Dsrna, use thereof, and preparation method therefor

Abstract

The present invention relates to modulators that can regulate, for example, inhibit the expression and / or activity of an inhibin subunit βE (INHBE), such as double-stranded RNA (dsRNA) active agents or antisense polynucleotide formulations. The present invention also relates to a method for inhibiting the expression and / or activity of INHBE by using such modulators, and a method for preventing and treating INHBE-related diseases in subjects (such as metabolic disorders or obesity or cardiovascular diseases, such as metabolic syndrome).

Description

A type of dsRNA, its application and preparation method

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on and claims priority to Chinese patent applications CN202410515535.6, filed on April 26, 2024, and 202410899508.3, filed on July 5, 2024, the entire contents of which are incorporated herein by reference.

[0003] This invention relates to modulators, such as double-stranded RNA (dsRNA) activators or antisense polynucleotide formulations, that can modulate, for example, the expression and / or activity of inhibin subunit βE (INHBE). The invention also relates to methods for inhibiting INHBE expression and / or activity using such modulators, and methods for preventing or treating INHBE-related diseases in subjects (such as metabolic disorders or obesity or cardiovascular diseases such as metabolic syndrome).

[0004] Background of the Invention

[0005] In modern society, many people are sedentary and frequently consume high-calorie, low-fiber fast food, while also reducing physical activity. This has led to the spread of metabolic disorders such as metabolic syndrome, type 2 diabetes, hypertension, cardiovascular disease, stroke, and other illnesses. In fact, in recent years, the number of people suffering from metabolic disorders (such as metabolic syndrome) has been increasing, and these individuals have multiple diseases and a rising risk of heart disease, diabetes, stroke, and other ailments.

[0006] Current treatments for lipid metabolism disorders include lifestyle modifications, diet, exercise, and the use of lipid-lowering drugs (such as statins) and other medications. However, these therapies and treatments are often limited by patient adherence, are not always effective, can even cause side effects, and lead to drug interactions. Therefore, there is a need in the field for alternative therapies for patients with metabolic disorders.

[0007] Inhibin subunit βE (INHBE) is a member of the transforming growth factor-β (TGF-β) family. INHBE is primarily expressed in the liver and is a liver factor that has been shown to be positively correlated with insulin resistance and body mass index in humans. Quantitative real-time PCR analysis has also shown increased INHBE gene expression in liver samples from insulin-resistant humans. Furthermore, increased INHBE gene expression has also been shown in the liver of a well-established animal model of metabolic disorder (i.e., type 2 diabetes, db / db mouse model). Inhibition of INHBE expression in db / db mice suppressed weight gain; the weight loss was due to a reduction in fat rather than lean muscle.

[0008] Therefore, INHBE can serve as an effective target for treating metabolic disorders. Currently, there is still a need for inhibitors with better activity that can effectively regulate INHBE expression and / or activity for the treatment of metabolic disorders. Attached Figure Description

[0009] Figure 1 shows the effect of INHBE siRNA on the body weight of humanized INHBE mice induced by a high-fat diet.

[0010] Figure 2 shows the effect of INHBE siRNA on body fat in humanized INHBE mice induced by a high-fat diet. Summary of the Invention

[0011] This invention provides specific RNAi inhibitors capable of effectively reducing the expression and / or activity of INHBE. The RNAi activator of this invention can affect RNA-induced silencing complex (RISC)-mediated cleavage of the INHBE gene mRNA, thereby inhibiting INHBE expression in cells. By designing dsRNAs targeting INHBE, this invention can specifically inhibit the expression of the INHBE gene (e.g., in hepatocytes and / or adipocytes), which may help prevent or treat INHBE-related diseases in subjects (such as metabolic disorders or obesity, or cardiovascular diseases such as metabolic syndrome). In some embodiments, the dsRNAs targeting INHBE of this invention can more effectively knock out INHBE (e.g., in hepatocytes and / or adipocytes), thereby specifically inhibiting INHBE gene expression.

[0012] This invention provides a double-stranded ribonucleic acid (dsRNA) activator for inhibiting the expression of INHBE target genes in cells, such as adipocytes and / or liver cells; wherein the dsRNA activator comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides, for example, 15, 16, 17, 18, or 19, that differ from any sense nucleotide sequence in Table 1 by no more than 0, 1, 2, or 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides, for example, 15, 16, 17, 18, 19, 20, or 21, that differ from any antisense nucleotide sequence in Table 1 by no more than 0, 1, 2, or 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides, for example, 15, 16, 17, 18, 19, 20, or 21, that differ from any antisense nucleotide sequence in Table 1 by no more than 0, 1, 2, or 3 nucleotides. In some embodiments, these dsRNA activators further include, for example, one or more ligands conjugated to at least one strand of the dsRNA, the ligands being capable of targeted delivery of the dsRNA to liver tissue or hepatocytes, for example, via a linker, such as a bivalent or trivalent branched linker conjugated to at least one strand of the dsRNA activator, such as a ligand targeting ASPGR, such as a GalNAc ligand comprising GalNAc or a derivative thereof.

[0013] The present invention also provides a cell comprising the dsRNA activator described herein.

[0014] The present invention provides a pharmaceutical composition comprising the dsRNA activator described herein and optionally a pharmaceutically acceptable carrier.

[0015] This invention provides a pharmaceutical combination comprising the dsRNA activator described herein and one or more other therapeutic agents, which are any therapeutic agents effective, for example, in preventing or treating INHBE-related diseases and / or conditions, covering a wide range of therapeutic agents for treating metabolic disorders or obesity or cardiovascular diseases.

[0016] This invention provides the use of the dsRNA activators and / or pharmaceutical compositions and / or drug combinations described herein in the preparation of medicaments for treating diseases and / or conditions caused by INHBE gene expression.

[0017] This invention provides the use of the dsRNA activator described herein in the preparation of a drug that inhibits INHBE gene expression in cells, preferably inhibiting INHBE expression in liver tissue or hepatocytes.

[0018] The present invention provides a method for preventing or treating diseases and / or conditions caused by INHBE gene expression, the method comprising administering an effective amount of the dsRNA activator and / or pharmaceutical composition and / or combination of drugs described herein to a subject in need.

[0019] On the other hand, the present invention provides a method for inhibiting INHBE gene expression, the method comprising contacting cells, preferably hepatocytes, with an effective amount of the dsRNA activator and / or pharmaceutical composition and / or drug combination described herein, and optionally maintaining the cells produced in this step for a period of time sufficient to degrade the mRNA transcript of the INHBE gene, thereby inhibiting the expression of the INHBE gene in cells such as hepatocytes.

[0020] Invention Details

[0021] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methodologies, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0022] I. Definition

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0025] When used in conjunction with a numerical value, the term "about" or "approximately" means to encompass a range of numerical values ​​having a lower limit of 1%, 2%, 3%, 4%, or 5% smaller than the specified numerical value and an upper limit of 1%, 2%, 3%, 4%, or 5% larger than the specified numerical value. It should be understood that the specific value referred to by the term "about" or "approximately" is itself specific and preferably disclosed.

[0026] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0027] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps. For example, when referring to a justice chain that “comprising” a specific sequence, it is also intended to cover a justice chain consisting of that specific sequence.

[0028] As used herein, "regulator" is a molecule that can reduce or increase INHBE expression and / or activity. Preferably, the regulator is a molecule that can reduce or inhibit INHBE expression and / or activity, such as an RNAi activator or a dsRNA activator.

[0029] The term "inhibin subunit βE" used in this article can be used interchangeably with "INHBE" and refers to a growth factor belonging to the transforming growth factor-β (TGF-β) family. INHBE mRNA is mainly expressed in the liver (Fang J. et al. Biochemical & Biophysical Res. Comm. 1997; 231(3):655-61), and INHBE remnants regulate stem cell growth and differentiation. INHBE is also known as inhibin βE chain, activin βE, inhibin βE subunit, inhibin βE and MGC4638. The sequence of human INHBE mRNA transcript can be found, for example, in GenBank Accession No. GL:1877089956 (NM_031479.5; SEQ ID NO:664; reverse complementary sequence SEQ ID NO:665). The sequence of mouse INHBE mRNA can be found, for example, in GenBank Accession No. GL:1061899809 (NM_008382.3). The sequence of rat INHBE mRNA can be found, for example, in GenBank Accession No. GL:1061899809 (NM_008382.3). No. GI: ​​148747589 (NM_031815.2). Examples of other INHBE mRNA sequences are available in publicly available databases such as GenBank, UniProt, and OMIM. For more information on INHBE, please visit www.ncbi.nlm.nih.gov / gene / ?term=INHBE. The full contents of the above GenBank and gene database numbers are incorporated herein by reference. The term INHBE as used herein also refers to variants of the INHBE gene, including those available in the SNP database. Many variants in the INHBE gene have been identified and can be found in databases such as NCBI dbSNP and UniProt (see www.ncbi.nlm.nih.gov / snp / ?term=INHBE, the full contents of which are incorporated herein by reference).

[0030] As used herein, a “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during INHBE gene transcription, containing the mRNA as a primary transcription product of RNA processing. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for dsRNA-directed cleavage at or near that portion of the nucleotide sequence of the mRNA molecule formed during INHBE gene transcription. For example, the length of the target sequence can be, for example, 15-36 nucleotides (“nt”), or any sub-length therein. As a non-limiting example, the length of the target sequence can be 15-30 nt, 15-26 nt, 15-23 nt, 15-22 nt, 15-21 nt, 15-20 nt, 15-19 nt, 15-18 nt, 15-17 nt, 18-30 nt, 18-26 nt, 18-23 nt, 18-22 nt, 18-21 nt, 18-20 nt, 18 nt, 19-30 nt, 19-26 nt, The target sequence is 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 19 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 20 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides. In some embodiments of the invention, the target sequence is preferably at least 18, 19, 20, or 21 nucleotides long. In some embodiments of the invention, the target sequence is about 19 to about 23 nucleotides long. In some embodiments of the invention, the target sequence is about 20 or 21 nucleotides long.

[0031] “G,” “C,” “A,” “T,” and “U” generally represent nucleotides containing guanine, cytosine, adenine, thymine, and uracil as bases, respectively, and unless otherwise specified, encompass both native and modified nucleotides. However, it is understood that the terms “ribonucleotide” or “nucleotide” can also refer to modified nucleotides or surrogate replacement moiety. Those skilled in the art will readily recognize that guanine, cytosine, adenine, and uracil can be substituted with other moieties without substantially altering the base-pairing properties of oligonucleotides including those containing such substitution moieties. For example, but not limited to, nucleotides containing inosine (a nucleoside compound formed by the combination of hypoxanthine and ribose) as a base can pair with nucleotides containing adenine, cytosine, or uracil. Therefore, in the nucleotide sequence of the dsRNA characteristic of this invention, nucleotides containing uracil, guanine, or adenine can be substituted with nucleotides containing, for example, inosine. In another example, adenine and cytosine at any position in the oligonucleotide can be replaced by guanine and uracil, respectively, to form a GU-wobbling base pairing with the target mRNA. Sequences containing such substitution moieties are suitable for the compositions and methods characteristic of this invention.

[0032] When this article refers to a "nucleotide sequence", it means a continuous nucleotide sequence, which can be a natural nucleotide or a modified nucleotide.

[0033] As used interchangeably herein, the terms “dsRNA,” “dsRNA activator,” “double-stranded RNA,” and “double-stranded RNA molecule” refer to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands having “sense” and “antisense” orientations relative to the target RNA (i.e., the INHBE gene). In some embodiments of the invention, the double-stranded RNA (dsRNA) triggers the degradation of the target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi. In some embodiments, the dsRNA of the invention is a small interfering RNA (siRNA). In some embodiments, when referred to herein as a dsRNA activator, it may also comprise a ligand linked to the double-stranded structure, the ligand facilitating the delivery of the dsRNA to a target tissue or target cell.

[0034] The term "siRNA" in this article refers to a class of double-stranded RNA molecules that can mediate the silencing of their complementary target RNA (e.g., mRNA, the transcript of a gene encoding a protein). siRNA is typically double-stranded, consisting of an antisense strand complementary to the target RNA and a sense strand complementary to that antisense strand. For convenience, such mRNA is also referred to herein as the mRNA to be silenced. Such genes are also called target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene.

[0035] The term "antisense strand" or "guide strand" refers to an oligonucleotide chain in dsRNA that contains a region substantially complementary to the target sequence (e.g., INHBE mRNA).

[0036] As used herein, the terms “sense chain” or “follower chain” or “sense chain” refer to an oligonucleotide chain containing a region substantially complementary to the antisense chain as defined herein, which can complement the antisense chain to form dsRNA, and which can complement the antisense chain to form the double-stranded region of dsRNA.

[0037] In this document, unless otherwise specified, the terms "complementarity" or "complementarity" refer to the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under certain conditions and form a double-stranded structure. Those skilled in the art can determine the optimal complementarity of the two sequences and the conditions used to determine this complementarity based on the intended application of the hybridized oligonucleotide or polynucleotide. Therefore, in this document, when describing the base pairing between the sense and antisense strands of RNAi, or between the antisense strand and the target sequence of RNAi, the terms "complementarity" or "complementarity" should be understood to cover not only 100% complementarity (i.e., perfect complementarity) but also less than 100% complementarity (i.e., substantially complementarity), that is, the presence of base mismatches in the complementary double-stranded nucleotide region that do not substantially affect the RNAi's intended function. As those skilled in the art will appreciate, in double-stranded nucleic acid molecules, when a base on one strand forms a Watson-Crick base pair with a corresponding base on the other strand in a complementary manner, the bases at that position on both strands are considered to be "complementarily paired" or "matched." For example, the purine base adenine (A) is complementary to the pyrimidine base thymine (T) or uracil (U); the purine base guanine (C) is complementary to the pyrimidine base cytosine (G). Correspondingly, a "mismatch" refers to a situation in double-stranded nucleic acids where corresponding bases on one strand are not complementary to each other. However, it should be understood that nucleotides modified in the base portion of RNA nucleosides should also be considered complementary if Watson-Crick base pairing is permitted. Therefore, in this paper, nucleoside base “complementarity” encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, for example, Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

[0038] In this document, for the purposes of this invention, the expression "complementary" or "complementarity" associated with double-stranded RNAi activators (such as siRNA as described herein) is preferably not less than 70%, meaning that at least 70% of the base positions in the double-stranded region formed by complementary hybridization are complementary, i.e., the number of mismatched positions in the continuous nucleotide sequence forming the double-stranded region is less than 30%. For example, for a 21-base-pair double-stranded region, not less than 70% complementarity means that the double-stranded region forms no more than 6, 5, 4, 3, 2, 1, or 0 mismatched base pairs during hybridization. Preferably, the presence of insertions and deletions is not allowed when calculating the complementarity of the continuous nucleotide sequence in the double-stranded region. Accordingly, in this document, the expression associated with RNAi activators, "complementary (antisense) sequence" to the target sequence, or "complementary (sense) sequence" to a portion of the antisense sequence, can be "completely complementary" or "substantially complementary." "Completely complementary" means that the two sequences have 100% complementarity. When the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may contain one or more, but typically no more than 30%, 20%, or 10%, mismatched base pairs in the hybridized duplex and still retain the ability to hybridize under conditions most relevant to their final application (e.g., repressing gene expression via a RISC pathway). As used herein, “perfect complementarity” means perfect complementarity between the two strands. It should be understood that when referring to perfect complementarity of complementary regions or duplexes, it means perfect complementarity between two identical nucleotide chains that match at alignment. Therefore, it should be understood that when the two oligonucleotides of an RNAi are designed to form one or more single-stranded overhangs during hybridization, such overhangs will not be considered mismatches when determining complementarity. For example, for the purposes described herein, an RNAi containing a 19-nucleotide-long sense oligonucleotide chain and a 21-nucleotide-long antisense oligonucleotide chain could still be considered “perfectly complementary” if the longer antisense oligonucleotide contains a 19-nucleotide sequence that is perfectly complementary to the shorter sense oligonucleotide.

[0039] As used herein, the term "complementary region" refers to a region on the antisense strand that is complementary (substantially complementary or perfectly complementary) to a sequence defined herein (e.g., a target sequence, such as the INHBE mRNA target sequence). In cases where the complementary region is not perfectly complementary (substantially complementary) to the target sequence, mismatches can be located within the molecule or in terminal regions. Typically, the most tolerable mismatches are in terminal regions, such as within 5, 4, 3, 2, or 1 nucleotides at the 5' or 3' end of the dsRNA; for example, the first nucleotide at the 5' end of the antisense strand can tolerate a mismatch. In some embodiments, the double-stranded RNA activator of the present invention comprises nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA activator of the present invention comprises no more than 4 mismatches with the target mRNA; for example, the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the 5' end nucleotide of the antisense strand of the double-stranded RNA activator of the present invention is mismatched with the target mRNA, for example, the 5' end of the antisense strand of the double-stranded RNA activator of the present invention is U, regardless of whether the 3' end of the target mRNA is A, which pairs with U. In some embodiments, the antisense strand of the double-stranded RNA activator of the present invention has no more than four mismatches with the sense strand; for example, the antisense strand contains four, three, two, one, or zero mismatches with the sense strand. In some embodiments, the nucleotide mismatch is, for example, within five, four, or three nucleotides from the 3' end of the antisense strand or the corresponding 5' end of the sense strand. In some embodiments, the nucleotide mismatch is, for example, within five, four, or three nucleotides from the 3' end of the sense strand or the corresponding 5' end of the antisense strand. In another embodiment, the nucleotide mismatch is, for example, at the 3' end nucleotide of the sense or antisense strand.

