Nucleic acid targeting inhibin subunit βe and use thereof

Abstract

Provided are a nucleic acid targeting inhibin subunit βE and a use thereof. The nucleic acid can effectively inhibit INHβE expression, and can thus treat and / or prevent diseases associated with increased INHβE expression.

Description

Nucleic acids targeting inhibin subunit βE and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This invention claims priority to Chinese patent application No. 202410359219.4 filed on March 27, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biotechnology, in particular to a nucleic acid targeting inhibin subunit βE and uses thereof. Background Art

[0004] Obesity is closely linked to a variety of chronic diseases. Currently, over 500 million people worldwide are overweight or obese. Obesity is thought to increase the risk of cardiovascular disease, hypertension, diabetes, gout, and other conditions (Yusuf, S. et al. Obesity and the risk of myocardial infarction in 27,000 participants from 52 countries: a case-control study. Lancet 366, 1640–1649 (2005).). Although the mechanisms of fat distribution are not yet fully understood, abdominal fat accumulation is associated with cardiovascular and metabolic diseases. An increase in waist-to-hip ratio adjusted for body mass has been shown to be associated with type 2 diabetes and cardiovascular disease, as well as other lipid and blood pressure abnormalities (Emdin, CA et al. Genetic association of waist-to-hip ratio with cardiometabolic traits, type 2 diabetes, and coronary heart disease. JAMA 317, 626–634 (2017). and Dale, CE et al. Causal associations of adiposity and body fat distribution with coronary heart disease, stroke subtypes, and type 2 diabetes mellitus: a mendelian randomization analysis. Circulation 135, 2373–2388 (2017).).

[0005] Recently, whole-exome sequencing of over 300,000 people revealed that a loss-of-function variant in the inhibin subunit βE (INHβE) was associated with a low body mass-adjusted waist-to-hip ratio (Deaton, AM et al. Rare loss of function variants in the hepatokine gene INHBE protect from abdominal obesity. Nat Commun 13, 4319 (2022). These data suggest that targeting INHβE has the potential to improve fat distribution and reduce the risk of cardiovascular disease, type 2 diabetes, and other metabolic diseases. The INHβE gene is primarily expressed in the liver and encodes a secreted hepatocyte factor, activin E.

[0006] RNA interference (RNAi) refers to the highly conserved phenomenon of double-stranded small interfering RNA (siRNA) that induces the efficient and specific degradation of homologous mRNA. RNAi drugs also have the advantage of longer-lasting efficacy compared to antibodies. Therefore, the research and development of siRNA targeting INHβE is of great significance. Summary of the Invention

[0007] The purpose of the present invention is to overcome the problems existing in the prior art and provide a new nucleic acid targeting INHβE and its use.

[0008] The first aspect of the present invention provides a nucleic acid comprising a sense strand and an antisense strand, wherein the sense strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides differ from any at least 15 consecutive nucleotides in the sequence shown in any one of SEQ ID Nos. 1 to 68 by no more than 3 nucleotides; or the antisense strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides differ from any at least 15 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID Nos. 69 to 136 by no more than 3 nucleotides.

[0009] A second aspect of the present invention provides a targeted drug delivery system, which comprises a targeting group, a linking group, and the nucleic acid as described above connected to the targeting group via the linking group.

[0010] The third aspect of the present invention provides an in vitro cell containing the nucleic acid.

[0011] The fourth aspect of the present invention provides a pharmaceutical composition comprising the nucleic acid or targeted drug delivery system as described above and a pharmaceutically acceptable carrier.

[0012] The fifth aspect of the present invention provides a method for inhibiting the expression of inhibin subunit βE in a cell, the method comprising: contacting the cell with the nucleic acid, the targeted drug delivery system or the pharmaceutical composition to inhibit the expression of inhibin subunit βE in the cell.

[0013] The sixth aspect of the present invention provides the use of the nucleic acid, the targeted drug delivery system or the pharmaceutical composition in any of the following aspects: 1) treating and / or preventing diseases related to the inhibin subunit βE; 2) preparing drugs for treating and / or preventing diseases related to the inhibin subunit βE.

[0014] The nucleic acid of the present invention can effectively reduce the level of INHβE, and can effectively improve fat distribution and reduce the risks of cardiovascular disease, diabetes and other metabolic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] FIG1 shows the results of the inhibition of INHBE gene expression by different targeted drug delivery systems in Example 2 of the present invention. DETAILED DESCRIPTION

[0017] The following describes specific embodiments of the present invention in detail. It should be understood that the specific embodiments described herein are intended only to illustrate and explain the present invention and are not intended to limit the present invention. Those skilled in the art may make various modifications and variations to the present invention without departing from the scope or spirit of the present invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0018] Terminology

[0019] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0020] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0021] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0022] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0023] Concentration values ​​used in this invention include fluctuations within a certain range. For example, fluctuations within a certain precision range are permitted. For example, for 2%, fluctuations within ±0.1% are permitted. For larger values ​​or values ​​that do not require overly precise control, greater fluctuations are permitted. For example, for 100 mM, fluctuations within ±1%, ±2%, ±5%, etc. are permitted. Regarding molecular weight, fluctuations within ±10% are permitted.

[0024] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0025] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0026] In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0027] In the present invention, the terms "optionally," "optional," "optionally," "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or constraints, each "optional" or "optional" term is independent of the others.

[0028] In the present invention, the term "nucleic acid" refers to a composition containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that can degrade or inhibit (e.g., degrade or inhibit under appropriate conditions) the translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner. The nucleic acid can act through an RNA interference mechanism (i.e., by inducing RNA interference through interaction with the RNA interference pathway mechanism of mammalian cells (RNA-induced silencing complex or RISC)), or through any alternative mechanism or pathway. The scope of the nucleic acids disclosed herein, including sense and antisense strands, includes, but is not limited to, short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates.

