Nucleic acids targeting statin subunit betaE and uses thereof

Abstract

The invention relates to the technical field of biology, and discloses nucleic acid targeting statin subunit betaE and application of the nucleic acid. The nucleic acid provided by the invention can effectively inhibit the expression of INH [beta] E, so that diseases related to the increase of the expression of INH [beta] E can be treated and / or prevented.

Description

[0001] Cross - reference to related applications

[0002] This invention claims the priority of a Chinese patent application with the application number 202410359219.4, filed on March 27, 2024, the entire content of which is incorporated herein by reference. Technical field

[0003] This invention relates to the field of biotechnology, and specifically, to nucleic acids targeting inhibin subunit βE and their uses. Background art

[0004] Obesity is closely related to a variety of chronic diseases, and currently, more than 500 million people worldwide are overweight or obese. Obesity is considered to increase the risk of developing diseases such as cardiovascular diseases, hypertension, diabetes, gout, etc. (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 mechanism of fat distribution is not very clear, the accumulation of abdominal fat is correlated with cardiovascular diseases and metabolic diseases. An increase in the waist - to - hip ratio adjusted according to the body mass index has been confirmed to be associated with type 2 diabetes, cardiovascular diseases, and other abnormal blood lipid and blood pressure conditions (Emdin, C. A. 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, C. E. 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, in the whole-exome sequencing of more than 300,000 people, it was found that a loss-of-function variant of inhibin subunit βE (INHβE) was associated with a lower body mass index-adjusted waist-to-hip ratio (Deaton, A.M. 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 risks of cardiovascular disease, type 2 diabetes, and other metabolic diseases. The INHβE gene is mainly expressed in the liver and encodes a secreted hepatokine, activin E.

[0006] RNA interference (RNAi) refers to a phenomenon in which highly conserved in the process of evolution, homologous mRNA is efficiently and specifically degraded by small interfering RNA (siRNA), and RNAi drugs also have the advantage of a longer drug effect time compared to antibodies. Therefore, it is of great significance to research and develop siRNA targeting INHβE. Summary of the Invention

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

[0008] In the first aspect of the present invention, a nucleic acid is provided, which comprises a sense strand and an antisense strand. Among them, the sense strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides have no more than 3 nucleotide differences compared with any at least 15 consecutive nucleotides in the sequences shown in SEQ ID No. 1 to 68; or the antisense strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides have no more than 3 nucleotide differences compared with any at least 15 consecutive nucleotides in the nucleotide sequences shown in SEQ ID No. 69 to 136.

[0009] In the second aspect of the present invention, a targeted drug delivery system is provided, which comprises a targeting group, a linking group, and the nucleic acid as described above linked to the targeting group through the linking group.

[0010] In the third aspect of the present invention, an in vitro cell is provided, which contains the nucleic acid.

[0011] In the fourth aspect of the present invention, a pharmaceutical composition is provided, which contains the nucleic acid or the 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 cells, the method comprising: contacting the cells with the nucleic acid, the targeted drug delivery system or the pharmaceutical composition as described above to inhibit the expression of inhibin subunit βE in the cells.

[0013] The sixth aspect of the present invention provides the use of the nucleic acid, the targeted drug delivery system or the pharmaceutical composition as described above in any of the following aspects: 1) treating and / or preventing diseases related to inhibin subunit βE; 2) preparing a drug for treating and / or preventing diseases related to 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 diseases, 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 will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0016] Figure 1 It is the inhibition result of different targeted drug delivery systems on the expression of INHBE gene in Example 2 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

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

[0018] TERMINOLOGY DESCRIPTION

[0019] Unless otherwise specified, the meanings of all terms (including technical and scientific terms) used to disclose the present invention are the same as those commonly understood by those of ordinary skill in the art to which the present invention pertains. Through further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments, and are not intended to limit the present invention.

[0020] As used herein, the terms "and / or", "or / and", and "and / or" include any one of two or more related listed items, and also include any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more than two 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, this technical solution undoubtedly includes the technical solution connected by "logical AND", and undoubtedly also includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another 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 connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations 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 connected by "logical AND").

[0021] The terms "comprising", "containing", and "including" used in the present invention are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0022] The numerical ranges represented by endpoints in the present invention include all the numerical values and fractions included within the range, as well as the recited endpoints.

[0023] Regarding the concentration values involved in the present invention, their meanings include fluctuations within a certain range. For example, they can fluctuate within the corresponding precision range. For example, for 2%, a fluctuation within the range of ±0.1% is allowed. For larger numerical values or those that do not require overly precise control, larger fluctuations in their meanings are also allowed. For example, for 100 mM, fluctuations within the ranges of ±1%, ±2%, ±5%, etc. are allowed. Regarding the molecular weight, fluctuations within the range of ±10% in its meaning are allowed.

[0024] In the present invention, descriptions such as "a plurality of" and "a variety of", without special limitations, refer to a quantity greater than or equal to 2.

[0025] In the present invention, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open technical solution containing the listed features.

[0026] In the present invention, "preferred", "better", "more preferred", and "preferably" are only used to describe the embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present invention.