[0040] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure or double-stranded region of dsRNA. A nucleotide overhang exists, for example, when the 3' end of one strand of dsRNA extends beyond the 5' end of the other strand, or vice versa. dsRNA may contain an overhang having at least one nucleotide; alternatively, the overhang may contain at least two, three, four, five, or more nucleotides. The nucleotide overhang may contain or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). One or more overhangs may be located on the sense strand, antisense strand, or any combination thereof. Additionally, one or more nucleotides of the overhang may be present at the 5' end, 3' end, or both ends of the antisense strand or sense strand of siRNA. In some embodiments, the overhang is located at the 3' end of the antisense strand, and is, for example, 1, 2, 3, 4, or 5 nucleotides, such as 2 nucleotides.

[0041] "Flat-ended" or "flat-ended" means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A "flat-ended" dsRNA is a double-stranded dsRNA along its entire length, meaning that there are no nucleotide overhangs at either end of the molecule. The dsRNA of this invention encompasses dsRNAs with flat ends at both the 5' and 3' ends.

[0042] As used herein, the terms “double-stranded region” or “double-stranded body” or “double-stranded body region” are used interchangeably to refer to the double-stranded structure formed by the hybridization of the sense and antisense strands in dsRNA.

[0043] Generally, most nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also contain one or more modified ribonucleotides, such as deoxyribonucleotides or chemically modified nucleotides. As used herein, “dsRNA” may contain chemically modified ribonucleotides; dsRNA may contain substantial modifications at multiple nucleotide sites. As used herein, the term “modified nucleotide” refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide bond, or a modified nucleobase, or any combination thereof. Thus, the term modified nucleotide encompasses substitution, addition, or removal of, for example, functional groups or atoms, of internucleotide bonds, sugar moieties, or nucleobases. Modifications of the active agents suitable for use in this invention include all types of modifications disclosed herein or known in the art.

[0044] For naturally occurring oligonucleotides, internucleotide bonds include phosphate groups that form phosphodiester bonds between adjacent nucleosides. Hereinafter, the term "modified internucleotide bond" is defined as a bond that covalently links two nucleosides together, other than a phosphodiester (PO) bond. The nucleotide chain of the RNAi according to the invention may contain one or more internucleotide bonds modified from natural phosphodiester bonds. Modified internucleotide bonds contemplated according to the invention include, but are not limited to: thiophosphate bonds, dithiophosphate bonds, methylphosphate bonds, selenophosphate bonds, phosphoramidite bonds, etc. In some embodiments, the modified internucleotide bond in the oligonucleotide used for the RNAi of the invention is a thiophosphate bond.

[0045] As used herein, "ligand moiety" refers to a chemical portion conjugated to the double strand of dsRNA that can alter the distribution, targeting, or half-life of dsRNA. When "dsRNA" or "dsRNA activator" is mentioned herein, it also encompasses dsRNA containing a ligand moiety unless the context explicitly contradicts this description. Similarly, when "siRNA" or "siRNA activator" is mentioned herein, it also encompasses siRNA containing a ligand moiety unless the context explicitly contradicts this description. In some embodiments of the invention, the ligand moiety is a "GalNAc ligand." Herein, "GalNAc ligand" refers to an asialic acid glycoprotein receptor (ASGPR) ligand containing a structural moiety of N-acetylgalactosamine (GalNAc) or a derivative thereof. This term encompasses monovalent, divalent, trivalent, tetravalent, and multivalent GalNAc ligands providing one, two, three, four, or more structural moieties of GalNAc or GalNAc derivatives.

[0046] When referring to the nucleotide sequences contained in the sense and / or antisense strands of dsRNA in this article, the nucleotide sequences conjugated with ligands are also included unless the context clearly indicates otherwise.

[0047] As used herein, the term “inhibition” is used interchangeably with “reduction,” “silence,” “downregulation,” and other similar terms, and includes any level of inhibition.

[0048] The expression "inhibit INHBE" refers to the inhibition of the activity or expression of any INHBE gene. The expression "inhibit INHBE expression" refers to the inhibition of the expression of any INHBE gene, as well as variants or mutants of the INHBE gene. Therefore, the INHBE gene can be a wild-type INHBE gene, a mutant INHBE gene, or a transgenic INHBE gene in the case of genetically manipulated cells, cell groups, or organisms.

[0049] "Inhibition of INHBE gene expression" includes inhibition of any level of the INHBE gene, such as at least partial repression of INHBE gene expression. INHBE gene expression can be assessed based on the level or level change of any variable associated with INHBE gene expression, such as INHBE mRNA level or INHBE protein level. This level can be assessed in individual cells or in a group of cells (including, for example, samples derived from an individual). Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with INHBE expression compared to a control level. The control level can be any type of control level utilized in the art, such as baseline levels before administration or levels determined from similar untreated or controlled (e.g., buffer-only control or inert agent control) individuals, cells, or samples.

[0050] As used herein, the terms "inhibin subunit beta E-related disease or condition" or "INHBE-related disease or condition" refer to a disease or condition caused by or associated with abnormal expression and / or activity of INHBE. The term "INHBE-related disease or condition" includes diseases, disorders, or conditions from which one may benefit from decreased INHBE gene expression, replication, or protein activity. In some embodiments, an INHBE-related disease or condition is a metabolic disorder or obesity or cardiovascular disease, such as obesity or metabolic syndrome.

[0051] As described in this article, “metabolic disorder” refers to any disease or condition that disrupts normal metabolism, which is the process of converting food into energy at the cellular level. Metabolic disorders affect a cell’s ability to carry out key biochemical reactions involving the processing or transport of proteins (amino acids), carbohydrates (sugars and starches), or lipids (fatty acids).

[0052] The term "effective amount" refers to such an amount or dose of the dsRNA active agent or composition or combination of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. Depending on the intended effect, it may include "therapeutic effective amount" and "preventive effective amount".

[0053] "Therapeutic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. Therapeutic effective amount is also a amount in which any toxic or harmful effects of the dsRNA active agent or composition or combination are less than the beneficial therapeutic effect. Relative to untreated subjects, "therapeutic effective amount" preferably inhibits a measurable parameter by at least about 30%, and more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0054] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered to individuals before or at an early stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0055] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which foreign nucleic acids have been introduced, including the progeny of such cells.

[0056] The terms “individual” or “subject” may be used interchangeably herein and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0057] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0058] The term "pharmaceutical composition" refers to a composition that is present in a form that allows for the biological activity of the active ingredient contained therein, and that does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition. In some embodiments, when referring to "pharmaceutical composition," it also encompasses pharmaceutical preparations formulated as formulations or articles.

[0059] The term "drug combination" refers to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that active ingredients (e.g., (i) the dsRNA active agent of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. In some embodiments, the dsRNA active agent of the present invention and other therapeutic agents used in the drug combination are administered at levels not exceeding those obtained when used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in a separate formulation, and their formulations may be the same or different.

[0060] The term "combination therapy" refers to the administration of two or more therapeutic agents or modes of treatment to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0061] The term "other therapeutic agents" encompasses any therapeutic agent, other than the dsRNA active agent of the present invention or pharmaceutical compositions comprising it, that is effective in preventing or treating INHBE-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the INHBE gene), and includes various therapeutic agents for treating metabolic disorders or obesity or cardiovascular diseases, such as obesity or metabolic syndrome.

[0062] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0063] When used in this article, "prevention" includes the suppression of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition.

[0064] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0065] "Subject / Patient / Individual Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds that are naturally occurring and do not mix with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0066] II. dsRNA activator

[0067] This invention provides RNAi activators for inhibiting INHBE, such as dsRNA activators. In some embodiments, the dsRNA activator is siRNA. In some embodiments, the siRNA comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the INHBE gene in cells such as hepatocytes in a subject (e.g., mammals, such as individuals susceptible to INHBE-related diseases or conditions).

[0068] Intrinsic RNAi (RNA interference) mechanisms in organisms typically involve a series of processes, including: Dicer processing long dsRNA into short 19-21 base pairs (bp) siRNA; siRNA binding to Ago protein to form an RNA-induced silencing complex (RISC); Ago protein cleaving the sense strand of the siRNA and releasing it; subsequently, the mature RISC bound to the antisense strand cleaves the mRNA that is anticomplementary to the antisense strand through a sequence complementation mechanism. Based on this RNA interference mechanism, various artificial RNAi molecules with different structures have been developed. These structures can enter the RNAi pathway at different stages to achieve sequence-specific cleavage of target gene transcripts. See, for example, Molecules 2019, 24, 2211; doi:10.3390 / molecules24122211 (which is hereby incorporated herein by reference in its entirety). Artificial RNAi molecules with such structures include, for example, siRNA molecules having a double-stranded region (and optionally one or two overhangs), long-chain siRNA molecules that can serve as substrates for the Dicer enzyme, short hairpin RNA (shRNA) that can be processed by Dicer to produce siRNA structures, and long single-stranded siRNA molecules containing only the antisense strand. It is understood that these molecular forms all fall within the scope of the RNAi activators of this invention.

[0069] In some embodiments, the dsRNA activators of this disclosure, such as siRNA (including siRNA with modified nucleotides and siRNA with modified nucleotides and ligands), inhibit the expression of the INHBE gene (e.g., the human INHBE gene) by at least about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, or about 94% in cells, such as in hepatocytes, such as in human primary hepatocytes, as determined by, for example, PCR or by protein-based methods (e.g., by immunofluorescence analysis, using, for example, Western blotting or flow cytometry). In some embodiments, inhibition of expression is determined in suitable biological cell lines using dsRNA, such as siRNA, at concentrations of, for example, about 10 nM, about 1 nM, about 0.1 nM, or about 0.01 nM, by the qPCR method provided herein. In some implementations, inhibition of expression is determined in suitable biological cell lines using the qPCR method provided herein, with, for example, serially diluted concentrations (e.g., starting at 100 nM) of dsRNA, such as siRNA.

[0070] In some embodiments, the dsRNA active agents of this disclosure, such as siRNAs (including siRNAs containing modified nucleotides and siRNAs containing modified nucleotides and ligands), have a low off-target risk, for example, both the sense and antisense strands have a low off-target risk. In some embodiments, the dsRNA active agents of this disclosure, such as siRNAs (including siRNAs containing modified nucleotides and siRNAs containing modified nucleotides and ligands), have a lower off-target risk than control siRNAs, for example, compared to known control siRNAs, such as AD-1708473 in WO2023003922A1.

[0071] In some embodiments, the dsRNA activators of this disclosure, such as siRNA (including siRNA with modified nucleotides and siRNA with modified nucleotides and ligands), can be freely taken up by hepatocytes and inhibit the expression of the INHBE gene in hepatocytes, such as human primary hepatocytes, for example, by determining the inhibition of expression in a suitable biological cell line using, for example, serially diluted concentrations (e.g., starting concentration of 500 nM) of dsRNA, such as siRNA, as provided herein by the qPCR method.

[0072] In some embodiments, the dsRNA activators of this disclosure, such as siRNAs (including siRNAs with modified nucleotides and siRNAs with modified nucleotides and ligands), are capable of effectively inhibiting the expression of the INHBE gene in vivo, for example, in liver tissue or hepatocytes. In some embodiments, the dsRNA activators of the present invention, such as siRNA activators (particularly siRNA activators with specific modifications), have better inhibitory effects on INHBE in vivo, for example, their inhibitory level on INHBE mRNA in vivo is higher than their expected inhibitory effect on INHBE mRNA in in vitro screening.

[0073] In some embodiments, the dsRNA activator of this disclosure, such as siRNA (including siRNA with modified nucleotides and siRNA with modified nucleotides and ligands), inhibits the expression of the INHBE gene (e.g., the human INHBE gene) in vivo (e.g., in liver tissue, such as in mouse liver tissue) by at least about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90%, for example by a single subcutaneous administration to mice or by detection of mouse liver tissue homogenate, as described in Example 10.

[0074] In some embodiments, the dsRNA active agents of this disclosure, such as siRNA (including siRNA with modified nucleotides and siRNA with modified nucleotides and ligands), exhibit long-lasting inhibitory effects on the target gene INHBE, for example, maintaining inhibition of the target gene INHBE 1 week, 2 weeks, 3 weeks, or 5 weeks after in vitro contact with cells or in vivo administration. In some embodiments, the dsRNA active agents of this disclosure, such as siRNA (including siRNA with modified nucleotides and siRNA with modified nucleotides and ligands), exhibit long-lasting inhibition (knockdown) of the target gene INHBE in vivo, for example, maintaining inhibition of the target gene INHBE 1 week, 2 weeks, 3 weeks, or 5 weeks after administration.

[0075] In some embodiments, the dsRNA active agents of this disclosure, such as siRNA (including siRNA with modified nucleotides and siRNA with modified nucleotides and ligands), are effective in treating or reducing obesity in patients, for example, maintaining or reducing patient weight and / or body fat, for example, their effects are superior to or comparable to known control siRNAs (e.g., AD-1708473 or AD-1708473.1 in WO2023003922A1). In some embodiments, the dsRNA active agents, such as siRNA (including siRNA with modified nucleotides and siRNA with modified nucleotides and ligands), have a long-lasting effect, for example, remaining effective in treating patient obesity, for example, maintaining or reducing patient weight and / or body fat, even 1 week, 2 weeks, 3 weeks, or 60 days after the last administration.

[0076] In some embodiments, the dsRNA activators of this disclosure, such as siRNA (including siRNA with modified nucleotides and siRNA with modified nucleotides and ligands), exhibit in vivo inhibitory and / or therapeutic effects (e.g., weight loss and / or body fat reduction) on the target gene INHBE that exceed expectations based on in vitro screening assays, i.e., they have better target gene inhibitory activity and / or therapeutic effects in vivo compared to the activity expected during in vitro screening.

[0077] In some embodiments, the dsRNA activator of the present invention has the properties described in (i) and (ii), (i) and (iii), or (i), (ii) and (iii):

[0078] (i) The dsRNA activator (particularly nucleotide-modified siRNA, such as XD000155 or XD000275) inhibits INHBE mRNA expression in hepatocytes, but at a level lower than that of a known control siRNA, such as AD-1708473 in WO2023003922A1, for example, by using the qPCR method provided herein, in suitable biological cell lines (e.g., in hepatocytes such as Hep3B), to determine the inhibition of expression with, for example, a concentration of about 1 nM or about 0.1 nM of dsRNA, such as siRNA (e.g., nucleotide-modified siRNA), as described in Example 3;

[0079] (ii) The dsRNA activator (particularly nucleotide-modified siRNA, particularly siRNA with nucleotide modification and ligand, such as XD000155.1 or XD000202.1) inhibits INHBE mRNA expression in hepatocytes, but at a level lower than that of a known control siRNA, such as AD-1708473.1 in WO2023003922A1, for example, by the qPCR method provided herein, in suitable biological cell lines (e.g., in hepatocytes such as human primary hepatocytes or monkey primary hepatocytes), with, for example, different concentrations such as serially diluted concentrations (e.g., 100 nM starting, 5-fold or 10-fold serial dilutions; or 500 nM 4-fold serial dilutions) or with, for example, 100 nM, 10 nM, 1 nM or 0.1 nM concentrations of dsRNA, such as siRNA (e.g., siRNA with nucleotide modification and ligand), as described in Examples 4 or 5 or 8 or 9.

[0080] (iii) The dsRNA activator (particularly nucleotide-modified siRNA, particularly siRNA with nucleotide modification and ligand, such as XD000155.1, XD000202.1, or XD00275.36) inhibits INHBE mRNA expression in vivo, and its level of inhibition of INHBE mRNA expression in vivo is higher than that of a known control siRNA, such as AD-1708473.1 in WO2023003922A1, for example by the in vivo detection methods described herein; in some embodiments, the method includes extracting liver tissue from animals, such as mice, that have been administered the dsRNA activator of the present invention, and determining the inhibition of expression by PCR methods provided herein (e.g., the method described in Example 10).

[0081] Therefore, in some embodiments, the dsRNA activators of this disclosure, such as siRNA (including siRNA with modified nucleotides and siRNA with modified nucleotides and ligands), exhibit lower in vitro inhibitory effects on the target gene INHBE than known control siRNAs, such as AD-1708473.1 in WO2023003922A1, but are superior to the same control in vivo in terms of inhibitory and / or therapeutic effects (e.g., reduction of weight and / or body fat) on the target gene INHBE.

[0082] In some embodiments, the dsRNA activator comprises an antisense strand containing a complementary region that is complementary (substantially complementary or fully complementary) to at least a portion (e.g., a target sequence) of the mRNA formed during INHBE gene expression. In some embodiments, the length of the complementary region is about 15 to 30 nucleotides, such as 16 to 30, 17 to 30, or 18 to 30 nucleotides (e.g., lengths of about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides). In some embodiments, the length of the complementary region is between 18 and 23 nucleotides. In some embodiments, the length of the complementary region is 19 to 23 nucleotides. In some embodiments, the length of the complementary region is 18 to 21 nucleotides. In some embodiments, the length of the complementary region is 18, 19, 20, or 21 nucleotides. In some embodiments, the length of the complementary region is 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the antisense strand is complementary to the mRNA target sequence starting from the second nucleotide from the 5' end. In some embodiments, the complementary region of the antisense strand comprises the second nucleotide from the 5' end to the 3rd, 2nd, or 1st nucleotide from the 3' end. In some embodiments, the complementary region of the antisense strand comprises all antisense strand nucleotides starting from the second nucleotide from the 5' end. In some embodiments, the complementary region of the antisense strand comprises at least nucleotides 2-16, 2-17, 2-18, 2-19, 2-20, or 2-21 from the 5' end of the antisense strand. In some embodiments, the complementary region of the antisense strand comprises at least 2-19 consecutive nucleotides from the 5' end of the antisense strand. In some implementations, the complementary region of the antisense strand comprises, or is composed of, consecutive nucleotides at positions 2-19, 2-20, or 2-21, starting from the 5' end of the antisense strand.