[0029] In the present invention, when it is mentioned that "the antisense strand (sense strand) contains at least 15 consecutive nucleotides, and the at least n consecutive nucleotides differ by no more than 3 nucleotides compared with any at least 15 consecutive nucleotides in a reference sequence (such as the sequence shown in SEQ ID No. X or the sequence of nucleotides 1 to 21 of the shown sequence), the comparison situation involved includes the comparison of at least 15 consecutive nucleotides starting from any position (such as position 1, 2, 3, ..., 7, or 8) in the reference sequence. As an example, in some embodiments, the antisense strand (sense strand) contains 21 consecutive nucleotides, wherein the sequence of nucleotides 1 to 15 has 1, 2 or 3 differences with the sequence of nucleotides 2 to 16 of the reference sequence, and nucleotides 16 to 20 in the antisense strand (sense strand) are identical or different from the sequence of nucleotides 17 to 21 of the reference sequence. Such sequences are also within the scope of the present invention. In some embodiments, the antisense strand (sense strand) contains 21 consecutive nucleotides, wherein the nucleotide sequences at positions 1 to 15 are identical to the nucleotide sequences at positions 1 to 15 of the reference sequence, and the nucleotide sequences at positions 16 to 21 of the antisense strand (sense strand) are identical to or different from the nucleotide sequences at positions 16 to 21 of the reference sequence. Such sequences are also within the scope of the present invention.

[0030] In the present invention, when referring to descriptions related to sequence alignment, the “difference” or “difference” mentioned includes one or more of substitution, insertion, and deletion.

[0031] In this invention, "inhibin βE," abbreviated as INHβE, is encoded by the INHBE gene and is a member of the transforming growth factor β (TGF-β) superfamily, belonging to the β subunit of the inhibin / activin family. It is primarily expressed in the liver, and its expression level is positively correlated with insulin resistance and body mass index in humans. INHβE plays a role in regulating various cellular processes, including cell proliferation, apoptosis, immune responses, and hormone secretion.

[0032] As used herein, the terms "silencing," "reducing," "inhibiting," "downregulating," or "knockdown" when referring to the expression of a given gene mean that the expression of the gene is reduced when the cell, cell population, tissue, organ, or subject is treated with a nucleic acid described herein, as measured by the level of RNA transcribed from the gene or the level of a polypeptide, protein, or protein subunit translated from mRNA in the cell, cell population, tissue, organ, or subject in which the gene is transcribed, compared to a second cell, cell population, tissue, organ, or subject that has not been so treated.

[0033] As used herein, "fully complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, all (100%) bases in the contiguous sequence of the first oligonucleotide hybridize to the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first nucleotide sequence or the second nucleotide sequence.

[0034] As used herein, "partially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 70%, but not all, of the bases in the contiguous sequence of the first oligonucleotide hybridize to the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first nucleotide sequence or the second nucleotide sequence.

[0035] As used herein, "substantially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 85%, but not all, of the bases in the contiguous sequence of the first oligonucleotide hybridize to the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first nucleotide sequence or the second nucleotide sequence.

[0036] In the present invention, the term "at least partially complementary" means that the first oligonucleotide and the second oligonucleotide are partially complementary, substantially complementary or completely complementary in a hybridization pair of nucleobase or nucleotide sequence molecules.

[0037] As used herein, the term "treating" refers to methods or steps taken to provide relief or alleviation of the number, severity, and / or frequency of one or more disease symptoms in a subject. Such treatment may include prevention, management, prophylactic treatment, and / or inhibition or reduction of the number, severity, and / or frequency of one or more disease symptoms in a subject.

[0038] In the present invention, the term "connection" means that two compounds or molecules are joined by a covalent bond. Unless otherwise indicated, as used herein, the term "connection" may refer to a connection between a first compound and a second compound with or without any intermediate atoms or groups of atoms.

[0039] As used herein, the term "nucleotides containing unnatural bases" refers to the replacement of natural bases (adenine, uracil, guanine, and cytosine) in RNA molecules with unnatural bases through chemical synthesis. The introduction of these unnatural bases is intended to optimize the performance of RNAi reagents, such as enhancing stability, improving specificity, reducing immunogenicity, or conferring new functions.

[0040] Nucleic Acids

[0041] The present invention first provides a nucleic acid comprising a sense strand and an antisense strand, wherein the sense strand contains at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides, and the at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides differ from any of the at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides in any of SEQ ID No. 1 to 68 by no more than 0, 1, 2 or 3 nucleotides; or the antisense strand contains at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides, and the at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides differ from any of the at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides in any of SEQ ID No. The nucleotide sequence of any one of Nos. 69 to 136 differs by no more than 0, 1, 2 or 3 nucleotides in at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides.

[0042] In some embodiments, the sense strand is identical to nucleotides 2 to 20 in any one of SEQ ID Nos. 1 to 68, and the antisense strand is identical to nucleotides 2 to 20 in any one of SEQ ID Nos. 69 to 136.

[0043] In some embodiments, the antisense strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides (bases).

[0044] In some embodiments, the sense strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides (bases).

[0045] In the present invention, the sense strand and the antisense strand may have the same length or different lengths.

[0046] In specific implementations, those skilled in the art can combine the sequences provided in the present invention in consideration of the complementarity of the sense strand and the antisense strand, thereby obtaining a combined nucleic acid (such as siRNA).

[0047] In a preferred embodiment of the present invention, as shown in Table 1, the nucleic acid is selected from at least one of siRNA-1 having a sense chain sequence of SEQ ID No. 1 and an antisense chain sequence of SEQ ID No. 69, siRNA-2 having a sense chain sequence of SEQ ID No. 2 and an antisense chain sequence of SEQ ID No. 70, siRNA-3 having a sense chain sequence of SEQ ID No. 3 and an antisense chain sequence of SEQ ID No. 71, siRNA-4 having a sense chain sequence of SEQ ID No. 4 and an antisense chain sequence of SEQ ID No. 72, siRNA-5 having a sense chain sequence of SEQ ID No. 5 and an antisense chain sequence of SEQ ID No. 73..., siRNA-66, siRNA-67, and siRNA-68.