[0027] In the present invention, "optionally", "optional", "option", "alternatively", "alternative", "alternate" mean having or not having, that is, any one selected from two parallel options of "having" or "not having". If there are multiple occurrences of "optionally" or "alternatively" in a technical solution, without special instructions, and without contradictions or mutual constraints, each "optionally" or "alternatively" is independent.

[0028] In the present invention, the term "nucleic acid" refers to a composition containing RNA or RNA-like (such as chemically modified RNA) oligonucleotide molecules, and the oligonucleotide molecules can degrade or inhibit (for example, 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 the RNA interference mechanism (that is, induce RNA interference by interacting with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) of mammalian cells), or through any alternative mechanism or pathway. The defined scope of nucleic acids including sense strands and antisense strands disclosed herein 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 referring to "the antisense strand (sense strand) contains at least 15 consecutive nucleotides, and the at least n consecutive nucleotides have no more than 3 nucleotide differences compared to any at least 15 consecutive nucleotides in the reference sequence (such as the sequence shown in SEQ ID No. X or the nucleotide sequence of positions 1 to 21 of the shown sequence)", the alignment situations involved include the alignment with at least 15 consecutive nucleotides starting from any position (such as positions 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, where there are 1, 2, or 3 differences between the nucleotide sequence of positions 1 to 15 and the nucleotide sequence of positions 2 to 16 in the reference sequence, and the nucleotides at positions 16 to 20 in the antisense strand (sense strand) are the same or different from the nucleotide sequence of positions 17 to 21 in the reference sequence. Such sequences are also within the scope defined by the present invention. In some embodiments, the antisense strand (sense strand) contains 21 consecutive nucleotides, where the nucleotide sequence of positions 1 to 15 is the same as the nucleotide sequence of positions 1 to 15 in the reference sequence, and the nucleotides at positions 16 to 21 in the antisense strand (sense strand) are the same or different from the nucleotide sequence of positions 16 to 21 in the reference sequence. Such sequences are also within the scope defined by the present invention.

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

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

[0032] In the present invention, when referring to the expression of a given gene, the terms "silence", "reduce", "inhibit", "downregulate", or "knockdown" mean that, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from mRNA in a cell, cell population, tissue, organ, or subject in which the gene is transcribed, compared with a second cell, cell population, tissue, organ, or subject not so treated, the expression of the gene is reduced when the cell, cell population, tissue, organ, or subject is treated with the nucleic acid described herein.

[0033] In the present invention, "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 with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence can comprise all or part of the first nucleotide sequence or the second nucleotide sequence.

[0034] In the present invention, "partially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 70% but not all bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence can comprise all or part of the first nucleotide sequence or the second nucleotide sequence.

[0035] In the present invention, "substantially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 85% but not all bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence can comprise all or part of the first nucleotide sequence or the second nucleotide sequence.

[0036] In the present invention, when referring to "at least partially complementary", it means that in a hybridization pair of nucleobase or nucleotide sequence molecules, the first oligonucleotide is partially complementary, substantially complementary, or fully complementary to the second oligonucleotide.

[0037] In the present invention, the term "treatment" refers to a method or step taken to provide relief or alleviation of the number, severity, and / or frequency of one or more disease symptoms in a subject. The 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 the subject.

[0038] In the present invention, the term "link" means the joining of two compounds or molecules by a covalent bond. Unless otherwise specified, as used herein, the term "link" may refer to a connection between a first compound and a second compound with or without any intervening atom or group of atoms.

[0039] In the present invention, the term "nucleotide containing an unnatural base" refers to the replacement of natural bases (adenine, uracil, guanine, cytosine) in an RNA molecule with unnatural bases by 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 acid

[0041] The present invention first provides a nucleic acid, which comprises 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 have no more than 0, 1, 2 or 3 nucleotide differences compared with any at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides in the sequence shown in any one of SEQ ID No. 1-68; 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 have no more than 0, 1, 2 or 3 nucleotide differences compared with any at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID No. 69-136.

[0042] In some embodiments, the sense strand is the same as the 2nd to 20th nucleotides in the sequence shown in any one of SEQ ID No. 1-68, and the antisense strand is the same as the 2nd to 20th nucleotides in the sequence shown in any one of SEQ ID No. 69-136.

[0043] In some embodiments, the antisense strand has 15 to 30 (such as 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 (such as 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 to obtain 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 with a sense strand sequence of SEQ ID No.1 and an antisense strand sequence of SEQ ID No.69, siRNA-2 with a sense strand sequence of SEQ ID No.2 and an antisense strand sequence of SEQ ID No.70, siRNA-3 with a sense strand sequence of SEQ ID No.3 and an antisense strand sequence of SEQ ID No.71, siRNA-4 with a sense strand sequence of SEQ ID No.4 and an antisense strand sequence of SEQ ID No.72, siRNA-5 with a sense strand sequence of SEQ ID No.5 and an antisense strand sequence of SEQ ID No.73..., siRNA-66, siRNA-67, siRNA-68.