[0083] In some implementations, the dsRNA comprises two complementary RNA strands that form a double-stranded structure (double-stranded region or double-stranded region) under conditions that will cause the dsRNA to hybridize, namely the antisense strand and the sense strand.

[0084] In some embodiments, one strand of the dsRNA (antisense strand) contains a complementary region (antisense complement) that is substantially or completely complementary to the target sequence. Therefore, the antisense complement of the dsRNA can be substantially or completely complementary to the target sequence. The target sequence can be derived from the sequence of mRNA formed during INHBE gene expression. In some embodiments, the antisense complement is substantially complementary to the target sequence, for example, it is mismatched with the target sequence at 1, 2, 3, 4, or 5 nucleotides (preferably 1 or 2 nucleotides at the 5' and / or 3' ends, e.g., the first nucleotide at the 5' end of the antisense strand). In some embodiments, the antisense complement is completely complementary to the target sequence.

[0085] The other chain (the justice chain) contains regions complementary to the antisense chain, allowing the two chains to hybridize and form a bistranded structure (bistranded region) when combined under appropriate conditions.

[0086] In some embodiments, the antisense strand of the dsRNA is completely complementary to the corresponding portion of the target sequence, starting from the second nucleotide from the 5' end. In some embodiments, the antisense strand of the dsRNA is completely complementary to the corresponding portion of the target sequence from the second nucleotide from the 5' end to the first, second, or third nucleotide from the 3' end. In some embodiments, the entire length of the antisense strand of the dsRNA, starting from the second nucleotide from the 5' end, is completely complementary to the corresponding portion of the target sequence. In some embodiments, nucleotides 2-16, 2-17, 2-18, 2-19, 2-20, or 2-21 of the antisense strand of the dsRNA, starting from the 5' end, are completely complementary to the corresponding portion of the target sequence. In some embodiments, consecutive nucleotides from positions 2-19, 2-20, or 2-21 of the antisense strand of the dsRNA, starting from the 5' end, are completely complementary to the corresponding portion of the target sequence. In some embodiments, the antisense strand of the dsRNA has the same number of nucleotides as the target sequence and is completely complementary to the target sequence in all nucleotide sequences except for the first nucleotide at the 5' end, wherein the first nucleotide of the antisense strand is U or A. In some embodiments, the full length of the antisense strand is completely complementary to the target sequence.

[0087] When "the corresponding portion of the target sequence" is mentioned in this document, it refers to a consecutive nucleotide sequence in the target sequence that is completely complementary to the antisense strand. For example, when the target sequence is 21 nucleotides and its consecutive nucleotides from position 1 to 20 are completely complementary to the nucleotides from position 2 to 21 of the antisense strand, the "corresponding portion of the target sequence" refers to the consecutive nucleotides from position 1 to 20 of the target sequence.

[0088] In some implementations, the dsRNA described herein targets the INHBE gene sequence at or near the location of the INHBE genome mRNA shown in Table 1 (e.g., NM_031479.5).

[0089] In some embodiments, the dsRNA described herein targets any 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive nucleotides, such as 19-23 consecutive nucleotides, in the INHBE genome (e.g., NM_031479.5) mRNA or the nucleic acid sequence shown in SEQ ID NO:664 or its complementary sequence. The nucleic acid sequence shown in NO:664 includes sequences 35-55, 36-56, 40-60, 501-521, 503-523, 504-524, 507-527, 638-658, 880-900, 978-998, 980-1000, 1096-1116, 1109-1129, 1202-1222, 1206-1226, 1220-1240, 1301-1321, 1308-1328, 1309-1329, 1310-1330, 1346-1366, and 1352-1372. 1355-1375, 1357-1377, 1387-1407, 1390-1410, 1393-1413, 1397-1417, 1436-1456, 1444-1464, 1445-1465, 1446-1466, 1447-1467, 1448-1468, 1449-1469, 1450-1470, 1462-1482, 1465-1485, 1588-1608, 1589-1609, 1618-1638, 1621-1641, 1622-1 642, 1623-1643, 1647-1667, 1862-1882, 1863-1883, 2044-2064, 2048-2068, 2053-2073, 2054-2074, 2055-2075, 2056-2076, 2057-2077, 2058-2078, 2059-2079, 2161-2181, 2162-2182, 2163-2183, 2164-2184, 2206-2226, 2207-2227, 2235-2255, 223 7-2257, 2238-2258, 2240-2260, 2241-2261, 2242-2262, 2243-2263, 2244-2264, 2245-2265, 2246-2266, 2247-2267, 2349-2369, 2376-2396, 2377-2397, 2404-2424, 2405-2425, 2406-2426, 2408-2428, 2410-2430, 2411-2431, 2412-2432, 2413-2433,2420-2440, 361-381, 365-385, 369-389, 506-526, 515-535, 516-536, 518-538, 882-902, 1036-1056, 1037-1057, 1107-1127, 1125-1145, 1299-1319, 1 The sequences corresponding to bits 350-1370, 1351-1371, 1353-1373, 1398-1418, 1399-1419, 1400-1420, 1403-1423, 1404-1424, 1443-1463, 1587-1607, 2160-2180, or 2296-2316.

[0090] In some implementations, the dsRNA described herein targets a nucleotide sequence (target sequence) of the mRNA of the INHBE gene selected from the following:

[0091] (i) A continuous sequence of INHBE mRNA at or near the location of the INHBE genome (e.g., NM_031479.5) mRNA as shown in Table 1, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 consecutive nucleotides at or near the location;

[0092] (ii) The 35th, 36th, 40th, 361st, 365th, 369th, 501st, 503rd, 504th, 506th, 507th, 515th, 516th, 518th, 638th, 880th, 882nd, 978th, 980th, 1036th, 1037th, 1096th, 1107th, 1109th, 1125th, 1202nd, 1206th, 1220th, 1299th, 13th of the INHBE genome (e.g., NM_031479.5) mRNA or the nucleic acid sequence shown in SEQ ID NO:664 or its complementary sequence. 01, 1308, 1309, 1310, 1346, 1350, 1351, 1352, 1353, 1355, 1357, 1387, 1390, 1393, 1397, 1398, 1399, 1400, 1403, 1404, 1436, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1462 1465, 1587, 1588, 1589, 1618, 1621, 1622, 1623, 1647, 1862, 1863, 2044, 2048, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2160, 2161, 2162, 2163, 2164, 2206, 2207, 2235, 2237 At least 15-35 consecutive nucleotides, such as 18, 19, 20 or 21 consecutive nucleotides, starting at position 2238, 2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247, 2296, 2349, 2376, 2377, 2404, 2405, 2406, 2408, 2410, 2411, 2412, 2413 or 2420;

[0093] (iii) INHBE genome (e.g., NM_031479.5) mRNA or SEQ ID The nucleic acid sequence shown in NO:664 or its complementary sequence contains the following sequences: 35-55, 36-56, 40-60, 501-521, 503-523, 504-524, 507-527, 638-658, 880-900, 978-998, 980-1000, 1096-1116, 1109-1129, 1202-1222, 1206-1226, 1220-1240, 1301-1321, 1308-1328, 1309-1329, 1310-1330, 1346-1366, 1352-1372, 1355-1375, 1357-1377, 138. 7-1407, 1390-1410, 1393-1413, 1397-1417, 1436-1456, 1444-1464, 1445-1465, 1446-1466, 1447-1467, 1448-1468, 1449-1469, 1450-1470, 1462-1482, 1465-1485, 1588-1608, 1589-1609, 1618-1638, 1621-1641, 1622-1642, 1623-1643, 1647-1667, 1862-1882, 1863-1883, 2044-2064, 204 8-2068, 2053-2073, 2054-2074, 2055-2075, 2056-2076, 2057-2077, 2058-2078, 2059-2079, 2161-2181, 2162-2182, 2163-2183, 2164-2184, 2206-2226, 2207-2227, 2235-2255, 2237-2257, 2238-2258, 2240-2260, 2241-2261, 2242-2262, 2243-2263, 2244-2264, 2245-2265, 2246-2266, 224 7-2267, 2349-2369, 2376-2396, 2377-2397, 2404-2424, 2405-2425, 2406-2426, 2408-2428, 2410-2430, 2411-2431, 2412-2432, 2413-2433, 2420-2440, 361-381, 365-385, 369-389, 506-526, 515-535, 516-536, 518-538, 882-902, 1036-1056, 1037-1057, 1107-1127, 1125-1145, 1299-1319,The 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, or 35th consecutive nucleotides of the sequence corresponding to positions 1350-1370, 1351-1371, 1353-1373, 1398-1418, 1399-1419, 1400-1420, 1403-1423, 1404-1424, 1443-1463, 1587-1607, 2160-2180, or 2296-2316;

[0094] (iv) INHBE genome (e.g., NM_031479.5) mRNA or SEQ ID The nucleic acid sequence shown in NO:664 includes sequences 35-55, 36-56, 40-60, 501-521, 503-523, 504-524, 507-527, 638-658, 880-900, 978-998, 980-1000, 1096-1116, 1109-1129, 1202-1222, 1206-1226, 1220-1240, 1301-1321, 1308-1328, 1309-1329, 1310-1330, 1346-1366, 1352-1372, 1355-1375, 1357-1377, and 1387-1407. 1390-1410, 1393-1413, 1397-1417, 1436-1456, 1444-1464, 1445-1465, 1446-1466, 1447-1467, 1448-1468, 1449-1469, 1450-1470, 1462-1482, 1465-1485, 1588-1608, 1589-1609, 1618-1638, 1621-1641, 1622-1642, 1623-1643, 1647-1667, 1862-1882, 1863-1883, 2044-2064, 2048-20 68, 2053-2073, 2054-2074, 2055-2075, 2056-2076, 2057-2077, 2058-2078, 2059-2079, 2161-2181, 2162-2182, 2163-2183, 2164-2184, 2206-2226, 2207-2227, 2235-2255, 2237-2257, 2238-2258, 2240-2260, 2241-2261, 2242-2262, 2243-2263, 2244-2264, 2245-2265, 2246-2266, 2247- 2267, 2349-2369, 2376-2396, 2377-2397, 2404-2424, 2405-2425, 2406-2426, 2408-2428, 2410-2430, 2411-2431, 2412-2432, 2413-2433, 2420-2440, 361-381, 365-385, 369-389, 506-526, 515-535, 516-536, 518-538, 882-902, 1036-1056, 1037-1057, 1107-1127, 1125-1145, 1299-1319,The sequence corresponding to positions 1350-1370, 1351-1371, 1353-1373, 1398-1418, 1399-1419, 1400-1420, 1403-1423, 1404-1424, 1443-1463, 1587-1607, 2160-2180, or 2296-2316, consisting of any 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides, preferably the nucleotide sequence corresponding to the positions mentioned above; or...

[0095] (v) A nucleotide sequence that contains or consists of the nucleotide sequences shown in any of SEQ ID NO:221-330.

[0096] In some implementations, the mRNA sequence targeted by the dsRNA described herein includes, or is composed of, the mRNA target sequence corresponding to the location of the INHBE gene shown in Table 1.

[0097] In some embodiments, the antisense strand of the dsRNA disclosed herein contains a complementary region that is completely, substantially, or at least partially complementary to the target sequence corresponding to the INHBE genome (e.g., NM_031479.5) mRNA location shown in Table 1. In some embodiments, the antisense strand of the dsRNA disclosed herein contains a core sequence (complementary region) that is completely, substantially, or at least partially complementary to the mRNA sequence (target sequence) disclosed in Table 2. In some embodiments, the INHBE mRNA target sequence targeted by the dsRNA described herein contains or is composed of the nucleotide sequence shown in any of SEQ ID NO: 221-330. In some embodiments, the target sequence contains a continuous sequence of INHBE mRNA at or near the INHBE genome (e.g., NM_031479.5) mRNA location shown in Table 1. In some embodiments, the antisense strand of the dsRNA has the same number of nucleotides as the INHBE target sequence (e.g., the target sequence shown in Table 2).

[0098] In some embodiments, the antisense strand of the dsRNA is fully complementary, substantially complementary, or at least partially complementary to the INHBE mRNA target sequence (e.g., the mRNA target sequence corresponding to the position shown in Table 1 or the mRNA target sequence shown in Table 2). In some embodiments, the antisense strand of the dsRNA has the same number of nucleotides as or differs from the INHBE mRNA target sequence (e.g., the mRNA target sequence corresponding to the position shown in Table 1 or the mRNA target sequence shown in Table 2) by 1, 2, or 3 nucleotides. In some embodiments, the antisense strand of the dsRNA is fully complementary to the INHBE mRNA target sequence (e.g., the mRNA target sequence corresponding to the position shown in Table 1 or the mRNA target sequence shown in Table 2). In some embodiments, the antisense strand contains 1, 2, 3, 4, or 5 non-complementary sites (mismatches), for example, 1-3 nucleotide mismatches.

[0099] In some embodiments, the antisense strand of the dsRNA is completely complementary to the INHBE gene target sequence (e.g., the target sequence corresponding to the position shown in Table 1 or the mRNA target sequence shown in Table 2) in the region excluding the first nucleotide at the 5' end, and to the region excluding the first or second nucleotide at the 3' end of the target sequence. In some embodiments, the first nucleotide at the 5' end of the antisense strand of the dsRNA is U or A, for example, U, to facilitate recognition by the Ago2 protein to form the RICS complex.

[0100] In some embodiments, the length of the positive and negative strands is independently 15-30 nucleotides, such as 17-27, 19-25, 18-24, 18-23, 19-22, or 19-21 nucleotides. In some embodiments, the length of the positive or negative strand is independently no more than 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides. In some embodiments, the length of the positive or negative strand is independently not less than 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the length of the positive or negative strand is 18-21 nucleotides (e.g., 18, 19, 20, or 21 nucleotides), and the length of the negative strand is 19-22 nucleotides (e.g., 19, 20, 21, or 22 nucleotides). In some embodiments, the length of the sense strand is 18 or 19 nucleotides, and the length of the antisense strand is 19-21 nucleotides. In some embodiments, the length of the sense strand is 19 nucleotides, and the length of the antisense strand is 21 nucleotides.

[0101] In some embodiments, the length of the double-stranded region is 15 to 30 nucleotide pairs. In some embodiments, the length of the double-stranded region is 15 to 25 nucleotide pairs or 16 to 25 nucleotide pairs. In some embodiments, the length of the double-stranded region is 16 to 24 nucleotide pairs or 17 to 24 nucleotide pairs. In some embodiments, the length of the double-stranded region is 17 to 23 nucleotide pairs or 18 to 23 nucleotide pairs. In some embodiments, the length of the double-stranded region is 16 to 22 nucleotide pairs, 17 to 22 nucleotide pairs, 18 to 22 nucleotide pairs, or 19 to 22 nucleotide pairs. In some embodiments, the length of the double-stranded region is 16 to 21 nucleotide pairs, for example, 16, 17, 18, 19, 20, or 21 nucleotide pairs. In some embodiments, the length of the double-stranded region is 19 to 21 nucleotide pairs. In some implementations, the length of the double-stranded region is 18, 19, 20, or 21 nucleotide pairs, for example, 19 nucleotide pairs.

[0102] In some embodiments, the double-stranded region formed by the sense and antisense strands is completely complementary. In other embodiments, the double-stranded region formed by the sense and antisense strands is substantially complementary, and may contain one, two, three, four, or five non-complementary sites (mismatches). In some embodiments, the length of the completely complementary double-stranded region is at least 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the length of the completely complementary double-stranded region is between 15 and 25 nucleotides, 16 and 25 nucleotide pairs, 16 and 24 nucleotide pairs, 17 and 24 nucleotide pairs, 17 and 23 nucleotide pairs, 18 and 23 nucleotide pairs, or 19 and 22 nucleotides. In some embodiments, the length of the completely complementary double-stranded region is 16, 17, 18, 19, 20, or 21 nucleotides, for example, 19 nucleotides.

[0103] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, for example, 1 to 4, 2 to 4, 1 to 3, 2 to 3, 1, 2, 3, or 4 nucleotides. In some embodiments, dsRNA having at least one nucleotide overhang has better repressive properties relative to its blunt-ended counterpart. The nucleotide overhang may include or consist of nucleotide / nucleoside analogs comprising deoxynucleotides / nucleosides. The overhang may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide may be present at the 5' end, 3' end, or both ends of the antisense strand or sense strand of the dsRNA.

[0104] In some embodiments, one or both of the sense strand and the antisense strand include a 3' overhang and / or a 5' overhang having at least 1, 2, or 3 nucleotides; for example, one or both of the sense strand and the antisense strand include a 3' overhang and / or a 5' overhang having at least 1 nucleotide. In some embodiments, at least one strand includes a 3' overhang or a 5' overhang having at least 1 nucleotide. In some embodiments, at least one strand includes a 3' overhang or a 5' overhang having at least 2 nucleotides. In some embodiments, at least one strand includes a 3' overhang or a 5' overhang having at least 3 nucleotides.