[0048] In some embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides, and the at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides differ from at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides in any of the sequences set forth in SEQ ID No. 115 or 120 by no more than 0, 1, 2, or 3 nucleotides; and the sense strand comprises a nucleotide sequence that is at least partially complementary (e.g., partially complementary, substantially complementary, or completely complementary) to the antisense strand. When the antisense strand has the above sequence, the double-stranded RNA has a significantly better inhibitory effect on INHβE.

[0049] In some preferred embodiments, the positive strand contains at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides, and the at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides differ from at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides in any of the sequences shown in SEQ ID No. 47 or 52 by no more than 0, 1, 2 or 3 nucleotides.

[0050] In some embodiments, the sense strand is identical to nucleotides 2 to 20 in the sequence shown in SEQ ID No. 47 or 52, and the antisense strand is identical to nucleotides 2 to 20 in the sequence shown in SEQ ID No. 115 or 120.

[0051] In some preferred embodiments, the positive strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 47 (5'-GGAGACAAGCAUUUAUACUUU-3') by 0, 1 or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 115 (5'-AAAGUAUAAAUGCUUGUCUCCUU-3') by 0, 1 or 2 nucleotides.

[0052] In some preferred embodiments, the positive strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 52 (5'-GCCUGGCUUAUACUUUCUUAA-3') by 0, 1 or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 120 (5'-UUAAGAAAGUAUAAGCCAGGCUU-3') by 0, 1 or 2 nucleotides.

[0053] In some preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 137 (5'-CGAGACAAGCAUUUAUACUUA-3') by 0, 1 or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 138 (5'-UAAGUAUAAAUGCUUGUCUCGUU-3') by 0, 1 or 2 nucleotides.

[0054] In some preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 139 (5'-CGAGACAAGCAUUUAUACUUG-3') by 0, 1 or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 140 (5'-CAAGUAUAAAUGCUUGUCUCGUU-3') by 0, 1 or 2 nucleotides.

[0055] All nucleotide groups in the above nucleic acid may be chemically unmodified, or may contain at least one modified nucleotide group, and the modification may be on the nucleotide at any position.

[0056] In some embodiments, the sense and antisense strands can be partially complementary, substantially complementary, or fully complementary to each other.

[0057] In some embodiments, when the sequence identity of the sense strand or antisense strand of the nucleic acid to the corresponding sequence mentioned in the present invention is less than 100% or differs by more than one nucleotide, it still has an inhibitory effect on INHβE that is similar to that of the corresponding sequence (e.g., still has an efficacy equivalent to 80-120%, 85-115%, or 90-110% of the corresponding sequence) or equivalent (e.g., still has an efficacy equivalent to 95-105% of the corresponding sequence). For example, the two bases at the 3' end of the antisense strand (such as the sequence shown in any of SEQ ID Nos. 69-136) are replaced with AA, CU, UC, AG, CC, GG, or UG, or a combination of any two nucleic acids. Such nucleic acid sequences also fall within the scope of protection of the present invention.

[0058] In some preferred embodiments, the nucleic acid has an inhibition efficiency of no less than 50% on INHβE (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).

[0059] Table 1

[0060] In Table 1, the first column indicates the position of the first base of the targeted gene in the coding sequence of INHβE, and so on; the numbers in columns 3 and 5 indicate the sequence number, for example, "1" indicates SEQ ID No. 1. The reference sequence of the targeted gene is the coding sequence of human INHβE, NM_031479.

[0061] The above-mentioned technical solutions for naked sequences (i.e., unmodified sequences) mentioned in the present invention have advantages in terms of effectiveness that are independent of the modification method or the selection of the targeting vector. The following describes applicable modification solutions and further preferred modification solutions.

[0062] In some embodiments, the nucleic acid contains a nucleotide group as a basic structural unit, wherein the nucleotide group contains a phosphate group, a ribose group and a base. Preferably, the nucleic acid contains at least one modified nucleotide group. The nucleic acid containing the modifying group has an inhibition efficiency of INHβE of not less than 50% (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).

[0063] In some embodiments, the modified nucleotide groups are phosphate and / or ribose groups. The modified sites can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 of nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 of the sense strand and / or antisense strand.

[0064] In some embodiments, modification of the phosphate group refers to modification of the oxygen in the phosphate group, including phosphorothioate and boranophosphate modifications. As shown in the following formulas, the oxygen in the phosphate group is replaced with sulfur, borane, amine, alkyl, or alkoxy groups. These modifications can stabilize the structure of nucleic acids and maintain high base pairing specificity and affinity.

[0065] In the above structural formula, BASE represents the base A, U, C, G, or T. X can be oxygen (O) or sulfur (S). R in the above structure can be the same or different, for example: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, amino, cyanoethyl, acetyl, etc. R' and R" can each independently be hydrogen (H), methyl (CH3), ethyl (CH2CH3), propyl (CH2CH2CH3), isopropyl (CH(CH3)2), allyl, propargyl, acyloxybenzyl, or acyloxyethyl.