[0048] In some embodiments, 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 have no more than 0, 1, 2, or 3 nucleotide differences compared to any at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides in the sequences shown in SEQ ID No.115 or 120; the sense strand contains a nucleotide sequence that is at least partially complementary (such as 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 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 by no more than 0, 1, 2 or 3 nucleotides from any of the at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides of the sequences shown in SEQ ID No. 47 or 52.

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

[0051] In some preferred embodiments, the sense 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 sense 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 can be unmodified or can contain at least one modified nucleotide group, and the modification can be on nucleotides at any position.

[0056] In some embodiments, the sense strand and the antisense strand 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 the antisense strand in the nucleic acid to the corresponding sequence mentioned in the present invention is less than 100% or differs by more than 1 nucleotide, it still has an inhibitory effect on INHβE that is similar (such as still having an efficacy equivalent to 80-120%, 85-115%, or 90-110% of the corresponding sequence) or equivalent (such as still having an efficacy equivalent to 95-105% of the corresponding sequence) to the corresponding sequence. For example, replace the two bases at the 3' end of the antisense strand (such as any of the sequences shown in SEQ ID No. 69-136) with AA, CU, UC, AG, CC, GG, or UG, etc., or any combination of two nucleic acids. Such nucleic acid sequences also fall within the protection scope of the present invention.

[0058] In some preferred embodiments, the inhibition efficiency of the nucleic acid on INHβE is not less than 50% (such as 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]

[0061]

[0062]

[0063] In Table 1, the first column indicates the position of the first base of the target gene in the coding sequence of INHβE, and so on; the numbers in the third and fifth columns represent the sequence numbers. For example, "1" represents SEQ ID No.1. Among them, the reference sequence of the target gene is the coding sequence NM_031479 of human INHβE.

[0064] For the technical solutions regarding the naked sequence (i.e., the unmodified sequence) mentioned in the present invention, the effect advantages do not depend on the modification method or the choice of the targeting vector. The applicable modification schemes and further preferred modification schemes will be introduced in detail below.

[0065] In some embodiments, the nucleic acid contains nucleotide groups as basic structural units, and the nucleotide groups contain phosphate groups, ribose groups, and bases. Preferably, the nucleic acid contains at least one modified nucleotide group. The nucleic acid containing the modified group has an inhibition efficiency of not less than 50% (such as 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%) on INHβE.

[0066] In some embodiments, the modified nucleotide group is a nucleotide group in which the phosphate group and / or the ribose group is modified. The modified sites can be at least one of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th nucleotides on the sense strand and / or the antisense strand.

[0067] In some embodiments, the modification of the phosphate group refers to the modification of the oxygen in the phosphate group, including phosphorothioate modification and boranophosphate modification, etc. As shown in the following formula, sulfur, borane, amino group, alkyl group or alkoxy group are respectively used to replace the oxygen in the phosphate group. These several modifications can all stabilize the structure of nucleic acid and maintain high specificity and high affinity of base pairing.

[0068]

[0069]

[0070] In the above structural formula, BASE represents the base A, U, C, G or T. X can be oxygen (O) or sulfur (S). R can be the same or different in the above structures, such as: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl group, allyl group, ethylamino group, propargyl group, amino group, cyanoethyl group, acetyl group, etc. R' and R'' can each independently be hydrogen (H), methyl (CH3), ethyl (CH2CH3), propyl (CH2CH2CH3), isopropyl (CH(CH3)2), allyl group, propargyl group, acyloxybenzyl group, acyloxyethyl group.

[0071] In some embodiments, the modification of the ribose group refers to the modification of the 2'-hydroxyl group (2'-OH) in the ribose group. After introducing certain substituents such as methoxy or fluorine at the 2'-hydroxyl position of the ribose group, the nucleic acid is less susceptible to cleavage by ribonucleases, thereby increasing the stability of the nucleic acid and endowing the nucleic acid with stronger resistance to nuclease hydrolysis. The modification of the 2'-hydroxyl group in the pentose of the nucleotide includes 2'-fluoromodification (such as 2'-arabino-fluoromodification), 2'-O-methoxymethyl modification (2'-OME), 2'-O-methoxyethyl modification (2'-MOE), 2'-2,4-dinitrophenyl modification (2'-DNP modification), 2',4'-constrained ethyl modification, 2'-amino modification, 2'-deoxy modification, bridged nucleic acid (BNA), acyclic nucleic acid modification, misaligned nucleic acid modification, L-form nucleic acid modification, etc. BNA (bridged nucleic acid) refers to a constrained or inaccessible nucleotide. BNA can contain a bridged structure with a "fixed" C3'-endo sugar puckering of a five-membered ring, six-membered ring, or seven-membered ring. Usually, the bridge is incorporated at the 2'- and 4'-positions of the ribose ring to provide 2',4'-BNA nucleotides, such as locked nucleic acid (LNA) modification, ethylene-bridged nucleic acid (ENA) modification, and cyclohexenyl-ethynyl-bridged nucleic acid (cET BNA) modification. 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. Misaligned nucleic acid modification refers to the replacement of the 3',5'-phosphodiester bond with a 2',5'-phosphodiester bond. L-form nucleic acid modification refers to the replacement of the naturally occurring D-form nucleic acid with its mirror stereoisomer, the L-form nucleic acid.