[0105] In some preferred embodiments, the antisense strand has a 3' overhang and / or a 5' overhang of at least one nucleotide, for example, the antisense strand comprises a 3' overhang and / or a 5' overhang of one nucleotide. In some preferred embodiments, the antisense strand has a 3' overhang and / or a 5' overhang of at least two nucleotides, for example, the antisense strand comprises a 3' overhang and / or a 5' overhang of two nucleotides. In some preferred embodiments, the antisense strand has a 3' overhang and / or a 5' overhang of at least three nucleotides, for example, the antisense strand comprises a 3' overhang and / or a 5' overhang of three nucleotides. In a preferred embodiment, the antisense strand has a 3' overhang of one, two, or three nucleotides at the 3' end, for example, a 3' overhang of two nucleotides.

[0106] In some embodiments, the sense strand includes a 5' overhang having at least 1, 2, or 3 nucleotides, and / or the antisense strand includes a 3' overhang having at least 1, 2, or 3 nucleotides.

[0107] In some embodiments, the antisense strand of the dsRNA has a 3' overhang, for example, a 2-nucleotide 3' overhang, and a blunt end at the 5' end.

[0108] In some embodiments, the present invention relates to a double-stranded RNA (dsRNA) activator for inhibiting the expression of repressin subunit βE (INHBE), wherein the dsRNA activator comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand is completely complementary to the corresponding portion of the target sequence of the INHBE gene at least from the 5' end at positions 2-19 (e.g., positions 2-20 or 2-21 or the full length), for example, wherein the first nucleotide at the 5' end of the antisense strand is A or U, such as U.

[0109] In some embodiments, the present invention relates to a double-stranded RNA (dsRNA) activator for inhibiting the expression of repressin subunit βE (INHBE), wherein the dsRNA activator comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand is at least partially complementary to the mRNA encoding INHBE. In some embodiments, the antisense strand is completely complementary to the target sequence of the INHBE gene in the region except for the first nucleotide from the 5' end, wherein the first nucleotide from the 5' end of the antisense strand is A or U, for example, U.

[0110] In some embodiments, the dsRNA activator of the present invention comprises a sense strand and an antisense strand, wherein the sense strand comprises 19 nucleotides and the antisense strand comprises 21 nucleotides, wherein the antisense strand comprises a 3' overhang of 2 nucleotides compared to the sense strand, and wherein the sense strand and the antisense strand are completely complementary at 19 nucleotides, for example, at 19 consecutive nucleotides (e.g., at the 1st to 19th consecutive nucleotides of the antisense strand counting from the 5' end).

[0111] In some embodiments, the dsRNA of the present invention comprises a sense strand and an antisense strand forming a double-stranded region, wherein

[0112] (i) The positive strand contains or is 19 nucleotides.

[0113] (ii) The antisense strand comprises or is 21 nucleotides and is completely complementary to the target sequence of the INHBE gene in the region except for the first nucleotide at the 5' end, wherein the first nucleotide from the 5' end of the antisense strand is A or U, for example U;

[0114] (iii) The antisense strand contains a 3' overhang of 2 nucleotides compared to the sense strand, and the sense strand and the antisense strand are completely complementary over 19 consecutive nucleotides, for example, completely complementary over 1-19 consecutive nucleotides starting from the 5' end of the antisense strand.

[0115] In one aspect of the invention, the invention relates to a double-stranded ribonucleic acid (dsRNA) activator for inhibiting the expression of repressin subunit βE (INHBE), wherein the dsRNA activator comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differing from any antisense strand nucleotide sequence in Table 1 by no more than 3, 2, or 1 nucleotide, wherein the first nucleotide at the 5' end of the antisense strand is A or U, for example, U.

[0116] In some embodiments, the dsRNA activator comprises a sense strand and an antisense strand, the sense strand comprising at least 15, 16, 17, 18, or 19 consecutive nucleotides differing by no more than 3 nucleotides from any nucleotide sequence of the sense strand in Table 1, and the antisense strand comprising at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differing by no more than 3 nucleotides from any nucleotide sequence of the antisense strand in Table 1 (e.g., the antisense strand corresponding to the sense strand (i.e., the antisense strand under the same siRNA name as the sense strand)), wherein the first nucleotide at the 5' end of the antisense strand is A or U, for example, U.

[0117] In some embodiments, the dsRNA activator comprises a sense strand and an antisense strand, the sense strand comprising at least 15, 16, 17, 18, or 19 consecutive nucleotides differing by no more than 2 nucleotides from any nucleotide sequence of the sense strand in Table 1, and the antisense strand (e.g., corresponding to the sense strand (i.e., the antisense strand under the same siRNA name as the sense strand)) comprising at least 15, 16, 17, 18, or 19, 20, or 21 consecutive nucleotides differing by no more than 2 nucleotides from any nucleotide sequence of the antisense strand in Table 1, wherein the first nucleotide at the 5' end of the antisense strand is A or U, for example, U.

[0118] In some embodiments, the dsRNA activator comprises a sense strand and an antisense strand, the sense strand comprising at least 15, 16, 17, 18, or 19 consecutive nucleotides differing by no more than one nucleotide from any nucleotide sequence of the sense strand in Table 1, and the antisense strand (e.g., corresponding to the sense strand (i.e., the antisense strand under the same siRNA name as the sense strand)) comprising at least 15, 16, 17, 18, or 19, 20, or 21 consecutive nucleotides differing by no more than one nucleotide from any nucleotide sequence of the antisense strand in Table 1, wherein the first nucleotide at the 5' end of the antisense strand is A or U, for example, U.

[0119] In some specific embodiments, the positive strand comprises a nucleotide sequence of at least 15, 16, 17, 18, or 19 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO:1-110. In some specific embodiments, the positive strand differs from the nucleotide sequence shown in any one of SEQ ID NO:1-110 by no more than 1, 2, or 3 nucleotides. In some specific embodiments, the positive strand comprises or is composed of the nucleotide sequence shown in SEQ ID NO:1-110.

[0120] In some specific embodiments, the antisense strand comprises a nucleotide sequence of at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 111-220. In some specific embodiments, the antisense strand differs from the nucleotide sequence shown in any one of SEQ ID NO: 111-220 by no more than 1, 2, or 3 nucleotides. In some specific embodiments, the antisense strand comprises or consists of the nucleotide sequence shown in any one of SEQ ID NO: 111-220.

[0121] In some embodiments, the dsRNA activator comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence selected from the nucleotide sequences of the sense strands in Table 1, and the antisense strand comprising a nucleotide sequence selected from the nucleotide sequences of the antisense strands in Table 1 (e.g., corresponding to the sense strand (i.e., the antisense strand under the same siRNA name as the sense strand)).

[0122] In some embodiments, the combination of the antisense and sense strands in the dsRNA activator is as shown in Table 1 for any combination of antisense and sense strands.

[0123] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand respectively comprise SEQ ID NO:1 / 111, SEQ ID NO:2 / 112, SEQ ID NO:3 / 113, SEQ ID NO:4 / 114, SEQ ID NO:5 / 115, SEQ ID NO:6 / 116, SEQ ID NO:7 / 117, SEQ ID NO:8 / 118, SEQ ID NO:9 / 119, SEQ ID NO:10 / 120, SEQ ID NO:11 / 121, SEQ ID NO:12 / 122, SEQ ID NO:13 / 123, SEQ ID NO:14 / 124, SEQ ID NO:15 / 125, SEQ ID NO:16 / 126, SEQ ID NO:17 / 127, SEQ ID NO:18 / 128, SEQ ID NO:19 / 129, SEQ ID NO:20 / 130, SEQ ID NO:19 / 129, SEQ ID NO:20 / 130, SEQ ID NO:19 / 129, SEQ ID NO:19 / 129, SEQ ID NO:19 / 120 ... NO:21 / 131, SEQ ID NO:22 / 132, SEQ ID NO:23 / 133, SEQ ID NO:24 / 134, SEQ ID NO:25 / 135, SEQ ID NO:26 / 136, SEQ ID NO:27 / 137, SEQ ID NO:28 / 138, SEQ ID NO:29 / 139, SEQ ID NO:30 / 140, SEQ ID NO:31 / 141, SEQ ID NO:32 / 142, SEQ ID NO:33 / 143, SEQ ID NO:34 / 144, SEQ ID NO:35 / 145, SEQ ID NO:36 / 146, SEQ ID NO:37 / 147, SEQ ID NO:38 / 148, SEQ ID NO:39 / 149, SEQ ID NO:40 / 150, SEQ ID NO:41 / 151, SEQ ID NO:42 / 152, SEQ ID NO:43 / 153, SEQ ID NO:44 / 154, SEQ ID NO:45 / 155, SEQ ID NO:46 / 156, SEQ ID NO:47 / 157, SEQ ID NO:48 / 158, SEQ ID NO:49 / 159, SEQ ID NO:50 / 160, SEQ ID NO:51 / 161, SEQ ID NO:52 / 162, SEQ ID NO:53 / 163, SEQ ID NO:54 / 164, SEQ ID NO:55 / 165, SEQ ID NO:56 / 166, SEQ IDNO:57 / 167、SEQ ID NO:58 / 168、SEQ ID NO:59 / 169、SEQ ID NO:60 / 170、SEQ ID NO:61 / 171、SEQ ID NO:62 / 172、SEQ ID NO:63 / 173、SEQ ID NO:64 / 174、SEQ ID NO:65 / 175、SEQ ID NO:66 / 176、SEQ ID NO:67 / 177、SEQ ID NO:68 / 178、SEQ ID NO:69 / 179、SEQ ID NO:70 / 180、SEQ ID NO:71 / 181、SEQ ID NO:72 / 182、SEQ ID NO:73 / 183、SEQ ID NO:74 / 184、SEQ ID NO:75 / 185、SEQ ID NO:76 / 186、SEQ ID NO:77 / 187、SEQ ID NO:78 / 188、SEQ ID NO:79 / 189、SEQ ID NO:80 / 190、SEQ ID NO:81 / 191、SEQ ID NO:82 / 192、SEQ ID NO:83 / 193、SEQ ID NO:84 / 194、SEQ ID NO:85 / 195、SEQ ID NO:86 / 196、SEQ ID NO:87 / 197、SEQ ID NO:88 / 198、SEQ ID NO:89 / 199、SEQ ID NO:90 / 200、SEQ ID NO:91 / 201、SEQ ID NO:92 / 202、SEQ ID NO:93 / 203、SEQ ID NO:94 / 204、SEQ ID NO:95 / 205、SEQ ID NO:96 / 206、SEQ ID NO:97 / 207、SEQ ID NO:98 / 208、SEQ ID NO:99 / 209、SEQ ID NO:100 / 210、SEQ ID NO:101 / 211、SEQ ID NO:102 / 212、SEQ ID NO:103 / 213、SEQ ID NO:104 / 214、SEQ ID NO:105 / 215、SEQ ID NO:106 / 216、SEQ ID NO:107 / 217、SEQ ID NO:108 / 218、SEQ ID NO:109 / 219、SEQ IDThe nucleotide sequence shown in NO:110 / 220 contains at least 15, 16, 17, 18 or 19 consecutive nucleotides, and optionally the antisense strand also has a 3' overhang of 1 or 2 nucleotides, and optionally the first nucleotide at the 5' end of the antisense strand is A or U, for example U.

[0124] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand respectively comprise SEQ ID NO:1 / 111, SEQ ID NO:2 / 112, SEQ ID NO:3 / 113, SEQ ID NO:4 / 114, SEQ ID NO:5 / 115, SEQ ID NO:6 / 116, SEQ ID NO:7 / 117, SEQ ID NO:8 / 118, SEQ ID NO:9 / 119, SEQ ID NO:10 / 120, SEQ ID NO:11 / 121, SEQ ID NO:12 / 122, SEQ ID NO:13 / 123, SEQ ID NO:14 / 124, SEQ ID NO:15 / 125, SEQ ID NO:16 / 126, SEQ ID NO:17 / 127, SEQ ID NO:18 / 128, SEQ ID NO:19 / 129, SEQ ID NO:20 / 130, SEQ ID NO:19 / 129, SEQ ID NO:20 / 130, SEQ ID NO:19 / 129, SEQ ID NO:19 / 129, SEQ ID NO:19 / 120 ... NO:21 / 131, SEQ ID NO:22 / 132, SEQ ID NO:23 / 133, SEQ ID NO:24 / 134, SEQ ID NO:25 / 135, SEQ ID NO:26 / 136, SEQ ID NO:27 / 137, SEQ ID NO:28 / 138, SEQ ID NO:29 / 139, SEQ ID NO:30 / 140, SEQ ID NO:31 / 141, SEQ ID NO:32 / 142, SEQ ID NO:33 / 143, SEQ ID NO:34 / 144, SEQ ID NO:35 / 145, SEQ ID NO:36 / 146, SEQ ID NO:37 / 147, SEQ ID NO:38 / 148, SEQ ID NO:39 / 149, SEQ ID NO:40 / 150, SEQ ID NO:41 / 151, SEQ ID NO:42 / 152, SEQ ID NO:43 / 153, SEQ ID NO:44 / 154, SEQ ID NO:45 / 155, SEQ ID NO:46 / 156, SEQ ID NO:47 / 157, SEQ ID NO:48 / 158, SEQ ID NO:49 / 159, SEQ ID NO:50 / 160, SEQ ID NO:51 / 161, SEQ ID NO:52 / 162, SEQ ID NO:53 / 163, SEQ ID NO:54 / 164, SEQ ID NO:55 / 165, SEQ ID NO:56 / 166, SEQ IDThe nucleotide sequences shown as SEQ ID NO:57 / 167, SEQ ID NO:58 / 168, SEQ ID NO:59 / 169, SEQ ID NO:60 / 170, SEQ ID NO:61 / 171, SEQ ID NO:62 / 172, SEQ ID NO:63 / 173, SEQ ID NO:64 / 174, SEQ ID NO:65 / 175, SEQ ID NO:66 / 176, SEQ ID NO:67 / 177, SEQ ID NO:68 / 178, SEQ ID NO:69 / 179, SEQ ID NO:70 / 180, SEQ ID NO:71 / 181, SEQ ID NO:72 / 182, SEQ ID NO:73 / 183, SEQ ID NO:74 / 184, SEQ ID NO:75 / 185, SEQ ID NO:76 / 186, SEQ ID NO:77 / 187, SEQ ID NO:78 / 188, SEQ ID NO:79 / 189, SEQ ID NO:80 / 190, SEQ ID NO:81 / 191, SEQ ID NO:82 / 192, SEQ ID NO:83 / 193, SEQ ID NO:84 / 194, SEQ ID NO:85 / 195, SEQ ID NO:86 / 196, SEQ ID NO:87 / 197, SEQ ID NO:88 / 198, SEQ ID NO:89 / 199, SEQ ID NO:90 / 200, SEQ ID NO:91 / 201, SEQ ID NO:92 / 202, SEQ ID NO:93 / 203, SEQ ID NO:94 / 204, SEQ ID NO:95 / 205, SEQ ID NO:96 / 206, SEQ ID NO:97 / 207, SEQ ID NO:98 / 208, SEQ ID NO:99 / 209, SEQ ID NO:100 / 210, SEQ ID NO:101 / 211, SEQ ID NO:102 / 212, SEQ ID NO:103 / 213, SEQ ID NO:104 / 214, SEQ ID NO:105 / 215, SEQ ID NO:106 / 216, SEQ ID NO:107 / 217, SEQ ID NO:108 / 218, SEQ ID NO:109 / 219, SEQ ID NO:110 / 220 or are respectively composed of the shown nucleotide sequences.

[0125] In some specific implementations, the dsRNA activator comprises a sense strand and an antisense strand, wherein

[0126] The sense strand contains or is composed of the nucleotide sequence shown in SEQ ID NO:60, and the antisense strand contains or is composed of the nucleotide sequence shown in SEQ ID NO:170; or

[0127] The sense strand contains or is composed of the nucleotide sequence shown in SEQ ID NO:101, and the antisense strand contains or is composed of the nucleotide sequence shown in SEQ ID NO:211.

[0128] For the purpose of inhibiting target mRNA expression, as those skilled in the art know, the oligonucleotide used as the sense strand does not participate in direct complementary binding to the target sequence, and does not need to have perfectly complementary base pairing with the antisense oligonucleotide in the duplex region. Therefore, in some aspects, the sense strand (passenger strand) according to the invention may include at least one or more of the following properties: substantially complementary to the consecutive nucleotides of the antisense strand in the duplex region with the antisense strand, for example, at least 70%, at least 80%, at least 90%, or 100% complementary; having one or more additional nucleotides forming a protrusion or loop relative to the consecutive nucleotides of the antisense strand in the duplex region; and having one or more nucleotide gaps or vacancies relative to the consecutive nucleotides of the antisense strand in the duplex region. Similarly, for the purpose of inhibiting target mRNA expression, as those skilled in the art will understand, the antisense strand, serving as a guide RNAi for the specific binding of the target mRNA, may also contain a sequence that is not 100% complementary to a consecutive nucleotide region of the target sequence; for example, the complementarity may be at least 80%, at least 90%, or 95% complementary; however, in some cases, 100% complementarity is preferred. According to the purpose of the invention, in some aspects, when considering the sequence motif of the antisense strand to be complementary to the consecutive nucleotide regions of the target sequence, the presence of insertions and deletions is preferably not permitted. With regard to the sense and antisense strands of the invention, in some aspects, when the complementary region is not perfectly complementary to the said consecutive nucleotide region, the mismatch may be located inside or at the end of that region, for example, a mismatch of 3, 2, or 1 nucleotides at the 5' and / or 3' ends. In a preferred embodiment, the antisense strand is complementary to the sense strand over at least 18 consecutive nucleotides. In a preferred embodiment, the antisense strand is complementary to the sense strand over 19 consecutive nucleotides (e.g., consecutive nucleotides 1-19 from the 5' end of the antisense strand), for example, perfectly complementary.

