0130] Table 11. Expression levels of INHBE protein in the serum of transgenic mice on day 14 after drug administration (normalized to 1 day before drug administration).

[0131] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any other changes, modifications, substitutions, combinations and simplifications made under the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A siRNA agent for regulating the level of inhibin subunit βE (INHBE), comprising a sense strand and an antisense strand; The antisense strand includes any one of the nucleotide sequences in SEQ ID NO 1 to SEQ ID NO 2444, or a sequence that differs from it by no more than 3 nucleotides, preferably 1, 2 or 3 nucleotides. The justice chain and the antisense chain are at least partially inversely complementary, forming a dual-chain region.

2. The siRNA agent of claim 1, wherein There are no more than 5 mismatches between the justice chain and the antisense chain, preferably no more than 4, no more than 3, no more than 2, and no more than 1 mismatch.

3. The siRNA agent of claim 1, wherein The sense strand and / or antisense strand independently contain one or more modified nucleotides.

4. The siRNA agent of claim 3, wherein The modified nucleotide is at least one of 2'-fluorine modified nucleotide, 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, bicyclic nucleic acid, deoxyribonucleotide, EVP, UNA and GNA.

5. The siRNA agent of claim 1, wherein The sense and / or antisense strands of the siRNA each independently contain up to three unpaired nucleotides.

6. The siRNA agent of claim 1, wherein The sense and / or antisense strands of the siRNA may be the same or different in length.

7. The siRNA agent of claim 1, wherein The length ratio of the justice chain to the antisense chain is 21 / 23.

8. The siRNA agent of claim 3, wherein Includes any one of the positive chains in SEQ ID NO 2540 to SEQ ID NO 2634, and / or any one of the negative chains in SEQ ID NO 2635 to SEQ ID NO 2729.

9. The siRNA agent of claim 1, wherein The nucleotide sequence is conjugated with a GalNAc ligand.

10. The siRNA agent of claim 9, wherein The GalNAc ligand is [Gal-6][Gal-6][Gal-6] or [Gal-6]s[Gal-6]s[Gal-6].

11. The siRNA agent of claim 9, wherein The GalNAc ligand is conjugated to the 5' end or the 3' end of the SS chain.

12. Use of the siRNA agent according to any one of claims 1-11 in the preparation of a medicament.

13. The use according to claim 12 for the manufacture of a medicament, characterized in that, The diseases or conditions mentioned include those caused by various lipid metabolism abnormalities.

14. The use according to claim 13, wherein the medicament is for the treatment of a disease selected from the group consisting of: cancer, autoimmune diseases, infectious diseases, and neurodegenerative diseases. The drug includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include liposomes, microcapsules, and microspheres.

15. A method for preventing or treating a disease or condition, the method comprising administering to a target subject an effective amount of an siRNA agent as described in any one of claims 1-11 or a pharmaceutical composition as described in any one of claims 12-14.

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