[0072]

[0073]

[0074] Wherein, 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 group, allyl group, ethylamino group, propargyl group, cyanoethyl group, acetyl group, etc.

[0075] In some embodiments, according to a particularly preferred embodiment of the present invention, in the sense strand of the RNAi reagent, the nucleotide group containing uracil base or cytosine base is a nucleotide group with a modified ribose group, that is, the 2'-OH of the ribose group in the nucleotide group containing uracil base or cytosine base in the sense strand of the RNAi reagent is substituted by methoxy or fluorine. More preferably, dTdT can be connected to the 3' ends of both the sense strand and the antisense strand of the RNAi reagent; or, AA or UU or a combination of any two nucleic acids (which can be, but is not limited to, CC, GG or UG) can be connected to the 3' end of the antisense strand of the RNAi reagent, so that the sequence has a specific inducement for mRNA degradation. The RNAi reagent with the above modifications shows a more excellent in vivo inhibitory effect, and the above modifications can further reduce the immunogenicity of the RNAi reagent of the present invention in vivo.

[0076] The RNAi reagent of the present invention can also include a modification of connecting a monophosphate nucleotide to the 5' end of the antisense strand. Since the 5'-monophosphate at the terminal of the siRNA guide strand is important for RISC recognition. The phosphorylation of the 5'-hydroxyl group plays a certain role in whether the siRNA can be effectively loaded onto Ago2 inside the cell. The 5'-monophosphate at the 5' end of the guide strand in the siRNA has an H-bond interaction with Argonaute-2 (Ago2), thus ensuring accurate positioning and precise cleavage of the mRNA target. The commonly used derivatives of 5'-monophosphate nucleotides are as follows. Such derivatives of phosphate nucleotides have been proven to have a certain stability in biological metabolic media and play a certain role in promoting the loading of the siRNA guide strand into Ago2 inside the cell (Nucleic Acids Research, 2015, 43, 2993–3011). According to the RNAi reagent of the present invention, preferably, trans-vinyl phosphate (VP) is the first choice, and it can also include derivatives of monophosphate nucleotides other than those described above.

[0077]

[0078]

[0079] In the above structures, BASE represents the bases A, U, C, G or T. R can be the same or different in the above structures, for example: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl group, allyl group, ethylamino group, propargyl group, cyanoethyl group, amino group, acetyl group, etc.

[0080] In the present invention, has the same meaning, referring to a chemical element X connected to any one or more groups.

[0081] In some embodiments, at least one nucleotide in the nucleic acid is a modified nucleotide or comprises a modified internucleoside bond.

[0082] In some embodiments, the modified nucleotide is preferably selected from one or more of 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2',3'-cyclic nucleotide analog, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted 2'-O-methyl nucleotide, inverted 2'-deoxy nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, Morpholino nucleotide, peptide nucleic acid (PNA), glycerol nucleic acid (GNA), tricyclo-DNA (tcDNA), nucleotide containing unnatural base, nucleotide containing vinyl phosphonate, nucleotide containing cyclopropyl phosphonate, and 3'-O-methyl nucleotide; the modified nucleotide is further preferably selected from one or two of 2'-O-methyl nucleotide and 2'-fluoro nucleotide.

[0083] In some embodiments, the modified internucleoside bond is preferably selected from one or more of phosphorothioate internucleoside bond and methylphosphonate internucleoside bond. In some embodiments, the modified internucleoside bond is further preferably selected from one or more of phosphorothioate monoester internucleoside bond and phosphorothioate diester internucleoside bond.

[0084] In some preferred embodiments, the antisense strand has a 2'-fluoro nucleotide at the 14th nucleotide position of the nucleotide sequence shown in any one of SEQ ID Nos. 69 to 136 and at least one (such as the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th or 9th) of the nucleotide positions selected from the 1st, 2nd, 3rd, 4th, 6th, 7th, 8th, 12th, 16th nucleotide positions. Compared with known modification methods, the above antisense strand modification scheme is further beneficial to enhancing the inhibitory effect of the nucleic acid on INHβE.

[0085] In some specific embodiments, the antisense strand has a 2'-fluoro nucleotide at the 2nd, 6th, 14th and 16th nucleotide positions of the nucleotide sequence shown in any one of SEQ ID Nos. 69 to 136 and at least one (such as the 1st, 2nd, 3rd, 4th, 5th or 6th) of the nucleotide positions selected from the 1st, 3rd, 4th, 7th, 8th, 12th nucleotide positions, and 2'-O-methyl nucleotides at other positions.

[0086] In some preferred embodiments, the sense strand has 2'-fluoro nucleotides at the 9th nucleotide position of the nucleotide sequence shown in any one of SEQ ID Nos. 1 to 68 and at least one (such as 1, 2, 3, 4, or 5) of the nucleotide positions selected from the 1st, 7th, 10th, 11th, and 12th nucleotide positions. Compared with known modification methods, the above sense strand modification scheme further helps to enhance the inhibitory effect of the nucleic acid on INHβE.