[0129] In some embodiments, the dsRNA activator is prepared or provided in the form of a salt, a mixed salt, or a free acid. In some embodiments, the dsRNA activator is prepared as a sodium salt. Such forms are within the scope of the invention disclosed herein.

[0130] Those skilled in the art will recognize that the dsRNA molecule according to the invention can be unmodified (i.e., containing naturally occurring RNA nucleosides), but can also be (and preferably) modified, as long as it retains the desired functional activity (i.e., capable of forming the desired double-stranded structure and allowing or mediating specific degradation of the target RNA via the RISC pathway). Such RNA modification can occur at the base moiety, sugar moiety, and / or phosphate ester linker of the nucleotide. As a non-limiting example, modified RNAi activators can be constructed using methods known in the art, employing chemical synthesis and enzymatic ligation reactions. For example, modified RNAi activators can be chemically synthesized using naturally occurring nucleotides or nucleotides with various modifications (designed to reduce off-target effects and / or increase the biological stability of the molecule, or increase the physical stability of the double-stranded structure formed between antisense and sense nucleic acids).

[0131] In some embodiments, at least one nucleotide of the dsRNA activator of the present invention is a modified nucleotide. In some embodiments, in the dsRNA activator of the present invention, substantially all nucleotides of the sense strand are modified nucleotides; or substantially all nucleotides of the antisense strand are modified nucleotides; or substantially all nucleotides of both the sense strand and the antisense strand are modified nucleotides.

[0132] In some embodiments, the dsRNA activator comprises one or more modified nucleotides. As used herein, "modified nucleotide" refers to a nucleotide other than a ribonucleotide (2'-hydroxynucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides.

[0133] In some embodiments, all or substantially all nucleotides of the dsRNA activator of the present invention are modified nucleotides. As described herein, a dsRNA activator in which substantially all nucleotides are modified nucleotides refers to a dsRNA activator having a total of 4 or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides that are native ribonucleotides in both the sense and antisense strands. As used herein, a sense strand in which substantially all nucleotides are modified nucleotides refers to a sense strand in which 2 or fewer (i.e., 0, 1, or 2) nucleotides are native ribonucleotides in the sense strand. As used herein, an antisense strand in which substantially all nucleotides are modified nucleotides refers to an antisense strand in which 2 or fewer (i.e., 0, 1, or 2) nucleotides are native ribonucleotides in the antisense strand.

[0134] In some embodiments, all nucleotides in the sense strand of the dsRNA activator are modified nucleotides and / or all nucleotides in the antisense strand are modified nucleotides; or all nucleotides in both the sense strand and the antisense strand are modified nucleotides.

[0135] In some embodiments, nucleotide modifications suitable for the dsRNA activator of the present invention encompass modifications to nucleoside bases, ribose moieties, and / or the phosphate backbone. Exemplary modifications can be found in PCT Publication WO 200370918, which is incorporated herein by reference in its entirety.

[0136] Examples of nucleoside base modifications that can be used to generate dsRNA activators include the substitution of nucleotides containing uracil, guanine, or adenine with nucleotides containing, for example, inosine; and the replacement of adenine and cytosine in oligonucleotides with guanine and uracil, respectively, to form GU Wobble base pairing with the target mRNA. In addition, other examples of modified nucleoside bases that can be used to generate RNAi activators include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7 -Methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl queosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. These modified nucleoside bases are all within the scope of this invention.

[0137] Examples of ribosome modifications that can be used to generate dsRNA activators include ribosome structures modified by replacing, for example, a hexose ring (HNA), a threonose ring (TNA), locked nucleic acid (LNA, a bicyclic ring with a bimolecular bridge between the C2 and C4 carbons on the ribosome), or a non-locked nucleic acid (UNA, a ribosome lacking a bond between the C2 and C3 carbons). Examples of usable sugar-modified nucleosides also include, for example, bicyclic hexose nucleic acids (WO 2011 / 017521) or tricyclic nucleic acids (WO 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar portion is replaced by a non-sugar portion, such as in the case of peptide nucleic acids (PNA) or morpholino nucleic acids. Sugar modification also includes modifications by replacing the naturally occurring 2'-OH group on the ribosome ring of the RNA nucleoside with other groups. Furthermore, substituents can be introduced, for example, at the 2', 3', 4', or 5' positions of the sugar ring.

[0138] In some embodiments, the dsRNA activator of the present invention may comprise a 2' sugar-modified nucleotide, such as a 2'-substituted nucleoside. Examples of 2'-substituted modified nucleosides are 2'-O-alkyl-RNA nucleoside, 2'-O-methyl-RNA nucleoside, 2'-alkoxy-RNA nucleoside, 2'-O-methoxyethyl-RNA nucleoside (MOE), 2'-amino-DNA nucleoside, 2'-fluoro-RNA nucleoside, and 2'-F-ANA nucleoside. Other examples may be found, for example, in Freier and Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213 and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. In some embodiments, the RNAi activator according to the present invention comprises at least one 2'-modified nucleotide. In some embodiments, the 2'-modification is selected from 2'-deoxy, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-allyl, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), and 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, the RNAi activator according to the invention comprises at least one 2'-modified nucleoside selected from the following: 2'-O-alkyl-RNA nucleoside, 2'-O-methyl-RNA nucleoside, 2'-alkoxy-RNA nucleoside, 2'-O-methoxyethyl-RNA nucleoside (MOE), 2'-amino-DNA nucleoside, 2'-fluoro-RNA nucleoside, and 2'-F-ANA nucleoside.

[0139] In some embodiments, the dsRNA activator according to the invention may optionally also comprise a chemical modification at the 5' and / or 3' ends, i.e., a non-nucleotide or nucleoside chemical moiety linked to the end of the oligonucleotide chain (sense and / or antisense strand) of RNAi. Examples of chemical moieties linked to the 3' end of the oligonucleotide chain can be found, for example, in WO 2005 / 021749 and WO 2007 / 128477. Examples of chemical moieties linked to the 5' end of the oligonucleotide chain may include, but are not limited to, 5'-terminal phosphate ester modifications, such as 5'-(E)-vinylphosphonate (5'-(E)-VP), 5'-methylphosphonate (5'-MP), (S)-5'-C-methyl analogues, and 5'-thiophosphate (5'-PS).

[0140] In some embodiments, at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, 2'-5'-linked ribonucleotides (3'-RNA), unlocked nucleotides, conformation-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'- O-alkyl modified nucleotides, morpholinonucleotides, aminophosphates, nucleotides including non-natural bases, tetrahydropyran modified nucleotides, 1,5-dehydrohexyl modified nucleotides, cyclohexenyl modified nucleotides, nucleotides including thiophosphate groups (e.g., nucleosides containing 5'-thiophosphate groups), nucleotides including methylphosphonate groups, nucleotides including 5'-phosphates, nucleotides including 5'-phosphate mimics, vinyl-phosphonate nucleotides, heat-labile nucleotides, ethylene glycol modified nucleotides (GNA), nucleotides including 2'-phosphates and 2-O-(N-methylacetamide) modified nucleotides; and combinations thereof.

[0141] In some embodiments, at least one of the modified nucleotides in the dsRNA activator is selected from the group consisting of: nonlocked nucleotides (UNA), locked nucleotides (LNA), HNA, threonucleotides (TNA), CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, and ethylene glycol; and combinations thereof. In some embodiments, at least one of the modified nucleotides in the dsRNA activator is selected from the group consisting of: deoxynucleotides, 2′-O-methyl modified nucleotides, 2′-fluoro modified nucleotides, 2′-deoxy modified nucleotides, nucleotides comprising 2′ phosphate groups, and nucleotides comprising thiophosphate groups; and combinations thereof.

[0142] In some embodiments, the nucleotides in the antisense strand of the dsRNA activator of the present invention comprise 2'-methoxy (2'-O-methyl) modified nucleotides, for example, all nucleotides are 2'-methoxy modified nucleotides or 1-21 nucleotides are 2'-methoxy modified nucleotides, for example, 16 or 17 nucleotides are 2'-methoxy modified nucleotides. In some embodiments, the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 from the 5' end of the antisense strand of the dsRNA activator of the present invention are 2'-methoxy modified nucleotides. In some embodiments, the nucleotides at positions 1, 3-6, 8-11, 13, 15, and 17-21 from the 5' end of the antisense strand of the dsRNA activator of the present invention are 2'-methoxy modified nucleotides. In some embodiments, the positive strand of the dsRNA activator of the present invention contains 2'-methoxy (2'-O-methyl) modified nucleotides, for example, all nucleotides are 2'-methoxy modified nucleotides or 1-19 nucleotides are 2'-methoxy modified nucleotides, for example, 15 or 16 nucleotides are 2'-methoxy modified nucleotides. In some embodiments, the positive strand of the dsRNA activator of the present invention has nucleotides 1-6 and 10-19 from the 5' end of the positive strand of the positive strand of the dsRNA activator of the present invention having nucleotides 1-6 and 11-19 from the 5' end of the positive strand of the positive strand of the dsRNA activator of the present invention having nucleotides 2'-methoxy modified nucleotides.

[0143] In some embodiments, the antisense strand of the dsRNA activator of the present invention has nucleotides 1, 3-5, 7-13, 15 and 17-21 from the 5' end that are 2'-methoxy modified nucleotides, and the sense strand has nucleotides 1-6 and 10-19 from the 5' end that are 2'-methoxy modified nucleotides.

[0144] In some embodiments, the nucleotides at positions 1, 3-6, 8-11, 13, 15, and 17-21 of the antisense strand of the dsRNA activator of the present invention, starting from the 5' end, are 2'-methoxy modified nucleotides, and the nucleotides at positions 1-6 and 11-19 of the sense strand, starting from the 5' end, are 2'-methoxy modified nucleotides.

[0145] In some embodiments, the antisense strand of the dsRNA activator of the present invention contains 2'-fluorinated nucleotides, for example, 1-5 nucleotides are 2'-fluorinated nucleotides, for example, 4 or 5 nucleotides are 2'-fluorinated nucleotides. In some embodiments, the 2nd, 6th, 14th, and 16th nucleotides from the 5' end of the antisense strand of the dsRNA activator of the present invention are 2'-fluorinated nucleotides. In some embodiments, the 2nd, 7th, 12th, 14th, and 16th nucleotides from the 5' end of the antisense strand of the dsRNA activator of the present invention are 2'-fluorinated nucleotides. In some embodiments, the sense strand of the dsRNA activator of the present invention contains 2'-fluorinated nucleotides, for example, 1-5 nucleotides are 2'-fluorinated nucleotides, for example, 3 or 4 nucleotides are 2'-fluorinated nucleotides. In some embodiments, the 7th-9th or 7th-10th nucleotides from the 5' end of the sense strand of the dsRNA activator of the present invention are 2'-fluorinated nucleotides.

[0146] In some embodiments, in the dsRNA activator of the present invention, the nucleotides at positions 2, 6, 14 and 16 of the antisense strand, counting from the 5' end, are 2'-fluorinated nucleotides, and the nucleotides at positions 7-9 of the sense strand, counting from the 5' end, are 2'-fluorinated nucleotides.

[0147] In some embodiments, in the dsRNA activator of the present invention, the nucleotides at positions 2, 7, 12, 14 and 16 of the antisense strand, counting from the 5' end, are 2'-fluorinated nucleotides, and the nucleotides at positions 7-10 of the sense strand, counting from the 5' end, are 2'-fluorinated nucleotides.

[0148] In some embodiments, one or more nucleotides of the dsRNA activator are linked by a non-standard bond or backbone (i.e., a modified nucleotide bond or a modified backbone). In some embodiments, the modified nucleotide bond is a covalent nucleotide bond containing a non-phosphate group. In some embodiments, the modified nucleoside internucleotide bond or skeleton includes, but is not limited to: a 5'-thiophosphate group (represented herein as lowercase "s"), a chiral thiophosphate, a thiophosphate, a dithiophosphate, a triphosphate, an aminoalkyl phosphate triester, an alkylphosphonate (e.g., a methylphosphonate or a 3'-alkylenephosphonate), a chiral phosphonate, a hypophosphonate, a phosphoramide (e.g., a 3'-aminophosphoramide, an aminoalkylphosphoramide, or a thiophosphoramide), a thioalkyl-phosphonate, a thioalkyl phosphate, a morpholino bond, a borophosphate having a normal 3'-5' bond, a borophosphate analog having a 2'-5' bond, or a borophosphate having an antipolarity wherein adjacent nucleoside unit pairs are 3'-5' to 5'-3' or 2'-5'-2' bonds. In some embodiments, the modified nucleoside internucleotide bond or skeleton does not contain a phosphorus atom. In some embodiments, the modified nucleoside interbonds that do not contain phosphorus atoms include, but are not limited to: short-chain alkyl or cycloalkyl sugar interbonds, mixed heteroatom and alkyl or cycloalkyl sugar interbonds, or one or more short-chain heteroatom or heterocyclic sugar interbonds. In some embodiments, the modified nucleoside interskeletons include, but are not limited to: siloxane skeletons, sulfide skeletons, sulfoxide skeletons, sulfone skeletons, formylacetyl and thioformylacetyl skeletons, methyleneformylacetyl and thioformylacetyl skeletons, olefin-containing skeletons, aminosulfonic acid skeletons, methyleneimine and methylenehydrazine skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and other skeletons having mixed N, O, S, and CH2 components.

[0149] In some embodiments, the sense strand of the dsRNA activator may contain 1, 2, 3, 4, 5, or 6 phosphate-thioester bonds. In some embodiments, the antisense strand of the dsRNA activator may contain 1, 2, 3, 4, 5, or 6 phosphate-thioester bonds. In some embodiments, both the sense and antisense strands may independently contain 1, 2, 3, 4, 5, or 6 phosphate-thioester bonds. In some embodiments, the sense strand of the dsRNA activator may contain 1, 2, 3, or 4 phosphate-thioester bonds. In some embodiments, the antisense strand of the dsRNA activator may contain 1, 2, 3, or 4 phosphate-thioester bonds. In some embodiments, both the sense and antisense strands may independently contain 1, 2, 3, or 4 phosphate-thioester bonds.

[0150] In some embodiments, the sense strand of the dsRNA activator may contain 1, 2, 3, 4, 5, or 6 phosphate-thioester bonds, and the antisense strand of the dsRNA activator may contain 1, 2, 3, 4, 5, or 6 phosphate-thioester bonds, and both the sense and antisense strands may independently contain 1, 2, 3, 4, 5, or 6 phosphate-thioester bonds. In some embodiments, the sense strand of the dsRNA activator may contain 1, 2, 3, or 4 phosphate-thioester bonds, and the antisense strand of the dsRNA activator may contain 1, 2, 3, or 4 phosphate-thioester bonds, and both the sense and antisense strands may independently contain 1, 2, 3, or 4 phosphate-thioester bonds.

[0151] In some embodiments, the dsRNA activator's sense strand contains two phosphate-thioester nucleoside bonds. In some embodiments, these two phosphate-thioester nucleoside bonds are located between nucleotides at positions 1-3 starting from the 5' end of the sense strand. In some embodiments, the dsRNA activator's antisense strand contains four phosphate-thioester nucleoside bonds. In some embodiments, these four phosphate-thioester nucleoside bonds are located between nucleotides at positions 1-3 starting from the 5' end and between nucleotides at positions 1-3 starting from the 3' end of the antisense strand. In some embodiments, the dsRNA activator contains two phosphate-thioester nucleoside bonds in the sense strand and four phosphate-thioester nucleoside bonds in the antisense strand. In some embodiments, the dsRNA activator contains two phosphate-thioester nucleoside bonds between nucleotides at positions 1-3 starting from the 5' end in the sense strand and four phosphate-thioester nucleoside bonds between nucleotides at positions 1-3 starting from the 5' end and 1-3 starting from the 3' end in the antisense strand.

[0152] Other modifications of the dsRNA activator can be found in, for example, those listed in WO2023044094A1, WO2023245060A2, or WO2018 / 027106, the entire contents of which are incorporated herein by reference.

[0153] In some implementations, the modified nucleotides in the sense and antisense strands of the dsRNA activator have the following modification patterns:

[0154] antisense chain:

[0155] NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm; and / or

[0156] Chain of Justice:

[0157] NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm

[0158] in,

[0159] Nf = any 2'-fluorine modified nucleotide

[0160] Nfs = any 2'-fluorine modified nucleoside-3' thiophosphate;

[0161] Nm = any 2'-methoxynucleotide;

[0162] Nms = any 2'-methoxynucleoside-3'-thiophosphate;

[0163] 's' indicates that the two nucleotides are linked by a phosphate thioester bond.

[0164] In some implementations, the modified nucleotides in the sense and antisense strands of the dsRNA activator have the following modification patterns:

[0165] antisense chain:

[0166] NmsNfsNmNmNmNmNfNmNmNmNmNfNmNfNmNfNmNmNmsNmsNm; and / or

[0167] Chain of Justice:

[0168] NmsNmsNmNmNmNmNfNfNfNfNmNmNmNmNmNmNmNmNm;

[0169] in,

[0170] Nf = any 2'-fluorine modified nucleotide

[0171] Nfs = any 2'-fluorine modified nucleoside-3' thiophosphate;

[0172] Nm = any 2'-methoxynucleotide;

[0173] Nms = any 2'-methoxynucleoside-3'-thiophosphate;

[0174] 's' indicates that the two nucleotides are linked by a phosphate thioester bond.