[0066] In some embodiments, modification of the ribose group refers to modification of the 2′-hydroxyl group (2′-OH) in the ribose group. Introducing certain substituents, such as methoxy or fluorine, at the 2′-hydroxyl position of the ribose group makes the nucleic acid less susceptible to cleavage by ribonucleases, thereby increasing the stability of the nucleic acid and making it more resistant to nuclease hydrolysis. Modifications of the 2′-hydroxyl group in nucleotide pentoses include 2′-fluoro modification (such as 2′-arabino-fluoro modification), 2′-methoxy modification (2′-OME), 2′-methoxyethyl modification (2′-MOE), 2′-2,4-dinitrophenol modification (2′-DNP modification), 2′,4′-constrained ethyl modification, 2′-amino modification, 2′-deoxy modification, BNA, acyclic nucleic acid modification, misaligned nucleic acid modification, and L-type nucleic acid modification. BNA (endocyclic bridged nucleotide) refers to a constrained or inaccessible nucleotide. BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C 3'-endo sugar condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose ring to provide a 2', 4'-BNA nucleotide, such as a locked ethyl modification (LNA), a ring locked ethyl modification (ENA) and an ethyl locked nucleic acid modification (cET BNA). Acyclic nucleic acids are nucleotides formed by opening the sugar ring of the nucleotide, such as unlocked nucleic acid (UNA) nucleotides and glycerol nucleic acid (GNA) nucleotides. Misplaced nucleic acid modification refers to the replacement of a 3', 5'-phosphate bond link by a 2', 5'-phosphate bond chain. L-type nucleic acid modification refers to the replacement of a naturally occurring D-type nucleic acid with its mirror image stereo equivalent, an L-type nucleic acid.

[0067] Wherein, BASE represents the base A, U, C, G or T. R in the above structure can be the same or different, such as hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, acetyl, etc.

[0068] In some embodiments, according to a particularly preferred embodiment of the present invention, the nucleotide group containing a uracil base or a cytosine base in the sense strand of the RNAi agent is a nucleotide group in which the ribose group is modified, that is, the 2'-OH of the ribose group in the nucleotide group containing a uracil base or a cytosine base in the sense strand of the RNAi agent is replaced by a methoxy group or a fluorine group. More preferably, the 3' end of both the sense strand and the antisense strand of the RNAi agent can be connected to dTdT; or, the 3' end of the antisense strand of the RNAi agent can be connected to AA or UU or a combination of any two nucleic acids (which can be but is not limited to CC, GG or UG), so that the sequence has a specific inducement for mRNA degradation. The RNAi agent with the above modifications exhibits a more excellent in vivo inhibitory effect, and the above modifications can further reduce the immunogenicity of the RNAi agent of the present invention in vivo.

[0069] The RNAi reagents of the present invention may also include a modification that includes a nucleoside monophosphate attached to the 5' end of the antisense strand. The 5'-monophosphate at the terminal end of the siRNA guide strand is important for RISC recognition. Phosphorylation of the 5'-hydroxyl group plays a role in the effective loading of siRNA onto Ago2 within cells. The monophosphate at the 5' end of the siRNA guide strand interacts with Argonaute-2 (Ago2) through H-bonding, ensuring accurate targeting and precise cleavage of the mRNA target. Commonly used 5'-monophosphate nucleoside derivatives include the following. These phosphate nucleoside derivatives have been shown to exhibit a certain degree of stability in biological metabolic media and to be effective in promoting the loading of siRNA guide strands onto Ago2 within cells (Nucleic Acids Research, 2015, 43, 2993–3011). In the RNAi reagents of the present invention, trans-vinyl phosphate (VP) is preferably used as the primary choice, but monophosphate nucleoside derivatives other than those mentioned above may also be included.

[0070] In the above structures, BASE represents the base A, U, C, G, or T. R in the above structures can be the same or different, such as hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, amino, acetyl, etc.

[0071] In the present invention, The meaning is consistent with that of , which means that a chemical element X is connected to any one or more groups.

[0072] In some embodiments, at least one nucleotide in the nucleic acid is a modified nucleotide or includes a modified linkage.

[0073] In some embodiments, the modified nucleotides are preferably selected from one or more of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-open ring nucleotide mimics, locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitols, inverted nucleotides, inverted 2'-O-methyl nucleotides, inverted 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides (Morpholino), peptide nucleic acids (PNA), glycerol nucleic acids (GNA), triazacyclic DNA (tcDNA), nucleotides containing non-natural bases, nucleotides containing vinyl phosphonate, nucleotides containing cyclopropyl phosphonate and 3'-O-methyl nucleotides; the modified nucleotides are further preferably selected from one or both of 2'-O-methyl nucleotides and 2'-fluoro nucleotides.

[0074] In some embodiments, the modified inter-linkage is preferably selected from one or more of phosphorothioate inter-nucleotide linkages and methylphosphonate inter-nucleotide linkages. In some embodiments, the modified inter-linkage is further preferably selected from one or more of phosphorothioate monoester inter-nucleotide linkages and phosphorothioate diester inter-nucleotide linkages.

[0075] In some preferred embodiments, the antisense strand comprises a 2'-fluoro nucleotide at nucleotide position 14 and at least one of nucleotide positions 1, 2, 3, 4, 6, 7, 8, 12, or 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) in the nucleotide sequence of any one of SEQ ID Nos. 69 to 136. Compared to known modification methods, the above-described antisense strand modification scheme further enhances the inhibitory effect of the nucleic acid on INHβE.

[0076] In some specific embodiments, the antisense strand has 2'-fluoro nucleotides at nucleotide positions 2, 6, 14, and 16, and at least one of nucleotide positions 1, 3, 4, 7, 8, and 12 (such as 1, 2, 3, 4, 5, or 6) in the nucleotide sequence of any one of SEQ ID Nos. 69 to 136, and the other positions are 2'-O-methyl nucleotides.

[0077] In some preferred embodiments, the sense strand comprises a 2'-fluoro nucleotide at nucleotide position 9 and at least one of nucleotide positions 1, 7, 10, 11, and 12 (e.g., 1, 2, 3, 4, or 5) in the nucleotide sequence of any one of SEQ ID Nos. 1 to 68. Compared to known modification methods, the above-described sense strand modification scheme further enhances the inhibitory effect of the nucleic acid on INHβE.