[0087] In some specific embodiments, the sense strand has 2'-fluoro nucleotides at the 7th, 9th, and 11th nucleotide positions of the nucleotide sequence shown in any one of SEQ ID Nos. 1 to 68 and at least one (such as 1, 2, or 3) of the nucleotide positions selected from the 1st, 10th, and 12th nucleotide positions, and 2'-O-methyl nucleotides at other positions.

[0088] In some embodiments, the antisense strand contains phosphorothioate internucleotide linkages between the last 2 to 4 (such as 2, 3, or 4) nucleotides at the 5'-end and / or 3'-end, and the sense strand contains phosphorothioate internucleotide linkages between the last 2 to 4 (such as 2, 3, or 4) nucleotides at the 5'-end and / or 3'-end.

[0089] In some specific embodiments, the antisense strand contains phosphorothioate internucleotide linkages between the last 3 nucleotides at the 5'-end and 3'-end, and the sense strand contains phosphorothioate internucleotide linkages between the last 3 nucleotides at the 5'-end.

[0090] In some embodiments, the antisense strand contains 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.

[0091] In some embodiments, the sense strand contains 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.

[0092] In some embodiments, the nucleic acid contains a duplex shown in any one of Table 2 or Table 3.

[0093] In some preferred embodiments, the nucleic acid contains 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, SN-256697.

[0094] Table 2

[0095]

[0096]

[0097] Table 3

[0098]

[0099]

[0100] In each modified sequence of the present invention, the nucleotide represented by a lowercase letter represents that the nucleotide is a 2'-O-methyl nucleotide; f represents that one nucleotide adjacent to its left is a 2'-fluoro nucleotide; s represents that the two nucleotides adjacent to the left and right are connected by a phosphorothioate internucleotide bond.

[0101] The nucleic acid described in the present invention can be obtained by conventional methods in the art, such as by solid-phase synthesis and liquid-phase synthesis. Solid-phase synthesis has commercial custom services, so it can be obtained by commercial purchase. The modified nucleotide groups can be introduced by nucleotide monomers with corresponding modifications.

[0102] Based on the nucleic acid (siRNA) synthesized as 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 this expression plasmid is well known to those skilled in the art and will not be elaborated herein.

[0103] Targeted drug delivery system

[0104] The present invention also provides a targeted drug delivery system, which includes a targeting group, a linking group, and the above-mentioned nucleic acid connected to the targeting group through the linking group.

[0105] When the nucleic acid (siRNA) of the present invention is applied to different targeted drug delivery systems in combination with common knowledge in the art, it has excellent inhibitory effects. In other words, the effect advantages of the naked sequence and the modified sequence in the present invention do not depend on the choice of the targeted vector. To further improve the bioavailability and therapeutic effect of siRNA, the present invention also optimizes the targeted drug delivery system and obtains the following technical solutions.

[0106] In some specific 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).

[0107] In some specific embodiments, the linking group can be selected from -O-[CH2CH2O]n-, -[CH2]m-CONH-[CH2]nO-, -O-[CH2CH2O]m-CONH-[CH2]nO-, -O-[CH2]m-CONH-[CH2H2O]nO-. Wherein, m and n can each independently be an integer from 1 to 10.

[0108] In some embodiments, the targeted drug delivery system includes a ligand and the nucleic acid linked to the ligand, and 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.

[0109] In some preferred embodiments, the ligand is a GalNAc derivative.

[0110] In some preferred embodiments, the ligand is one or more GalNAc derivatives linked by a single-stranded, double-stranded or triple-stranded branched linker.

[0111] In some further preferred embodiments, the RNAi reagent contains a compound having the following structural formula I:

[0112]

[0113] In the formula, Nu represents the duplex. The targeted drug delivery system can improve the cell penetration ability of the nucleic acid drug (Nu), enhance its stability in cells, and has a simple preparation process and strong practicability by virtue of its structural characteristics on the left side.

[0114] In specific implementation, the ligand moiety can be contacted with nucleotide monomers or nucleic acids linked to a solid support in the presence of coupling reaction conditions and coupling reagents, so that the compound moiety is linked to the nucleic acid through a coupling reaction.

[0115] Cell

[0116] The present invention also provides an isolated cell, which contains the nucleic acid described above.

[0117] In some embodiments, the cell can be used for gene function research, disease model research, drug screening and other purposes.

[0118] In some embodiments, the cell will not develop into an animal individual. In some specific embodiments, the cell can be a microbial cell or an animal cell, but the animal cell is not an embryonic stem cell of an animal and cells at each formation and development stage thereof (such as germ cells, fertilized egg cells, etc.).

[0119] Drug composition

[0120] The present invention also provides a drug composition, which contains the nucleic acid or the targeted drug delivery system described above and a pharmaceutically acceptable carrier.

[0121] The drug composition can be prepared from the nucleic acid and the pharmaceutically acceptable carrier by conventional methods. For example, the drug composition can be an injection solution. The injection solution can be used for subcutaneous, intramuscular or intravenous injection.

[0122] For the drug composition according to the present invention, there is no particular requirement for the amounts of the nucleic acid or the 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 based on the nucleic acid), the content of the pharmaceutically acceptable carrier can be 1-100000 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, 1000 parts by weight, 5000 parts by weight, 10000 parts by weight, 50000 parts by weight, 100000 parts by weight or any value between any two of the above values).