[0175] In some embodiments, specific modification patterns of the present invention are particularly suitable for the dsRNA activators of the present disclosure. For example, dsRNA activators of the present invention with specific modification patterns exhibit superior levels of inhibition against the target gene INHBE mRNA in vivo compared to control siRNA, although their levels of inhibition against the target gene INHBE mRNA are lower than those against control siRNA in in vitro screening. In some embodiments of the dsRNA activators of the present disclosure, specific nucleotide sequences of the sense and antisense strands combined with specific modification patterns (optionally also including ligands of the present disclosure) yield dsRNA activators having one or more of the following characteristics:

[0176] (i) It exhibits superior inhibition of INHBE mRNA expression in vivo compared to control siRNAs (e.g., AD-1708473 or AD-1708473.1 in WO2023003922A1);

[0177] (ii) It has a long-lasting effect on INHBE gene knockdown and INHBE mRNA expression inhibition in vivo, for example, it still inhibits the target gene INHBE 1, 2, 3 or 5 weeks after administration.

[0178] (iii) It has superior therapeutic effects in vivo compared to control siRNAs (e.g., AD-1708473 or AD-1708473.1 in WO2023003922A1), such as reducing weight and / or body fat;

[0179] (iv) It has a long-lasting therapeutic effect in vivo, such as a therapeutic effect on obesity, such as the ability to maintain a long-lasting effect on reducing the patient's weight and / or body fat; in some embodiments, it can still effectively treat the patient's obesity 1 week, 2 weeks, 3 weeks or 60 days after the last administration, such as maintaining the patient's weight and / or body fat, or reducing the patient's weight and / or body fat.

[0180] In some embodiments, the antisense strand of the dsRNA activator comprises any modified nucleotide sequence of the antisense strand in Table 2 of the specification, and / or the sense strand comprises any modified nucleotide sequence of the sense strand in Table 2 of the specification.

[0181] In some implementations, the combination of modified antisense and sense strands in the dsRNA activator is shown in Table 2 as any combination of antisense and sense strands.

[0182] In some embodiments, the dsRNA activator is any of the dsRNA activators shown in Table 2.

[0183] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand respectively comprise SEQ ID NO:331 / 441, SEQ ID NO:332 / 442, SEQ ID NO:333 / 443, SEQ ID NO:334 / 444, SEQ ID NO:335 / 445, SEQ ID NO:336 / 446, SEQ ID NO:337 / 447, SEQ ID NO:338 / 448, SEQ ID NO:339 / 449, SEQ ID NO:340 / 450, SEQ ID NO:341 / 451, SEQ ID NO:342 / 452, SEQ ID NO:343 / 453, SEQ ID NO:344 / 454, SEQ ID NO:345 / 455, SEQ ID NO:346 / 456, SEQ ID NO:347 / 457, SEQ ID NO:348 / 458, SEQ ID NO:347 / 457, SEQ ID NO:348 / 458, SEQ ID NO:331 / 441, SEQ ID NO:332 / 442, SEQ ID NO:333 / 443, SEQ ID NO:334 / 444, SEQ ID NO:335 / 457, SEQ ID NO:348 / 458, SEQ ID NO:347 / 457, SEQ ID NO:348 / 458, SEQ ID NO:349 / 449, SEQ ID NO:340 / 450, SEQ ID NO:341 / 451, SEQ ID NO:342 / 452, SEQ ID NO:343 / 453, SEQ ID NO:344 / 454, SEQ ID NO:345 / 455, SEQ ID NO:346 / 456, SEQ ID NO:347 NO:349 / 459, SEQ ID NO:350 / 460, SEQ ID NO:351 / 461, SEQ ID NO:352 / 462, SEQ ID NO:353 / 463, SEQ ID NO:354 / 464, SEQ ID NO:355 / 465, SEQ ID NO:356 / 466, SEQ ID NO:357 / 467, SEQ ID NO:358 / 468, SEQ ID NO:359 / 469, SEQ ID NO:360 / 470, SEQ ID NO:361 / 471, SEQ ID NO:362 / 472, SEQ ID NO:363 / 473, SEQ ID NO:364 / 474, SEQ ID NO:365 / 475, SEQ ID NO:366 / 476, SEQ ID NO:367 / 477、SEQ ID NO:368 / 478、SEQ ID NO:369 / 479, SEQ ID NO:370 / 480, SEQ ID NO:371 / 481, SEQ ID NO:372 / 482, SEQ ID NO:373 / 483, SEQ ID NO:374 / 484, SEQ ID NO:375 / 485, SEQ ID NO:376 / 486, SEQ ID NO:377 / 487, SEQ ID NO:378 / 488, SEQ ID NO:379 / 489, SEQ ID NO:380 / 490, SEQ ID NO:381 / 491, SEQ ID NO:382 / 492, SEQ IDNO:383 / 493、SEQ ID NO:384 / 494、SEQ ID NO:385 / 495、SEQ ID NO:386 / 496、SEQ ID NO:387 / 497、SEQ ID NO:388 / 498、SEQ ID NO:389 / 499、SEQ ID NO:390 / 500、SEQ ID NO:391 / 501、SEQ ID NO:392 / 502、SEQ ID NO:393 / 503、SEQ ID NO:394 / 504、SEQ ID NO:395 / 505、SEQ ID NO:396 / 506、SEQ ID NO:397 / 507、SEQ ID NO:398 / 508、SEQ ID NO:399 / 509、SEQ ID NO:400 / 510、SEQ ID NO:401 / 511、SEQ ID NO:402 / 512、SEQ ID NO:403 / 513、SEQ ID NO:404 / 514、SEQ ID NO:405 / 515、SEQ ID NO:406 / 516、SEQ ID NO:407 / 517、SEQ ID NO:408 / 518、SEQ ID NO:409 / 519、SEQ ID NO:410 / 520、SEQ ID NO:411 / 521、SEQ ID NO:412 / 522、SEQ ID NO:413 / 523、SEQ ID NO:414 / 524、SEQ ID NO:415 / 525、SEQ ID NO:416 / 526、SEQ ID NO:417 / 527、SEQ ID NO:418 / 528、SEQ ID NO:419 / 529、SEQ ID NO:420 / 530、SEQ ID NO:421 / 531、SEQ ID NO:422 / 532、SEQ ID NO:423 / 533、SEQ ID NO:424 / 534、SEQ ID NO:425 / 535、SEQ ID NO:426 / 536、SEQ ID NO:427 / 537、SEQ ID NO:428 / 538、SEQ ID NO:429 / 539、SEQ ID NO:430 / 540、SEQ ID NO:431 / 541、SEQ ID NO:432 / 542、SEQ ID NO:433 / 543、SEQ ID NO:434 / 544、SEQ ID NO:435 / 545、SEQ ID NO:436 / 546、SEQ ID NO:437 / 547、SEQ IDAt least 15, 16, 17, 18, or 19 consecutive modified nucleotides in the nucleotide sequences shown in NO:438 / 548, SEQ ID NO:439 / 549, SEQ ID NO:440 / 550, and SEQ ID NO:661 / 662, optionally the antisense strand also having a 3' overhang of 1 or 2 nucleotides, optionally the first nucleotide at the 5' end of the antisense strand being a modified A or U, such as a modified U, such as Um, or Am, such as Um.

[0184] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand respectively comprise SEQ ID NO:331 / 441, SEQ ID NO:332 / 442, SEQ ID NO:333 / 443, SEQ ID NO:334 / 444, SEQ ID NO:335 / 445, SEQ ID NO:336 / 446, SEQ ID NO:337 / 447, SEQ ID NO:338 / 448, SEQ ID NO:339 / 449, SEQ ID NO:340 / 450, SEQ ID NO:341 / 451, SEQ ID NO:342 / 452, SEQ ID NO:343 / 453, SEQ ID NO:344 / 454, SEQ ID NO:345 / 455, SEQ ID NO:346 / 456, SEQ ID NO:347 / 457, SEQ ID NO:348 / 458, SEQ ID NO:347 / 457, SEQ ID NO:348 / 458, SEQ ID NO:331 / 441, SEQ ID NO:332 / 442, SEQ ID NO:333 / 443, SEQ ID NO:334 / 444, SEQ ID NO:335 / 457, SEQ ID NO:348 / 458, SEQ ID NO:347 / 457, SEQ ID NO:348 / 458, SEQ ID NO:349 / 449, SEQ ID NO:340 / 450, SEQ ID NO:341 / 451, SEQ ID NO:342 / 452, SEQ ID NO:343 / 453, SEQ ID NO:344 / 454, SEQ ID NO:345 / 455, SEQ ID NO:346 / 456, SEQ ID NO:347 NO:349 / 459, SEQ ID NO:350 / 460, SEQ ID NO:351 / 461, SEQ ID NO:352 / 462, SEQ ID NO:353 / 463, SEQ ID NO:354 / 464, SEQ ID NO:355 / 465, SEQ ID NO:356 / 466, SEQ ID NO:357 / 467, SEQ ID NO:358 / 468, SEQ ID NO:359 / 469, SEQ ID NO:360 / 470, SEQ ID NO:361 / 471, SEQ ID NO:362 / 472, SEQ ID NO:363 / 473, SEQ ID NO:364 / 474, SEQ ID NO:365 / 475, SEQ ID NO:366 / 476, SEQ ID NO:367 / 477、SEQ ID NO:368 / 478、SEQ ID NO:369 / 479, SEQ ID NO:370 / 480, SEQ ID NO:371 / 481, SEQ ID NO:372 / 482, SEQ ID NO:373 / 483, SEQ ID NO:374 / 484, SEQ ID NO:375 / 485, SEQ ID NO:376 / 486, SEQ ID NO:377 / 487, SEQ ID NO:378 / 488, SEQ ID NO:379 / 489, SEQ ID NO:380 / 490, SEQ ID NO:381 / 491, SEQ ID NO:382 / 492, SEQ IDNO:383 / 493、SEQ ID NO:384 / 494、SEQ ID NO:385 / 495、SEQ ID NO:386 / 496、SEQ ID NO:387 / 497、SEQ ID NO:388 / 498、SEQ ID NO:389 / 499、SEQ ID NO:390 / 500、SEQ ID NO:391 / 501、SEQ ID NO:392 / 502、SEQ ID NO:393 / 503、SEQ ID NO:394 / 504、SEQ ID NO:395 / 505、SEQ ID NO:396 / 506、SEQ ID NO:397 / 507、SEQ ID NO:398 / 508、SEQ ID NO:399 / 509、SEQ ID NO:400 / 510、SEQ ID NO:401 / 511、SEQ ID NO:402 / 512、SEQ ID NO:403 / 513、SEQ ID NO:404 / 514、SEQ ID NO:405 / 515、SEQ ID NO:406 / 516、SEQ ID NO:407 / 517、SEQ ID NO:408 / 518、SEQ ID NO:409 / 519、SEQ ID NO:410 / 520、SEQ ID NO:411 / 521、SEQ ID NO:412 / 522、SEQ ID NO:413 / 523、SEQ ID NO:414 / 524、SEQ ID NO:415 / 525、SEQ ID NO:416 / 526、SEQ ID NO:417 / 527、SEQ ID NO:418 / 528、SEQ ID NO:419 / 529、SEQ ID NO:420 / 530、SEQ ID NO:421 / 531、SEQ ID NO:422 / 532、SEQ ID NO:423 / 533、SEQ ID NO:424 / 534、SEQ ID NO:425 / 535、SEQ ID NO:426 / 536、SEQ ID NO:427 / 537、SEQ ID NO:428 / 538、SEQ ID NO:429 / 539、SEQ ID NO:430 / 540、SEQ ID NO:431 / 541、SEQ ID NO:432 / 542、SEQ ID NO:433 / 543、SEQ ID NO:434 / 544、SEQ ID NO:435 / 545、SEQ ID NO:436 / 546、SEQ ID NO:437 / 547、SEQ IDThe modified nucleotide sequences shown in NO:438 / 548, SEQ ID NO:439 / 549, SEQ ID NO:440 / 550, and SEQ ID NO:661 / 662, or each of the modified nucleotide sequences shown.

[0185] In some specific implementations, the dsRNA activator comprises a sense strand and an antisense strand, wherein

[0186] The sense strand comprises or is composed of the modified nucleotide sequence shown in SEQ ID NO:390, and the antisense strand comprises or is composed of the modified nucleotide sequence shown in SEQ ID NO:500.

[0187] The sense strand comprises or is composed of the modified nucleotide sequence shown in SEQ ID NO:431, and the antisense strand comprises or is composed of the modified nucleotide sequence shown in SEQ ID NO:541.

[0188] The sense strand comprises or consists of the modified nucleotide sequence shown in SEQ ID NO:661, and the antisense strand comprises or consists of the modified nucleotide sequence shown in SEQ ID NO:662.

[0189] As used herein (e.g., in Tables 1, 2, and 3), the following symbols are used to denote modified nucleotides, targeting groups, and linking groups. Unless otherwise specified in the sequence, it will be readily understood by those skilled in the art that, when present in oligonucleotides, these monomers are interconnected by 5'-3'-phosphodiester bonds (where 's' represents a phosphothioester bond connecting the two nucleotides when the first nucleotide of the two nucleotides is a phosphothioester (s)):

[0190] Nf = any 2'-fluorine modified nucleotide

[0191] Nfs = any 2'-fluorine-modified nucleoside-3'-thiophosphate ester

[0192] Af = 2'-Fluoroadenosine-3'-phosphate

[0193] Afs = 2'-Fluoroadenosine-3'-Thiophosphate

[0194] Cf = 2'-Fluorocytidine-3'-phosphate

[0195] Cfs = 2'-Fluorocytidine-3'-Thiophosphate

[0196] Gf = 2'-Fluoroguanosine-3'-phosphate

[0197] Gfs = 2'-Fluoroguanosine-3'-Thiophosphate

[0198] Tf = 2'-Fluoro-5'-methyluridine-3'-phosphate

[0199] Tfs = 2'-Fluoro-5'-methyluridine-3'-thiophosphate

[0200] Uf = 2'-fluorouridine-3'-phosphate

[0201] Ufs = 2'-fluorouridine-3'-thiophosphate

[0202] Nm = any 2'-methoxynucleotide

[0203] Nms = any 2'-methoxynucleoside-3'-thiophosphate

[0204] Am = 2'-methoxyadenosine-3'-phosphate

[0205] Ams = 2'-methoxyadenosine-3'-thiophosphate

[0206] Tm = 2'-methoxythymidine-3'-phosphate

[0207] Tms = 2'-methoxythymidine-3'-thiophosphate

[0208] Um = 2'-methoxyuridine-3'-phosphate

[0209] Ums = 2'-methoxyuridine-3'-thiophosphate

[0210] Gm = 2'-methoxyguanosine-3'-phosphate

[0211] Gms = 2'-methoxyguanosine-3'-thiophosphate

[0212] Cm = 2'-methoxycytidine-3'-phosphate

[0213] Cms = 2'-methoxycytidine-3'-thiophosphate.

[0214] In some embodiments, the dsRNA activator of the present invention further includes a ligand. When the present invention refers to "dsRNA" or "dsRNA activator," it also encompasses dsRNA conjugated with a ligand, also known as a dsRNA-ligand conjugate.

[0215] As described herein, a "ligand" refers to a chemical moiety conjugated to an oligonucleotide of dsRNA that can alter the distribution, targeting, or half-life of the dsRNA. In a preferred embodiment, such a ligand provides enhanced affinity for selected targets (e.g., molecules, cells or cell types, compartments (e.g., cell or organ compartments, tissues, organs, or regions of the body) compared to, for example, dsRNA without the ligand.

[0216] In some implementations, ligands modulate or enhance the pharmacokinetic properties of dsRNA by improving the cellular distribution, bioavailability, metabolism, excretion, permeability, and / or cellular uptake of nucleotides. Specifically, ligands can direct oligonucleotides to specific organs, tissues, or cell types and thus enhance the effectiveness of dsRNA in such organs, tissues, or cell types. Simultaneously, ligands can reduce the activity of dsRNA in non-target cell types, tissues, or organs (e.g., off-target activity or activity in non-target cell types, tissues, or organs). Regarding ligands and conjugation modifications suitable for dsRNA, see Vajinder Kumar, Targeted delivery of oligonucleotides using multivalent protein–carbohydrate interactions, Cite this: Chem. Soc. Rev., 2023, 52, 1273; Rosemary Kanasty, Delivery materials for siRNA therapeutics, NATURE MATERIALS, VOL 12, NOVEMBER 2013; Wanyi Tai, Current Aspects of siRNA Bioconjugate for In Vitro and In Vivo Delivery, Molecules 2019, 24, 2211; doi:10.3390 / molecules24122211; and WO 93 / 07883, WO Descriptions are provided in 2013 / 033230, WO2023044094A1, WO2023245060A2, WO2018 / 027106, WO2012083185A2, WO2015021092, WO2018044350A1, WO2024 / 197017A2 or WO2023 / 076451A1, which are incorporated herein by reference.

[0217] In some embodiments, the ligands used in the RNAi activator of the present invention, such as the dsRNA activator, may be selected from sugars, cell surface receptor ligands, antibodies, drugs, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

[0218] In some embodiments, the ligands used in the RNAi activator of the present invention, such as the dsRNA activator, are sugars, including but not limited to galactose, lactose, N-acetylgalactosamine, mannose, and mannose-6-phosphate. Sugar ligands can be used to enhance delivery or activity in a range of tissues such as the liver and / or muscle.