[0078] In some specific embodiments, the sense strand has 2'-fluoro nucleotides at nucleotide positions 7, 9, and 11, and at least one (e.g., 1, 2, or 3) selected from nucleotide positions 1, 10, and 12 of the nucleotide sequence shown in any one of SEQ ID Nos. 1 to 68, and the other positions have 2'-O-methyl nucleotides.

[0079] In some embodiments, the last 2 to 4 (e.g., 2, 3, or 4) nucleotides at the 5' end and / or 3' end of the antisense strand contain a phosphorothioate internucleotide bond, and the last 2 to 4 (e.g., 2, 3, or 4) nucleotides at the 5' end and / or 3' end of the sense strand contain a phosphorothioate internucleotide bond.

[0080] In some specific embodiments, the last three nucleotides at the 5' end and the 3' end of the antisense strand contain a phosphorothioate internucleotide bond, and the last three nucleotides at the 5' end of the sense strand contain a phosphorothioate internucleotide bond.

[0081] In some embodiments, the antisense strand comprises a nucleotide sequence that differs from the antisense strand shown in any one of Tables 2 or 3 by 0, 1, or 2 nucleotides.

[0082] In some embodiments, the sense strand comprises a nucleotide sequence that differs from the sense strand shown in any one of Tables 2 or 3 by 0, 1, or 2 nucleotides.

[0083] In some embodiments, the nucleic acid comprises a duplex as shown in any one of Tables 2 or 3.

[0084] In some preferred embodiments, the nucleic acid comprises a duplex selected from any one of SN-254866, SN-254861, SN-255250, SN-256698, SN-256699, SN-256690, SN-256691, SN-255259, SN-256692, SN-256603, SN-256604, SN-256605, SN-256674, SN-256675, SN-256689, SN-256680, SN-256681, SN-256687, SN-256682, SN-256693, SN-256694, SN-256688, SN-256695, SN-256696, and SN-256697.

[0085] Table 2

[0086] Table 3

[0087] In each modified sequence of the present invention, a nucleotide represented by a lowercase letter indicates that the nucleotide is a 2'-O-methyl nucleotide; f indicates that the nucleotide adjacent to the left is a 2'-fluoro nucleotide; s indicates that the two adjacent nucleotides on the left and right are connected by a phosphorothioate diester internucleotide bond.

[0088] The nucleic acids described herein can be obtained by conventional methods in the art, such as solid-phase synthesis and liquid-phase synthesis. Solid-phase synthesis is commercially available on a custom-built basis and can therefore be purchased commercially. The modified nucleotide groups can be introduced by correspondingly modified nucleotide monomers.

[0089] Based on the nucleic acid (siRNA) synthesized above, the present invention can further construct an shRNA expression plasmid having the same or similar function as the above nucleic acid. The method for constructing the expression plasmid is well known to those skilled in the art and will not be described in detail here.

[0090] Targeted drug delivery system

[0091] The present invention also provides a targeted drug delivery system, which comprises a targeting group, a linking group, and the nucleic acid as described above connected to the targeting group via the linking group.

[0092] In conjunction with common sense in the art, nucleic acid (siRNA) of the present invention, when applied to different targeted drug delivery systems, all has preferably inhibitory effect. In other words, the effect advantage of naked sequence and modified sequence in the present invention does not depend on the selection of targeting vector. In order to further improve the bioavailability and therapeutic effect of siRNA, the present invention has also been optimized to the targeted drug delivery system, and has obtained the following technical scheme.

[0093] In some embodiments, the targeting group can further improve the targeting of small nucleic acids and can be provided by monosaccharides (such as glucose, mannose, allose, altrose, galactose, galactosamine, N-acetylgalactosamine, talose, fructose, idose, etc.) and / or polypeptides (such as proteins, monoclonal antibodies, nanobodies).

[0094] In some embodiments, the linking group may be selected from -O-[CH2CH2O]n-, -[CH2]m-CONH-[CH2]nO-, -O-[CH2CH2O]m-CONH-[CH2]nO-, and -O-[CH2]m-CONH-[CH2H2O]nO-, wherein m and n may each independently be an integer from 1 to 10.

[0095] In some embodiments, the targeted drug delivery system comprises a ligand and the nucleic acid linked to the ligand, wherein the ligand is linked to one or more of the 5' end of the antisense strand, the 3' end of the antisense strand, the 5' end of the sense strand, and the 3' end of the sense strand.

[0096] In some preferred embodiments, the ligand is a GalNAc derivative.

[0097] In some preferred embodiments, the ligand is one or more GalNAc derivatives connected by single-stranded, double-stranded or triple-stranded branched linkers.

[0098] In some further preferred embodiments, the RNAi agent comprises a compound having a structure shown in the following formula I:

[0099] In the formula, Nu represents the duplex. This targeted drug delivery system utilizes the structural characteristics on the left side to improve the cell penetration ability of the nucleic acid drug (Nu) and enhance its intracellular stability. It also has a simple preparation process and strong practicality.

[0100] In a specific implementation, the ligand portion can be contacted with a nucleotide monomer or a nucleic acid attached to a solid support under coupling reaction conditions and in the presence of a coupling reagent, so that the compound portion is attached to the nucleic acid through a coupling reaction.

[0101] cell

[0102] The present invention also provides an in vitro cell containing the nucleic acid.

[0103] In some embodiments, the cells can be used for gene function research, disease model research, drug screening, and the like.

[0104] In some embodiments, the cells will not develop into an animal individual. In some specific embodiments, the cells can be microbial cells or animal cells, but the animal cells are not animal embryonic stem cells and cells in various formation and development stages (e.g., germ cells, fertilized egg cells, etc.).

[0105] Pharmaceutical composition

[0106] The present invention also provides a pharmaceutical composition comprising the nucleic acid or targeted drug delivery system as described above and a pharmaceutically acceptable carrier.