[0123] The pharmaceutical composition according to the present invention, wherein the pharmaceutically acceptable carrier can be various carriers conventionally used in the art. For example, it can include at least one of a pH buffer, a protecting agent, and an osmotic pressure regulator. The pH buffer can be a tris(hydroxymethyl)aminomethane 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 protecting agent can be at least one of inositol, sorbitol, and sucrose. Based on the total weight of the pharmaceutical composition, the content of the protecting agent can be 0.01 - 30% by weight (such as 0.01% by weight, 0.05% by weight, 0.1% by weight, 0.5% by weight, 1% by weight, 5% by weight, 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by 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 200 - 700 milliosmoles per kilogram. Those skilled in the art can determine the content of the osmotic pressure regulator according to the required osmotic pressure.

[0124] 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 it can include, but is not limited to, cholesterol and its analogs or derivatives.

[0125] The dosage of the pharmaceutical composition according to the present invention can be a conventional dosage in the art, and the dosage can be determined according to various parameters, especially according to the age, weight, and gender of the subject. For example, for female mice at 3 - 4 months of age with a weight of 25 - 30 g, based on the amount of the nucleic acid in the pharmaceutical composition, the dosage of the pharmaceutical composition can be 0.01 - 100 mg / kg body weight, preferably 1 - 10 mg / kg body weight.

[0126] Methods and uses

[0127] The present invention also provides a method for inhibiting the expression of inhibin subunit βE in cells, which includes: 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.

[0128] In some embodiments, the cells are in a subject, for example, a human subject, such as a subject suffering from a disease related to inhibin subunit βE, or a subject in need of preventing the risk of a disease related to inhibin subunit βE.

[0129] In some embodiments, the cells are located in vitro. The method is based on research purposes or for constructing an animal model.

[0130] In some embodiments, contacting the cells with the RNAi reagent or the pharmaceutical composition inhibits the expression of inhibin subunit βE by at least 50%, 60%, 70%, 80%, 90%, 95% (e.g., compared to the expression level of inhibin subunit βE before the cells first contacted the RNAi reagent or the pharmaceutical composition; e.g., before administering the first dose of the RNAi reagent or the pharmaceutical composition to the subject). In certain embodiments, inhibiting the 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 expression level of inhibin subunit βE before the cells first contacted the RNAi reagent or the pharmaceutical composition.

[0131] The present invention also provides the use of the nucleic acid, the targeted drug delivery system or the pharmaceutical composition in the treatment and / or prevention of diseases associated with inhibin subunit βE. That is, a method for treating and / or preventing a disease associated with inhibin subunit βE, which comprises: administering the nucleic acid, the targeted drug delivery system or the pharmaceutical composition to a subject.

[0132] The present invention also provides the use of the nucleic acid, the targeted drug delivery system or the pharmaceutical composition in the preparation of a medicament for treating and / or preventing a disease associated with inhibin subunit βE.

[0133] In some embodiments, the disease is: (i) a disease associated with enhanced or elevated inhibin subunit βE; or (ii) a disease that would benefit from a reduction in the expression of inhibin subunit βE.

[0134] In some embodiments, the disease is selected from the related diseases caused by abnormal fat distribution.

[0135] In some embodiments, the disease is selected from at least one of cardiovascular diseases, diabetes, lipid metabolism disorders, hypertension, obesity, and metabolic syndrome.

[0136] In the present invention, the subject can be a mammal, including primates (such as humans, non-human primates, e.g., monkeys and chimpanzees), non-primates (such as cows, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats or mice) or birds. In some embodiments, the subject is preferably a primate, more preferably a human.

[0137] In some embodiments, administration can be carried out by various routes, depending on whether local treatment or systemic treatment is required. The dosage can be referred to as described above and will not be elaborated here.

[0138] In some embodiments, administration can be local (e.g., transdermal patch), pulmonary, e.g., inhalation or insufflation via powder or spray, including via nebulizer; intratracheal, nasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subepidermal, e.g., via an implantation device; or intracranial, e.g., via intracerebral, intrathecal or intraventricular administration.

[0139] In some embodiments, the nucleic acid, the targeted drug delivery system or the pharmaceutical composition is administered to a subject by subcutaneous administration, intravenous administration and / or intramuscular administration.

[0140] Examples

[0141] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following examples, the guidance given in the present invention is preferably referred to, and it can also be in accordance with the experimental manuals or conventional conditions in the art, and other experimental methods known in the art can also be referred to, or in accordance with the conditions recommended by the manufacturers.