[0219] In some implementations, the ligand is a monosaccharide.

[0220] In some embodiments, the ligand, for example, a monosaccharide, is N-acetylgalactosamine (GalNAc) or a derivative thereof. GalNAc ligands comprising one or more N-acetylgalactosamine (GalNAc) or derivatives thereof are described, for example, in US 8,106,022, the full contents of which are hereby incorporated herein by reference. In some embodiments, GalNAc ligands are used as ligands to target dsRNA activators to specific cells. In some embodiments, GalNAc ligands target dsRNA to hepatocytes, for example, by acting as ligands for desialylate glycoprotein receptors of hepatocytes (e.g., hepatocytes). Exemplary GalNac ligands suitable for delivering dsRNA include, for example, Alnylam's three-touch GalNAc delivery system (see, for example, PCT / US2008 / 085574, US8828956B2) or "(1+1+1) trivalent GalNAc" (see, for example, US986788B2, Rajeev, Kallanthottathil G et al. "Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo." Chembiochem: a European journal of chemical biology vol. 16, 6(2015): 903-8. doi: 10.1002 / cbic.201500023); Dicerna's GalXC TM Platform or GalXC-PlusTM (Involving tetraloop structures with unit-priced GalNAc coupling) (see, for example, WO2016100401A1), or Arrowhead's Dynamic PolyConjugates (DPC) TM It contains polymers of butyl aminovinyl ether (PBAVE) or TriM TM (Targeted RNNI molecule). All cited references are included in full in this paper.

[0221] Commonly used ligands conjugated with dsRNA can also be found in, for example, those disclosed in WO2023044094A1, WO2023245060A2, or WO2018 / 027106, or those disclosed in WO2012083185A2, WO2015021092, WO2018044350A1, WO2024 / 197017A2, or WO2023 / 076451A1, the full contents of which are hereby incorporated herein by reference.

[0222] In some embodiments, the ligand used for the RNAi activator of the present invention, such as the dsRNA activator, is an asialic acid glycoprotein receptor (ASPGR) ligand. Specifically, the ASPGR ligand comprises a moiety selected from galactose or galactose derivatives (e.g., galactosamine, N-formylgalactosamine, N-acetylgalactosamine (GalNAc), N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, etc.). The ASPGR ligand may or may not have a linker group (also referred to as a "connector").

[0223] In this document, when the galactose derivative in the ligand is N-acetylgalactosamine (GalNAc) or a GalNAc derivative, the ligand is also referred to as a GalNAc ligand, encompassing monovalent, divalent, trivalent, or tetravalent GalNAc ligands capable of providing 1, 2, 3, or 4 structural moieties of GalNAc or GalNAc derivatives.

[0224] In some embodiments, in the dsRNA activator formed by the dsRNA oligonucleotide of the present invention and a ligand containing a galactose derivative (e.g., GalNAc) as a targeting group, the molar ratio of the dsRNA oligonucleotide to the galactose derivative (e.g., GalNAc) can be any suitable ratio, for example, 1:1.

[0225] In some embodiments, the dsRNA activator of the present invention comprises GalNAc or GalNAc derivatives linked to an oligonucleotide of dsRNA. In some embodiments, the dsRNA activator of the present invention comprises one or more (e.g., two, three, four, five, or six) GalNAc or GalNAc derivatives, each of which is independently linked to multiple nucleotides of the dsRNA activator via multiple monovalent linkers.

[0226] In some embodiments, the ligand is an N-acetylgalactosamine (GalNAc) derivative. In some embodiments, the ligand is one or more GalNAc derivatives linked via monovalent, divalent, or trivalent branched linkers. In some embodiments, the dsRNA, for example, its nucleotides, are conjugated to a ligand portion containing N-acetylgalactosamine via a phosphate ester group or a thiophosphate ester group. In some embodiments, the ligand is conjugated to the 5' or 3' end of the sense and / or antisense strand of the dsRNA activator.

[0227] In some embodiments, the RNAi activator of the present invention, such as the dsRNA activator, comprises one or more GalNAc or GalNAc derivatives as ligands. The GalNAc or GalNAc derivative can be linked to the oligonucleotide of the RNAi via a linker group, such as a divalent, trivalent, or tetravalent branching point linker group. In some embodiments, the ligand is conjugated to the 5' or 3' end of the sense and / or antisense strand of the dsRNA activator. In some embodiments, the GalNAc ligand is conjugated to the 3' end of the sense strand of the RNAi. In some embodiments, the GalNAc ligand is linked to the 3' end of the sense strand of the RNAi oligonucleotide via a linker group. In some embodiments, the GalNAc ligand binds to the 5' end of the sense strand. In some embodiments, the GalNAc ligand is linked to the 5' end of the sense strand of the RNAi oligonucleotide via a linker group. In some embodiments, the GalNAc ligand binds to the 3' end of the antisense strand. In some embodiments, the GalNAc ligand is linked to the 3' end of the antisense oligonucleotide of the RNAi via a linker group. In some embodiments, GalNAc or a GalNAc derivative is linked to the oligonucleotide of the RNAi of the present invention via a divalent linker. In other embodiments, GalNAc or a GalNAc derivative is linked to the oligonucleotide of the RNAi of the present invention via a trivalent linker. In still other embodiments, GalNAc or a GalNAc derivative is linked to the oligonucleotide of the RNAi of the present invention via a tetravalent linker.

[0228] In some embodiments, one or more of GalNAc or GalNAc derivatives may be individually linked to the RNAi oligonucleotide via a linker group, independent of any other GalNAc or GalNAc derivative. In some embodiments, any two or more of GalNAc or GalNAc derivatives may be linked to the RNAi oligonucleotide in a tandem cluster via a common linker group moiety. Furthermore, dsRNA activators comprising multiple GalNAc or GalNAc derivatives linked thereto, either independently or in a tandem cluster, are also included in this invention. In some embodiments, the dsRNA oligonucleotide is conjugated to a ligand moiety comprising N-acetylgalactosamine via a phosphate ester group or a thiophosphate ester group.

[0229] In some embodiments, the ligand is a lipid or lipid-based molecule. In one embodiment, such a lipid or lipid-based molecule binds to a serum protein, such as human serum albumin (HSA). The HSA-binding ligand allows the conjugate to be distributed to a target tissue, such as a non-renal target tissue of the body. For example, the target tissue could be the liver, containing hepatic parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipids or lipid-based ligands can (a) increase resistance to conjugate degradation, (b) increase targeting or transport to target cells or cell membranes, or (c) be used to modulate binding to serum proteins (e.g., HSA). In some embodiments, the ligand is a lipid nanoparticle, such as a lipid nanoparticle (LNP). Exemplary LNP delivery systems include DLin-DMA, DLin-MC3-DMA, L319, PNP (peptide nanoparticles) delivery platforms, and the EDV (EnGeneIC Dream Vector) endogenous delivery carrier nanocell platform.

[0230] In some implementations, antibodies can also be used as ligands for the delivery of dsRNA, such as antibody-oligonucleotide conjugates (AOCs).

[0231] In some implementations, polymer matrix copolymerization can also be used to deliver dsRNA, such as the LODER (Local Drug EluteR) delivery platform.

[0232] In some embodiments, a ligand suitable for the dsRNA activator of the present invention is attached to the 5' and / or 3' terminal nucleotide of the sense strand and optionally the 5' and / or 3' terminal nucleotide of the antisense strand of the dsRNA oligonucleotide of the present invention, optionally via a phosphate ester group or a phosphate ester group. In some embodiments, the dsRNA activator is conjugated to the ligand via a phosphate ester group or a phosphate ester group, for example, the phosphate ester nucleotide bond is located at the 3' end of the sense strand or antisense strand; or the phosphate ester nucleotide bond is located at the 5' end of the sense strand or antisense strand; or the phosphate ester nucleotide bond is located at both the 5' and 3' ends of the sense strand, and / or the phosphate ester nucleotide bond is located at both the 5' and 3' ends of the antisense strand.

[0233] In some cases, the ligands of the present invention can also be conjugated to the internal sequence of the oligonucleotide of dsRNA. In some embodiments, the ligand can be attached to the phosphate group, the 2′-hydroxyl group, or a base of the nucleotide. In other embodiments, the ligand can be attached to the 3′-hydroxyl group of the nucleotide, in which case the nucleotides are linked by a 2′-5′ phosphodiester bond. In some embodiments, when the ligand is attached to the end of the dsRNA (such as siRNA) nucleotide chain, the ligand is typically attached to the phosphate group of the nucleotide; when the ligand is attached to the internal sequence of the dsRNA (such as siRNA) nucleotide, the ligand is typically attached to the sugar ring of the ribose or a base.

[0234] The ligands of the present invention can be directly linked to the nucleotide double strand of the dsRNA of the present invention or linked via a linker portion (e.g., a adapter or linker group, such as a linker group contained in the ligand). In some embodiments, the adapter is a biocleavable adapter. In some embodiments, the adapter need not be biocleavable. In some embodiments, the adapter may include a branching region. Hereinafter, the term "branching region" means a compound portion capable of covalently coupling two or more entities together. In some embodiments, adapters having branching regions can be used to conjugate multiple entities, such as N-acetylgalactosamine moieties, to the oligonucleotide of the dsRNA of the present invention. Adapters having branching regions that can be used for this purpose are known in the art and include, but are not limited to, amino acids (including natural and non-natural amino acids), peptides and their derivatives, glycounits and their derivatives, aromatic-substituted compounds and their derivatives, substituted hydrocarbon groups and their derivatives, triazole-containing derivatives, etc. See, for example, CN104651408A, CN113286888A, WO2015 / 173208, and WO2023 / 076451. In some embodiments of the present invention, when referring to "ligand" or "ligand portion", it may also refer to a ligand or ligand portion that includes a connector.

[0235] In some embodiments, an RNAi activator, such as a dsRNA activator, is provided comprising one or more (e.g., one) ligands linked to the dsRNA oligonucleotide of the present invention, wherein each ligand independently has the structure of formula (I):

[0236] in,

[0237] Gal represents terminal galactose derivatives independently;

[0238] L indicates a connector;

[0239] n is an integer selected from 1, 2, 3, and 4; and

[0240] The wavy line indicates the oligonucleotide linked to the dsRNA of the present invention via this valence bond. It is understood that the ligand is linked to the 5' and / or 3' ends of the sense and / or antisense strands of the oligonucleotide of the dsRNA of the present invention, preferably via a phosphate ester bond or a thiophosphate ester bond.

[0241] In some embodiments, the connector L may or may not have branching components. In some embodiments, the branching components may be in the form of two antennas, three antennas, or other multi-branched shapes.

[0242] In some embodiments, each Gal is individually linked to the oligonucleotide of the dsRNA of the present invention via a linker L, independently of the other Gals. In some embodiments, Gals are linked to the oligonucleotide of the dsRNA of the present invention via linker L in a tandem cluster.

[0243] Therefore, in some embodiments, the ligands of formula (I) of the present invention each independently have the structure of formula (Ia):

[0244] in,

[0245] L A1 This indicates the connection used to link the Gal section to L. A2 Partial linker;

[0246] L A2 This indicates a 2-valent, 3-valent, 4-valent, or 5-valent connector, used to connect n Gal-L... A1 - Partially linked to the RNAi of this invention;

[0247] Gal independently represents terminal galactose derivatives; and

[0248] n is an integer selected from 1, 2, 3, and 4; and

[0249] The wavy line indicates the oligonucleotide linked to the dsRNA of the present invention via this valence bond. It will be understood that the ligand is linked to the 5' and / or 3' ends of the sense and / or antisense strands of the oligonucleotide of the dsRNA of the present invention, and optionally the 3' end of the antisense strand, preferably via a phosphate ester bond or a thiophosphate ester bond.

[0250] In the dsRNA activators of the present invention comprising formula (I) or its subforms such as (Ia), Gal independently represents GalNAc (N-acetylgalactosamine) or a GalNAc derivative. In some embodiments, Gal independently represents a galactose derivative moiety having the following structure:

[0251] in,

[0252] R1 is an H or hydroxyl protecting group,

[0253] R2 is selected from hydrogen, hydroxyl group, C 1-20 Alkyl, C 2-20 alkenyl, C 1-20 Alkoxy, C 1-20 Alkylthio, -NR a R b C 6-20 Aryl-C 0-8 Alkylene-O-, C 6-20 Aryl-C 0-8 Alkylene-S- and CH3O-(CH2CH2O) q -CH2CH2O-, where R a and R b Each is independently H or C 1-20 Alkyl, q represents an integer from 1 to 16, and wherein the aryl group is optionally surrounded by one or more C... 1-8 Alkyl substitution; and

[0254] A wavy valence bond indicates that the bond is connected to the rest of the molecule.

[0255] In this document, suitable hydroxyl protecting groups are known to those skilled in the art, including but not limited to acetyl (Ac), benzoyl (Bz), phenoxyacetyl, tertvalyl, monomethoxytriphenylmethyl (MMTr), dimethoxytriphenylmethyl (DMTr), isobutyryl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl and isopropyldimethylsilyl.

[0256] Therefore, in some embodiments, R1 is independently H, acetyl (Ac), benzoyl (Bz), monomethoxytriphenylmethyl (MMTr), dimethoxytriphenylmethyl (DMTr), or tert-butyldiphenylsilyl (TBDPS). Preferably, R1 is independently H, acetyl (Ac), benzoyl (Bz), dimethoxytriphenylmethyl (DMTr), or tert-butyldiphenylsilyl (TBDPS). In some embodiments, R1 is independently H or benzoyl (Bz). In some embodiments, R1 is independently H.

[0257] In some implementations, R2 is independently selected from: hydrogen, hydroxyl, C 1-6 Alkyl, C 2-6 alkenyl, C 1-16 Alkoxy groups (e.g., C) 1-6 alkoxy), C 1-6 Alkylthio, -NR a R b , phenyl-C 0-4 alkylene-O-, phenyl-C 0-4 Alkylene-S- and CH3O-(CH2CH2O) q -CH2CH2O-, where R a and R b Each is independently H or C 1-6 Alkyl groups such as C 1-4 Alkyl, q represents an integer from 1 to 12, and the phenyl group is optionally constituting one or more C atoms. 1-4 Alkyl substitution. In some embodiments, R2 is independently C10. 1-16 Alkoxy groups, such as C 1-6 Alkyl group.

[0258] In some implementations, R2 is independently H, OH,

[0259] Preferably, R2 is OH, More preferably, R2 is independently OH or in particular

[0260] In some implementations, Gal is represented independently. Preferred The variables are defined as described in this paper, and the wavy valence bond represents the connection to the rest of the molecule via this valence bond.

[0261] In some implementations, Gal is represented independently. Preferred The variables are defined as described in this paper, and the wavy valence bond represents the connection to the rest of the molecule via this valence bond.

[0262] In some implementations, Gal is represented independently. The wavy valence bond indicates that the valence bond is connected to the rest of the molecule.

[0263] In some implementations, in formula (Ia), L A1 Each can be independently represented as a linking base having the following structure:

[0264] -(CH2) m1 -C(O)-NH-(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0265] -(CH2) m1 -C(O)-NH-(CH2) m2 -C(O)-NH-(CH2) m3 -;

[0266] -(CH2) m1 -NH-C(O)-(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0267] -(CH2) m1 -NH-C(O)-(CH2) m2 -C(O)-NH-(CH2) m3 -;

[0268] -(CH2) m1 -O-(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0269] -(CH2CH2O) m4 -(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0270] Wherein, m1, m2, m3 and m4 are each independently 1, 2, 3, 4, 5, 6, 7 or 8; and the left side of the group is connected to Gal, and the right side is connected to the rest of the molecule.

[0271] In some implementations, m1 is preferably 3, 4, 5, 6, 7 or 8, more preferably 3, 4, 5 or 6, and most preferably 4.

[0272] In some implementations, m2 is preferably 1, 2, 3, 4, 5 or 6, more preferably 1, 2 or 3.

[0273] In some implementations, m3 is preferably 1, 2, 3, 4, 5 or 6, more preferably 1, 2 or 3, and most preferably 2.

[0274] In some implementations, m4 is preferably 1, 2, 3, 4, 5 or 6, more preferably 1, 2, 3 or 4, and most preferably 1 or 3.

[0275] In some implementations, L A1 Each of these independently represents a linker base selected from the following:

[0276] Wherein, m1, m2 and m4 are each independently 1, 2, 3, 4, 5, 6, 7 or 8; and wherein the 1 position of the group is connected to Gal, and the 2 position is connected to the rest of the molecule.

[0277] In some implementations, L A1 Each of these independently represents a linker base selected from the following:

[0278] Preferred is

[0279] The group is connected to Gal at position 1 and to the rest of the molecule at position 2.

[0280] In some implementations, L A2 This refers to a divalent, trivalent, or tetravalent connector comprising monohydroxymethylmethane, dihydroxymethylmethane, or trihydroxymethylmethane components, wherein the L... A2 via the oxygen atom in the hydroxymethyl group and L A1 The oligonucleotides are partially linked by ether bonds and are linked (directly or indirectly) to the dsRNA of the present invention via methane carbon atoms.

[0281] In some implementations, L A2 This indicates a joint having a structure selected from the following:

[0282] Where L A3 This indicates the absence of a spacer group; the oxygen atom on the left side of the group is related to L. A1 Partially linked via ether bonds, the right side is linked to an oligonucleotide of the dsRNA of this invention. The carbon atom marked with an asterisk can be considered as an L... A2 Some branch points.