[0107] The pharmaceutical composition can be prepared from the nucleic acid and the pharmaceutically acceptable carrier by conventional methods. For example, the pharmaceutical composition can be an injection. The injection can be used for subcutaneous, intramuscular, or intravenous injection.

[0108] According to the pharmaceutical composition of the present invention, there are no special requirements for the amount of the nucleic acid or targeted drug delivery system and the pharmaceutically acceptable carrier. Generally, relative to 1 part by weight of the nucleic acid (or 1 part by weight of the targeted drug delivery system calculated as nucleic acid), the content of the pharmaceutically acceptable carrier can be 1-100,000 parts by weight (such as 1 part by weight, 5 parts by weight, 10 parts by weight, 50 parts by weight, 100 parts by weight, 500 parts by weight, 1,000 parts by weight, 5,000 parts by weight, 10,000 parts by weight, 50,000 parts by weight, 100,000 parts by weight or any value between any two of the above values).

[0109] According to the pharmaceutical composition of the present invention, wherein the pharmaceutically acceptable carrier can be various carriers conventionally used in the art, for example, can include at least one of a pH buffer, a protective agent and an osmotic pressure regulator. The pH buffer can be a tris hydroxymethylaminomethane hydrochloride buffer with a pH of 7.5-8.5 and / or a phosphate buffer with a pH of 5.5-8.5, preferably a phosphate buffer with a pH of 5.5-8.5. The protective agent can be at least one of inositol, sorbitol and sucrose. Based on the gross weight of the pharmaceutical composition, the content of the protective agent can be 0.01-30 weight % (such as 0.01 weight %, 0.05 weight %, 0.1 weight %, 0.5 weight %, 1 weight %, 5 weight %, 10 weight %, 15 weight %, 20 weight %, 25 weight %, 30 weight % or any value between any two of the above values). The osmotic pressure regulator can be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition be 200-700 mOsmole / kg. According to the desired osmotic pressure, those skilled in the art can determine the content of the osmotic pressure regulator.

[0110] According to a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is a liposome. The liposome can be any liposome capable of encapsulating nucleic acid, and its diameter can be 25-1000 nm, and can include but not limited to cholesterol and its analogs or derivatives.

[0111] The dosage of the pharmaceutical composition of the present invention can be a conventional dosage in the art, and can be determined based on various parameters, particularly the age, weight, and sex of the subject. For example, for female mice aged 3-4 months and weighing 25-30 g, the dosage of the pharmaceutical composition can be 0.01-100 mg / kg body weight, preferably 1-10 mg / kg body weight, based on the amount of the nucleic acid in the pharmaceutical composition.

[0112] Methods and uses

[0113] The present invention also provides a method for inhibiting the expression of inhibin subunit βE in cells, the method comprising: contacting the cells with the nucleic acid, the targeted drug delivery system or the pharmaceutical composition to inhibit the expression of inhibin subunit βE in the cells.

[0114] In some embodiments, the cell is in a subject, eg, a human subject, eg, a subject having an inhibin subunit [beta]E-associated disease, or a subject at risk of an inhibin subunit [beta]E-associated disease in need of prevention.

[0115] In some embodiments, the cells are located in vitro. The methods are for research purposes or for constructing animal models.

[0116] In some embodiments, contacting the cell with the RNAi agent or the pharmaceutical composition inhibits expression of inhibin subunit βE by at least 50%, 60%, 70%, 80%, 90%, or 95% (e.g., compared to the level of inhibin subunit βE expression before the cell is first contacted with the RNAi agent or the pharmaceutical composition; e.g., before the first dose of the RNAi agent or the pharmaceutical composition is administered to the subject). In certain embodiments, inhibiting expression of inhibin subunit βE reduces the level of inhibin subunit βE protein in a serum sample of the subject by at least 50%, 60%, 70%, 80%, 90%, or 95%, e.g., compared to the level of inhibin subunit βE expression before the cell is first contacted with the RNAi agent or the pharmaceutical composition.

[0117] The present invention also provides the use of the nucleic acid, the targeted drug delivery system, or the pharmaceutical composition in treating and / or preventing diseases associated with the inhibin subunit βE. Specifically, a method for treating and / or preventing diseases associated with the inhibin subunit βE comprises administering the nucleic acid, the targeted drug delivery system, or the pharmaceutical composition to a subject.

[0118] The present invention also provides use of the nucleic acid, the targeted drug delivery system or the pharmaceutical composition in preparing a drug for treating and / or preventing diseases associated with inhibin subunit βE.

[0119] In some embodiments, the disease is: (i) a disease associated with enhanced or elevated inhibin subunit [beta]E; or (ii) a disease that would benefit from decreased expression of inhibin subunit [beta]E.

[0120] In some embodiments, the disease is selected from diseases related to abnormal fat distribution.

[0121] In some embodiments, the disease is selected from at least one of cardiovascular disease, diabetes, lipid metabolism disorder, hypertension, obesity, and metabolic syndrome.

[0122] In the present invention, the subject can be a mammal, including a primate (such as a human, a non-human primate, such as a monkey and a chimpanzee), a non-primate (such as a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse), or a bird. In some embodiments, the subject is preferably a primate, more preferably a human.

[0123] In some embodiments, the drug can be administered via a variety of routes, depending on whether local or systemic treatment is desired. The dosage can be referred to above and will not be repeated here.

[0124] In some embodiments, administration can be topical (e.g., transdermal patch), pulmonary, e.g., via inhalation or insufflation of a powder or spray, including via a nebulizer; intratracheal, nasal, epidermal, and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, e.g., via an implant device; or intracranial, e.g., via intraparenchymal, intrathecal, or intraventricular administration.

[0125] In some embodiments, the nucleic acid, the targeted drug delivery system, or the pharmaceutical composition is administered to the subject by subcutaneous administration, intravenous administration, and / or intramuscular administration.