[0142] In the following specific examples, for the measurement parameters of raw material components, if there is no special description, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0143] Example 1

[0144] Dilute the siRNA in Table 2 with ddH2O to a final concentration of 1 nM and 10 nM, and perform the Reneilla luciferase assay in HEK293T cells. The specific steps are as follows: After subculturing HEK293T cells for 2 - 3 passages in DMEM (Gibco, 11995065) medium containing 10% FBS (Hyclone, SH30406.05), seed them on a 96-well plate (Greiner #655098) coated with 0.1% gelatin so that the cell density in each well is 30K, and culture overnight. The next day, dissolve and dilute the siRNA sample with ddH2O, and then mix 10 μL of the siRNA sample with a final concentration of 1 nM or 10 nM with 10 μL of serum-free medium (Absin, abs9461) containing 100 ng of psiCHECK TM2-INHBE plasmid and 0.5 μL of lipofectamine2000 (Thermo Fisher, 11668019). Add the mixture to the 96-well plate containing HEK293T cells, place it in an incubator with 5% CO2, and culture at 37°C for 24 h. After 24 h, add 75 μL of glo-luciferase reagent (Promega, E2940) to each well, incubate at room temperature for 15 minutes, and read the firefly luciferase luminescence at OD 590 nm on a microplate reader; add stop&glo-luciferase reagent (Promega, E2940) to each well, incubate at room temperature for 15 minutes, and read the Renilla luciferase luminescence at OD 528 nm on a microplate reader. Calculate %KD by calculating Rluc / Fluc. %KD = 1 - (sample Rluc / Fluc - blank) / (plasmid alone Rluc / Fluc - blank) to obtain the knockdown data of the siRNA sample. The results are shown in Table 4.

[0145] Table 4

[0146]

[0147]

[0148] It can be seen that the siRNAs containing the duplex sequences of the present invention all have inhibitory effects on the expression of INHβE to varying degrees.

[0149] Example 2

[0150] The targeted drug delivery systems conjugated with TriGalNAc (the structure of this compound is shown in Formula I) in Table 5 were separately dissolved in 100 μL of enzyme-free and sterile water to form 1000 μM solutions, which were correspondingly used as working solutions of 1000 nM. The primary monkey hepatocytes were taken out from liquid nitrogen, thawed and resuscitated at 37 °C, rinsed, counted and centrifuged with PMonH plating medium. After removing the supernatant, the cells were diluted to 250 k / mL with fresh PMonH plating medium, and then 100 μL of the diluted cell suspension was plated onto a 96-well cell culture plate so that the number of cells per well was 25 k. 10 μL of 1000 mM compound solution was pipetted into the corresponding cell wells, and double replicates were made for each gradient concentration of the compounds; they were placed in a 5% carbon dioxide incubator and cultured at a constant temperature of 37 °C, and samples were collected after 24 h. After 24 hours, all the culture media in the 96-well culture plate were aspirated, washed with 1×PBS buffer, 50 μL of the prepared Cells to CT lysis solution (as recommended by the manufacturer) was added and mixed well, and after standing for 10 min, 2.5 μL of the termination solution was added to terminate for 2 min. RT-PCR was performed according to the recommendations of the High Capacity cDNA Reverse Transcription Kits (Thermo Fisher, catalog number: 4368814), and each reaction contained 10 μL of the lysed liquid. The gene expression quantification was measured by real-time fluorescence PCR. The TaqMan probe for monkey INHBE was Mf02820386_g1, and the probe for the reference gene (monkey PPIB) was Mf02802985_m1 (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions were 1 cycle of 95 °C for 20 seconds, and 40 cycles of 95 °C for 1 second and 60 °C for 20 seconds. The real-time fluorescence PCR instrument was the QuantStudio TM 6 Pro real-time fluorescence quantitative PCR system (Thermo Fisher). The INHBE gene expression was calculated as 2^-ΔΔCt, and the monkey PPIB gene expression was used as the reference. The INHBE gene expression level was expressed as a percentage relative to the cell group with only culture medium as the control. The results are shown in Figure 1 .

[0151] Table 5

[0152]

[0153]

[0154] Example 3

[0155] The siRNAs in Table 6 were serially diluted with enzyme-free sterile water and subjected to the Reneilla luciferase assay in HEK293T cells. The specific method was referred to Example 1. The IC50 values were calculated using Prism GraphPad, and the results are shown in Table 7.

[0156] Table 6

[0157] Number Sense strand (5’-3’) Antisense strand (5’-3’) SN-255250 gscscuggCfuUfAfUfacuuucuuaa usUfsaAfgAfaaguauaAfgCfcaggcsusu SN-256698 gscscuggCfuUfAfUfacuuucuuaa UfsUfsaAfgAfaaguauaAfgCfcaggcsusu SN-256699 gscscuggCfuUfAfUfacuuucuuaa usUfsaagAfaaguauaAfgCfcaggcsusu SN-256690 gscscuggCfuUfAfUfacuuucuuaa usUfsaAfgAfAfaguauaAfgCfcaggcsusu SN-256691 gscscuggCfuUfAfUfacuuucuuaa usUfsaAfgAfaAfguauaAfgCfcaggcsusu SN-255259 gsgsagacAfaGfCfAfuuuauacuuu asAfsaGfuAfuaaaugcUfuGfucuccsusu SN-256692 gsgsagacAfaGfCfAfuuuauacuuu AfsAfsaGfuAfuaaaugcUfuGfucuccsusu SN-256603 gsgsagacAfaGfCfAfuuuauacuuu asAfsaguAfuaaaugcUfuGfucuccsusu SN-256604 gsgsagacAfaGfCfAfuuuauacuuu asAfsaGfuAfUfaaaugcUfuGfucuccsusu SN-256605 gsgsagacAfaGfCfAfuuuauacuuu asAfsaGfuAfuAfaaugcUfuGfucuccsusu

[0158] Table 7

[0159] Number IC50, nM SN-255250 0.063 SN-256698 0.032 SN-256699 0.103 SN-256690 0.282 SN-256691 0.142 SN-255259 0.016 SN-256692 0.023 SN-256603 0.078 SN-256604 0.039 SN-256605 0.021

[0160] Example 4

[0161] The siRNAs in Table 8 were serially diluted with enzyme-free sterile water and subjected to the Reneilla luciferase assay in HEK293T cells. The specific method was referred to Example 1. The results are shown in Table 9.

[0162] Table 8

[0163]

[0164]

[0165] Table 9

[0166] Number SN-255259 SN-256674 SN-256675 2 nM 10.88 9.98 19.36 10 nM 10.43 6.18 13.99

[0167] Example 5

[0168] The siRNAs in Table 10 were serially diluted with enzyme-free sterile water and subjected to the Reneilla luciferase assay in HEK293T cells. The specific method was referred to Example 1. The IC50 values were calculated using Prism GraphPad, and the results are shown in Table 11.

[0169] Table 10

[0170] Number Sense strand (5’-3’) Antisense strand (5’-3’) SN-256674 csgsagacAfaGfCfAfuuuauacuua usAfsaGfuAfuaaaugcUfuGfucucgsusu SN-256689 csgsagacAfaGfCfAfuuuauacuua UfsAfsAfGfuAfuaaaugcUfuGfucucgsusu SN-256680 csgsagacAfaGfCfAfuuuauacuua usAfsAfGfuAfuaaauGfcUfuGfucucgsusu SN-256681 csgsagacAfaGfCfAfuuuauacuua UfsAfsaGfuAfuaaauGfcUfuGfucucgsusu SN-256687 CfsgsagacAfaGfCfAfuuuauacuua usAfsaGfuAfuaaaugcUfuGfucucgsusu SN-256682 CfsgsagacAfaGfCfAfuuuauacuua UfsAfsAfGfuAfuaaaugcUfuGfucucgsusu SN-256693 CfsgsagacAfaGfCfAfuuuauacuua usAfsAfGfuAfuaaauGfcUfuGfucucgsusu SN-256694 CfsgsagacAfaGfCfAfuuuauacuua UfsAfsaGfuAfuaaauGfcUfuGfucucgsusu SN-256688 CfsgsagacAfaGfCfAfUfuuauacuua usAfsaGfuAfuaaaugcUfuGfucucgsusu SN-256695 CfsgsagacAfaGfCfAfUfuuauacuua UfsAfsAfGfuAfuaaaugcUfuGfucucgsusu SN-256696 CfsgsagacAfaGfCfAfUfuuauacuua usAfsAfGfuAfuaaauGfcUfuGfucucgsusu SN-256697 CfsgsagacAfaGfCfAfUfuuauacuua UfsAfsaGfuAfuaaauGfcUfuGfucucgsusu SN-256601 csgsagacAfagCfAfuuuauacuua usAfsaGfuAfuaaaugcUfuGfucucgsusu SN-256602 csgsagacAfaGfCfAfuuuauacuua usAfsaGfuAfuaaaugcuuGfucucgsusu

[0171] Table 11

[0172]

[0173]

[0174] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection 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 No.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 No.69 to 136.

2. The nucleic acid according to claim 1, wherein The sense strand is identical to the 2nd to 20th nucleotides in any sequence shown in SEQ ID No. 1 to 68, and the antisense strand is identical to the 2nd to 20th nucleotides in any sequence shown in SEQ ID No. 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 contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 47 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 by 0, 1 or 2 nucleotides; or, The sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 52 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 by 0, 1 or 2 nucleotides; or, The sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 137 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 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 internucleotide; The modified nucleotide is preferably selected from 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2',3'-open ring nucleotide mimic, locked nucleotide, 2'-F-arabinose nucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, reverse nucleotide, reverse 2'-O-methyl nucleotide, reverse 2'-deoxy nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide (Morpholino), peptide nucleic acid (PNA), glycerol nucleic acid (GNA), trinitrogen ring DNA (tcDNA), nucleotide containing non-natural base, nucleotide containing vinyl phosphonate, nucleotide containing cyclopropyl phosphonate and 3'-O-methyl nucleotide. One or more; the modified nucleotide is further preferably selected from 2'-O-methyl nucleotide and 2'-fluoro nucleotide. 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 has a 2'-fluoro nucleotide at the 14th nucleotide position and at least one of the 1st, 2nd, 3rd, 4th, 6th, 7th, 8th, 12th and 16th 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 has 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 contains 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 contains 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 contains 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 of any one of claims 1 to 10, the targeted drug delivery system of any one of claims 11 to 13, or the pharmaceutical composition of 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 a subject by subcutaneous administration, intravenous administration and / or intramuscular administration.

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