[0126] Example

[0127] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0128] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0129] Example 1

[0130] The siRNAs listed in Table 2 were diluted with ddH2O to final concentrations of 1 nM and 10 nM, and a Reneilla luciferase assay was performed in HEK293T cells. The specific steps were as follows: HEK293T cells were passaged for 2-3 generations in DMEM (Gibco, 11995065) medium containing 10% FBS (Hyclone, SH30406.05), then plated on 0.1% gelatin-coated 96-well plates (Greiner #655098) at a cell density of 30K per well and cultured overnight. The next day, siRNA samples were dissolved and diluted in ddH2O. Then, 10 μL of siRNA sample (at a final concentration of 1 nM or 10 nM) was mixed with 10 μL of reduced-serum medium (Absin, abs9461) containing 100 ng of psiCHECK TM2-INHBE plasmid and 0.5 μL of lipofectamine 2000 (Thermo Fisher, 11668019). The mixture was added to a 96-well plate containing HEK293T cells and incubated in a 5% CO2 incubator at 37°C for 24 h. After 24 hours, 75 μL of glo-luciferase reagent (Promega, E2940) was added to each well and incubated at room temperature for 15 minutes. Firefly luciferase luminescence was measured on a microplate reader at an OD of 590 nm. Stop&glo-luciferase reagent (Promega, E2940) was added to each well and incubated at room temperature for 15 minutes. Renilla luciferase luminescence was measured on a microplate reader at an OD of 528 nm. %KD was calculated by calculating Rluc / Fluc. %KD = 1 - (sample Rluc / Fluc - blank) / (simple plasmid Rluc / Fluc - blank) to obtain knockdown data for the siRNA samples. Results are shown in Table 4.

[0131] Table 4

[0132] It can be seen that the siRNA containing the duplex sequence of the present invention has different degrees of inhibitory effect on the expression of INHβE.

[0133] Example 2

[0134] Each of the targeted drug delivery systems conjugated with TriGalNAc (see Formula I for its structure) listed in Table 5 was dissolved in 100 μL of enzyme-free sterile water to a 1000 μM solution, corresponding to a 1000 nM working solution. Monkey primary hepatocytes were removed from liquid nitrogen, thawed and revived at 37°C, rinsed, counted, and centrifuged using PMonH plating medium. After removing the supernatant, the cells were diluted to 250 kb / mL using fresh PMonH plating medium. 100 μL of this diluted cell solution was plated onto a 96-well cell culture plate, with 25 kb cells per well. 10 μL of the 1000 mM compound solution was pipetted into the corresponding wells, with a gradient of compound concentrations performed in duplicate. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours, and samples were harvested. After 24 hours, all medium was removed from the 96-well culture plate, and the cells were washed with 1× PBS buffer. 50 μL of prepared Cells to CT lysis buffer (as recommended by the manufacturer) was added and mixed thoroughly. After incubation for 10 minutes, 2.5 μL of stop buffer was added for 2 minutes. RT-PCR was performed according to the High Capacity cDNA Reverse Transcription Kits (Thermo Fisher, Cat. No. 4368814), with 10 μL of lysis buffer per reaction. Gene expression was quantified using real-time fluorescence PCR. The TaqMan probe for monkey INHBE was Mf02820386_g1, and the probe for the internal reference gene (monkey PPIB) was Mf02802985_m1 (Thermo Fisher Scientific, Waltham, MA, USA). PCR conditions were 1 cycle at 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. The real-time PCR instrument was QuantStudio. TM 6 Pro Real-Time PCR System (Thermo Fisher). INHBE gene expression was calculated using 2-ΔΔCt, with monkey PPIB gene expression serving as an internal reference. INHBE gene expression was expressed as a percentage of the control group containing cells cultured only. Results are shown in Figure 1.

[0135] Table 5

[0136] Example 3

[0137] The siRNAs listed in Table 6 were serially diluted with enzyme-free sterile water and assayed using a Reneilla luciferase assay in HEK293T cells. The specific method was as described in Example 1. The IC50 values ​​were calculated using Prism GraphPad, as shown in Table 7.

[0138] Table 6

[0139] Table 7

[0140] Example 4

[0141] The siRNAs listed in Table 8 were serially diluted with enzyme-free sterile water and assayed using a Reneilla luciferase assay in HEK293T cells. The specific method was as described in Example 1. The results are shown in Table 9.

[0142] Table 8

[0143] Table 9

[0144] Example 5

[0145] The siRNAs listed in Table 10 were serially diluted with enzyme-free sterile water and assayed in HEK293T cells using a Reneilla luciferase assay. The specific method was as described in Example 1. The IC50 values ​​were calculated using Prism GraphPad, as shown in Table 11.

[0146] Table 10

[0147] Table 11

[0148] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that variations and modifications are possible without departing from the spirit of the present invention, and all such variations and modifications fall within the scope of the present invention.

Claims

1. A nucleic acid comprising a sense strand and an antisense strand, characterized in that: The sense strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides differ by no more than 3 nucleotides compared with any at least 15 consecutive nucleotides in the sequence shown in any one of SEQ ID Nos. 1 to 68; or the antisense strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides differ by no more than 3 nucleotides compared with any at least 15 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID Nos. 69 to 136.

2. The nucleic acid according to claim 1, wherein The sense strand is identical to nucleotides 2 to 20 in any one of SEQ ID Nos. 1 to 68, and the antisense strand is identical to nucleotides 2 to 20 in any one of SEQ ID Nos. 69 to 136.

3. The nucleic acid according to claim 1 or 2, wherein The antisense strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides differ by no more than 3 nucleotides compared with at least 15 consecutive nucleotides in any of the sequences shown in SEQ ID No. 115 or 120; optionally, the positive strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides differ by no more than 3 nucleotides compared with at least 15 consecutive nucleotides in any of the sequences shown in SEQ ID No. 47 or 52.

4. The nucleic acid according to claim 1, wherein The sense strand comprises a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 47 by 0, 1, or 2 nucleotides, and the antisense strand comprises a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 115 by 0, 1, or 2 nucleotides; or, The sense strand comprises a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 52 by 0, 1, or 2 nucleotides, and the antisense strand comprises a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 120 by 0, 1, or 2 nucleotides; or, The sense strand comprises a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 137 by 0, 1, or 2 nucleotides, and the antisense strand comprises a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 138 by 0, 1, or 2 nucleotides; or, The sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 139 by 0, 1 or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 140 by 0, 1 or 2 nucleotides.

5. The nucleic acid according to claim 1, wherein At least one nucleotide in the nucleic acid is a modified nucleotide or includes a modified linkage; The modified nucleotides are preferably selected from one or more of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-open ring nucleotide mimics, locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitols, inverted nucleotides, inverted 2'-O-methyl nucleotides, inverted 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides (Morpholino), peptide nucleic acids (PNA), glycerol nucleic acids (GNA), triazacyclic DNA (tcDNA), nucleotides containing non-natural bases, nucleotides containing vinyl phosphonate, nucleotides containing cyclopropyl phosphonate and 3'-O-methyl nucleotides; the modified nucleotides are further preferably selected from one or both of 2'-O-methyl nucleotides and 2'-fluoro nucleotides; The modified internucleotide bond is preferably selected from one or more of phosphorothioate internucleotide bonds and methylphosphonate internucleotide bonds; the modified internucleotide bond is further preferably selected from one or more of phosphorothioate monoester internucleotide bonds and phosphorothioate diester internucleotide bonds.

6. The nucleic acid according to claim 5, wherein The antisense strand comprises a 2'-fluoro nucleotide at the 14th nucleotide position and at least one of the 1, 2, 3, 4, 6, 7, 8, 12, and 16 nucleotide positions of the nucleotide sequence shown in any one of SEQ ID Nos. 69 to 136.

7. The nucleic acid according to claim 5 or 6, wherein The sense strand comprises a 2'-fluoro nucleotide at the 9th nucleotide position and at least one of the 1st, 7th, 10th, 11th and 12th nucleotide positions of the nucleotide sequence shown in any one of SEQ ID Nos. 1 to 68.

8. The nucleic acid according to any one of claims 5 to 7, wherein The last 2 to 4 nucleotides at the 5' end and / or 3' end of the antisense strand contain a phosphorothioate internucleotide bond, and the last 2 to 4 nucleotides at the 5' end and / or 3' end of the sense strand contain a phosphorothioate internucleotide bond.

9. The nucleic acid according to claim 1, wherein The antisense strand comprises a nucleotide sequence that differs from the antisense strand shown in any one of Table 2 or Table 3 by 0, 1, or 2 nucleotides; Preferably, the sense strand comprises a nucleotide sequence that differs from the sense strand shown in any one of Table 2 or Table 3 by 0, 1 or 2 nucleotides; Preferably, the nucleic acid comprises a duplex as shown in any one of Table 2 or Table 3.

10. The nucleic acid according to claim 9, wherein The nucleic acid comprises a duplex selected from any one of SN-254866, SN-254861, SN-255250, SN-256698, SN-256699, SN-256690, SN-256691, SN-255259, SN-256692, SN-256603, SN-256604, SN-256605, SN-256674, SN-256675, SN-256689, SN-256680, SN-256681, SN-256687, SN-256682, SN-256693, SN-256694, SN-256688, SN-256695, SN-256696, and SN-256697.

11. A targeted drug delivery system, characterized in that: The targeted drug delivery system comprises a targeting group, a linking group, and the nucleic acid according to any one of claims 1 to 10 connected to the targeting group via the linking group.

12. The targeted drug delivery system according to claim 11, wherein The targeted drug delivery system comprises a ligand and the nucleic acid connected to the ligand, wherein the ligand is connected to one or more of the 5' end of the antisense strand, the 3' end of the antisense strand, the 5' end of the sense strand and the 3' end of the sense strand; preferably, the ligand is a GalNAc derivative; more preferably, the ligand is one or more GalNAc derivatives connected by single-stranded, double-stranded or triple-stranded branched linkers.

13. The targeted drug delivery system according to claim 11, wherein The structure of the targeted drug delivery system is shown below: In the formula, Nu represents the nucleic acid.

14. An isolated cell, characterized in that The cell contains the nucleic acid according to any one of claims 1 to 10.

15. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the nucleic acid according to any one of claims 1 to 10 or the targeted drug delivery system according to any one of claims 11 to 13 and a pharmaceutically acceptable carrier.

16. A method for inhibiting the expression of inhibin subunit βE in a cell, the method comprising: The cell is contacted with the nucleic acid according to any one of claims 1 to 10, the targeted drug delivery system according to any one of claims 11 to 13, or the pharmaceutical composition according to claim 15 to inhibit the expression of inhibin subunit βE in the cell.

17. Use of the nucleic acid according to any one of claims 1 to 10, the targeted drug delivery system according to any one of claims 11 to 13, or the pharmaceutical composition according to claim 15 in any of the following aspects: 1) Treating and / or preventing diseases related to inhibin subunit βE; 2) Preparation of drugs for treating and / or preventing diseases associated with inhibin subunit βE.

18. The use according to claim 17, wherein The diseases are: (i) a disease associated with an increase or elevation of the inhibin subunit βE; or (ii) Diseases that would benefit from reduced expression of the inhibin subunit βE.

19. The use according to claim 17, wherein The disease is selected from related diseases caused by abnormal fat distribution, preferably at least one of cardiovascular disease, diabetes, lipid metabolism disorder, hypertension, obesity, and metabolic syndrome.

20. The use according to claim 17, wherein The nucleic acid, the targeted drug delivery system or the pharmaceutical composition is administered to the subject by subcutaneous administration, intravenous administration and / or intramuscular administration.

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