[0283] In some implementations, L A3 Represents spacer bases with the following structure:

[0284] Preferred

[0285] Wherein, q is an integer selected from 1 to 16, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16; preferably, q is an integer selected from 1 to 12, for example, an integer selected from 8 to 12; the 1 position of the group is related to L. A2 The branch points in the part are connected, and the 2-position is connected to the oligonucleotide of the dsRNA of the present invention, for example, through a phosphate ester bond or a thiophosphate bond.

[0286] In some implementations, L A3 Represents spacer bases with the following structure:

[0287] Preferred

[0288] Wherein, the 1 position of the group is related to L A2 The branch points in the part are connected, and the 2-position is connected to the oligonucleotide of the dsRNA of the present invention, for example, through a phosphate ester bond or a thiophosphate bond.

[0289] In some implementations, the ligands of formula (I) or (Ia) each independently have the structure of formula (Ia-i):

[0290] In this document, each variable is defined as such, for example, as defined in formula (I) or (Ia); the wavy line indicates the oligonucleotide linked to the dsRNA via the valence bond. It is understood that the ligand is linked to the 5' and / or 3' ends of the sense and / or antisense strands of the oligonucleotide of the dsRNA of the present invention, preferably via a phosphate ester bond or a thiophosphate ester bond. In a preferred embodiment, R1 is independently H; m1 and m2 are independently 1, 2, 3, 4, 5, 6, 7, or 8, preferably 3 or 4; q is an integer selected from 1 to 16, preferably an integer selected from 1 to 12, more preferably an integer from 8 to 12, such as 8, 9, 10, 11, or 12.

[0291] In particular, the ligands of formula (I) or (Ia) are each independently an L96 moiety having the following structure:

[0292] The wavy line indicates the linking of the ligand to the oligonucleotide of the dsRNA via this valence bond. It is understood that the ligand is linked to the 5' and / or 3' end of the sense and / or antisense strands of the oligonucleotide of the dsRNA of the present invention, preferably via a phosphate ester bond or a thiophosphate ester bond. In some embodiments, the ligand is linked to the 3' end of the sense strand of the oligonucleotide of the dsRNA of the present invention.

[0293] In some embodiments, the dsRNA activators of the present invention having ligands of formula (Ia-i) are shown below:

[0294] in, The '3' represents the oligonucleotide double strand of the dsRNA described herein, '3' represents the 3' end of the RNAi positive strand, X represents oxygen or sulfur, and other variables are as defined herein.

[0295] In particular, the dsRNA activator of the present invention having a ligand with an L96 structure is shown below:

[0296] in, The '3' represents the oligonucleotide double strand of the dsRNA described herein, '3' represents the 3' end of the positive strand of the oligonucleotide of the dsRNA, and 'X' represents oxygen or sulfur.

[0297] In some embodiments, in the dsRNA activator of the present invention, the dsRNA oligonucleotide duplex is linked to an ASPGR ligand, wherein the ASPGR ligand has any structure selected from formula (I) or any subform thereof, for example, a structure selected from (Ia), (Ia-i), or L96, and the ASPGR ligand is linked to the 5' or 3' end of the sense and / or antisense strands of the oligonucleotide of the dsRNA of the present invention. Preferably, the ligand is linked via a phosphate ester bond or a thiophosphate ester bond.

[0298] In some embodiments, in the dsRNA activator of the present invention, the dsRNA oligonucleotide duplex is linked to an ASPGR ligand, wherein the ASPGR ligand has a structure of formula (Ia), preferably (Ia-i), more preferably L96, and the ASPGR ligand is linked to the 3' end of the sense strand of the oligonucleotide of the dsRNA of the present invention. Preferably, the ligand is linked via a phosphate ester bond or a thiophosphate ester bond.

[0299] In some embodiments, the ligand is L96, see, for example, WO2009073809 and WO2009082607, which are incorporated herein by reference in their entirety.

[0300] In some embodiments, in the dsRNA activator of the present invention, the dsRNA oligonucleotide double strand is linked to a GalNac ligand, such as a ligand having an L96 structure, and the ligand is linked to the 3' end of the sense strand of the oligonucleotide of the dsRNA of the present invention, wherein the ligand is linked by a phosphate ester bond or a thiophosphate ester bond (preferably a phosphate ester bond).

[0301] In some embodiments, the antisense strand of the dsRNA activator comprises any modified nucleotide sequence of the antisense strand in Table 2 of the specification, and / or the sense strand comprises any modified nucleotide sequence of the sense strand in Table 2 of the specification, wherein the sense strand is conjugated at its 3' end to a ligand of the present invention, such as an ASGPR ligand or a GalNac ligand, having, for example, the structure of formula (Ia), preferably (Ia-i), more preferably L96, and preferably, the ligand is linked by a phosphate ester bond or a thiophosphate ester bond.

[0302] In some embodiments, the combination of antisense and sense strands in the dsRNA activator is shown in Table 2 as any combination of antisense and sense strands, wherein the sense strand is conjugated at the 3' end to a GalNac ligand of the present invention, which has, for example, an L96 structure, and preferably, the ligand is linked by a phosphate ester bond or a thiophosphate bond.

[0303] In some embodiments, the dsRNA activator is any of the dsRNA activators shown in Table 2, and wherein the positive strand is conjugated at the 3' end with a GalNac ligand of the present invention, which has, for example, an L96 structure, and preferably, the ligand is linked by a phosphate ester bond or a thiophosphate ester bond.

[0304] In some embodiments, the antisense strand of the dsRNA activator comprises any modified nucleotide sequence of the antisense strand in Table 3 of the specification, and / or the sense strand comprises any modified nucleotide sequence of the sense strand in Table 3 of the specification that is conjugated to an L96 structural ligand at the 3' end via a phosphate ester bond.

[0305] In some implementations, the combination of the antisense and sense strands in the dsRNA activator is shown in Table 3 as any combination of antisense and sense strands.

[0306] In some embodiments, the dsRNA activator is any of the dsRNA activators shown in Table 3.

[0307] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand respectively comprise SEQ ID NO:551 / 441, SEQ ID NO:552 / 442, SEQ ID NO:553 / 443, SEQ ID NO:554 / 444, SEQ ID NO:555 / 445, SEQ ID NO:556 / 446, SEQ ID NO:557 / 447, SEQ ID NO:558 / 448, SEQ ID NO:559 / 449, SEQ ID NO:560 / 450, SEQ ID NO:561 / 451, SEQ ID NO:562 / 452, SEQ ID NO:563 / 453, SEQ ID NO:564 / 454, SEQ ID NO:565 / 455, SEQ ID NO:566 / 456, SEQ ID NO:567 / 457, SEQ ID NO:568 / 458, SEQ ID NO:551 / 441, SEQ ID NO:552 / 442, SEQ ID NO:553 / 443, SEQ ID NO:554 / 444, SEQ ID NO:555 / 455, SEQ ID NO:566 / 456, SEQ ID NO:567 / 457, SEQ ID NO:568 / 458, SEQ ID NO:569 / 449, SEQ ID NO:560 / 450, SEQ ID NO:561 / 451, SEQ ID NO:562 / 452, SEQ ID NO:563 / 453, SEQ ID NO:564 / 454, SEQ ID NO:565 / 455, SEQ ID NO:566 / 456, SEQ ID NO:567 / 457, SEQ ID NO:568 / 458, SEQ ID NO:569 NO:569 / 459, SEQ ID NO:570 / 460, SEQ ID NO:571 / 461, SEQ ID NO:572 / 462, SEQ ID NO:573 / 463, SEQ ID NO:574 / 464, SEQ ID NO:575 / 465, SEQ ID NO:576 / 466, SEQ ID NO:577 / 467, SEQ ID NO:578 / 468, SEQ ID NO:579 / 469, SEQ ID NO:580 / 470, SEQ ID NO:581 / 471, SEQ ID NO:582 / 472, SEQ ID NO:583 / 473, SEQ ID NO:584 / 474, SEQ ID NO:585 / 475, SEQ ID NO:586 / 476, SEQ ID NO:587 / 477、SEQ ID NO:588 / 478、SEQ ID NO:589 / 479, SEQ ID NO:590 / 480, SEQ ID NO:591 / 481, SEQ ID NO:592 / 482, SEQ ID NO:593 / 483, SEQ ID NO:594 / 484, SEQ ID NO:595 / 485, SEQ ID NO:596 / 486, SEQ ID NO:597 / 487, SEQ ID NO:598 / 488, SEQ ID NO:599 / 489, SEQ ID NO:600 / 490, SEQ ID NO:601 / 491, SEQ ID NO:602 / 492, SEQ IDNO:603 / 493、SEQ ID NO:604 / 494、SEQ ID NO:605 / 495、SEQ ID NO:606 / 496、SEQ ID NO:607 / 497、SEQ ID NO:608 / 498、SEQ ID NO:609 / 499、SEQ ID NO:610 / 500、SEQ ID NO:611 / 501、SEQ ID NO:612 / 502、SEQ ID NO:613 / 503、SEQ ID NO:614 / 504、SEQ ID NO:615 / 505、SEQ ID NO:616 / 506、SEQ ID NO:617 / 507、SEQ ID NO:618 / 508、SEQ ID NO:619 / 509、SEQ ID NO:620 / 510、SEQ ID NO:621 / 511、SEQ ID NO:622 / 512、SEQ ID NO:623 / 513、SEQ ID NO:624 / 514、SEQ ID NO:625 / 515、SEQ ID NO:626 / 516、SEQ ID NO:627 / 517、SEQ ID NO:628 / 518、SEQ ID NO:629 / 519、SEQ ID NO:630 / 520、SEQ ID NO:631 / 521、SEQ ID NO:632 / 522、SEQ ID NO:633 / 523、SEQ ID NO:634 / 524、SEQ ID NO:635 / 525、SEQ ID NO:636 / 526、SEQ ID NO:637 / 527、SEQ ID NO:638 / 528、SEQ ID NO:639 / 529、SEQ ID NO:640 / 530、SEQ ID NO:641 / 531、SEQ ID NO:642 / 532、SEQ ID NO:643 / 533、SEQ ID NO:644 / 534、SEQ ID NO:645 / 535、SEQ ID NO:646 / 536、SEQ ID NO:647 / 537、SEQ ID NO:648 / 538、SEQ ID NO:649 / 539、SEQ ID NO:650 / 540、SEQ ID NO:651 / 541、SEQ ID NO:652 / 542、SEQ ID NO:653 / 543、SEQ ID NO:654 / 544、SEQ ID NO:655 / 545、SEQ ID NO:656 / 546、SEQ ID NO:657 / 547、SEQ IDAt least 15, 16, 17, 18, or 19 consecutive modified nucleotides in the nucleotide sequences shown in NO:658 / 548, SEQ ID NO:659 / 549, SEQ ID NO:660 / 550, or SEQ ID NO:663 / 662, wherein the modified nucleotide of the sense strand is Um or Am at the 3' end and is linked to L96 via a phosphate ester bond, optionally the antisense strand also has a 3' overhang of 1 or 2 nucleotides, optionally the first nucleotide at the 5' end of the antisense strand is a modified A or U, such as a modified U, such as Um or Am.

[0308] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand respectively comprise SEQ ID NO:551 / 441, SEQ ID NO:552 / 442, SEQ ID NO:553 / 443, SEQ ID NO:554 / 444, SEQ ID NO:555 / 445, SEQ ID NO:556 / 446, SEQ ID NO:557 / 447, SEQ ID NO:558 / 448, SEQ ID NO:559 / 449, SEQ ID NO:560 / 450, SEQ ID NO:561 / 451, SEQ ID NO:562 / 452, SEQ ID NO:563 / 453, SEQ ID NO:564 / 454, SEQ ID NO:565 / 455, SEQ ID NO:566 / 456, SEQ ID NO:567 / 457, SEQ ID NO:568 / 458, SEQ ID NO:551 / 441, SEQ ID NO:552 / 442, SEQ ID NO:553 / 443, SEQ ID NO:554 / 444, SEQ ID NO:555 / 455, SEQ ID NO:566 / 456, SEQ ID NO:567 / 457, SEQ ID NO:568 / 458, SEQ ID NO:569 / 449, SEQ ID NO:560 / 450, SEQ ID NO:561 / 451, SEQ ID NO:562 / 452, SEQ ID NO:563 / 453, SEQ ID NO:564 / 454, SEQ ID NO:565 / 455, SEQ ID NO:566 / 456, SEQ ID NO:567 / 457, SEQ ID NO:568 / 458, SEQ ID NO:569 NO:569 / 459, SEQ ID NO:570 / 460, SEQ ID NO:571 / 461, SEQ ID NO:572 / 462, SEQ ID NO:573 / 463, SEQ ID NO:574 / 464, SEQ ID NO:575 / 465, SEQ ID NO:576 / 466, SEQ ID NO:577 / 467, SEQ ID NO:578 / 468, SEQ ID NO:579 / 469, SEQ ID NO:580 / 470, SEQ ID NO:581 / 471, SEQ ID NO:582 / 472, SEQ ID NO:583 / 473, SEQ ID NO:584 / 474, SEQ ID NO:585 / 475, SEQ ID NO:586 / 476, SEQ ID NO:587 / 477、SEQ ID NO:588 / 478、SEQ ID NO:589 / 479, SEQ ID NO:590 / 480, SEQ ID NO:591 / 481, SEQ ID NO:592 / 482, SEQ ID NO:593 / 483, SEQ ID NO:594 / 484, SEQ ID NO:595 / 485, SEQ ID NO:596 / 486, SEQ ID NO:597 / 487, SEQ ID NO:598 / 488, SEQ ID NO:599 / 489, SEQ ID NO:600 / 490, SEQ ID NO:601 / 491, SEQ ID NO:602 / 492, SEQ IDNO:603 / 493、SEQ ID NO:604 / 494、SEQ ID NO:605 / 495、SEQ ID NO:606 / 496、SEQ ID NO:607 / 497、SEQ ID NO:608 / 498、SEQ ID NO:609 / 499、SEQ ID NO:610 / 500、SEQ ID NO:611 / 501、SEQ ID NO:612 / 502、SEQ ID NO:613 / 503、SEQ ID NO:614 / 504、SEQ ID NO:615 / 505、SEQ ID NO:616 / 506、SEQ ID NO:617 / 507、SEQ ID NO:618 / 508、SEQ ID NO:619 / 509、SEQ ID NO:620 / 510、SEQ ID NO:621 / 511、SEQ ID NO:622 / 512、SEQ ID NO:623 / 513、SEQ ID NO:624 / 514、SEQ ID NO:625 / 515、SEQ ID NO:626 / 516、SEQ ID NO:627 / 517、SEQ ID NO:628 / 518、SEQ ID NO:629 / 519、SEQ ID NO:630 / 520、SEQ ID NO:631 / 521、SEQ ID NO:632 / 522、SEQ ID NO:633 / 523、SEQ ID NO:634 / 524、SEQ ID NO:635 / 525、SEQ ID NO:636 / 526、SEQ ID NO:637 / 527、SEQ ID NO:638 / 528、SEQ ID NO:639 / 529、SEQ ID NO:640 / 530、SEQ ID NO:641 / 531、SEQ ID NO:642 / 532、SEQ ID NO:643 / 533、SEQ ID NO:644 / 534、SEQ ID NO:645 / 535、SEQ ID NO:646 / 536、SEQ ID NO:647 / 537、SEQ ID NO:648 / 538、SEQ ID NO:649 / 539、SEQ ID NO:650 / 540、SEQ ID NO:651 / 541、SEQ ID NO:652 / 542、SEQ ID NO:653 / 543、SEQ ID NO:654 / 544、SEQ ID NO:655 / 545、SEQ ID NO:656 / 546、SEQ ID NO:657 / 547、SEQ IDThe modified nucleotide sequences shown in NO:658 / 548, SEQ ID NO:659 / 549, SEQ ID NO:660 / 550 or SEQ ID NO:663 / 662, or each of the modified nucleotide sequences shown, wherein the 3' end of the positive strand nucleotide sequence shown in any of SEQ ID Nos:551-663 is conjugated with an L96 structural ligand via a phosphate ester bond.

[0309] In some specific implementations, the dsRNA activator comprises a sense strand and an antisense strand, wherein

[0310] The sense strand comprises or is composed of a modified nucleotide sequence with an L96 ligand conjugated to the 3' end as shown in SEQ ID NO:610, and the antisense strand comprises or is composed of a modified nucleotide sequence as shown in SEQ ID NO:500.

[0311] The sense strand comprises or is composed of the modified nucleotide sequence shown in SEQ ID NO:651, which has an L96 structure ligand attached to its 3' end, and the antisense strand comprises or is composed of the modified nucleotide sequence shown in SEQ ID NO:541.

[0312] The sense strand comprises or is composed of a modified nucleotide sequence with an L96 structural ligand conjugated to the 3' end as shown in SEQ ID NO:663, and the antisense strand comprises or is composed of a modified nucleotide sequence as shown in SEQ ID NO:662.

[0313] In some specific implementations, the dsRNA activator comprises a sense strand and an antisense strand, wherein

[0314] The sense strand consists of a modified nucleotide sequence with an L96 ligand attached to the 3' end, as shown in SEQ ID NO:610, and the antisense strand consists of a modified nucleotide sequence, as shown in SEQ ID NO:500.

[0315] The sense strand consists of a modified nucleotide sequence with an L96 ligand attached to the 3' end, as shown in SEQ ID NO:651, and the antisense strand consists of a modified nucleotide sequence, as shown in SEQ ID NO:541.

[0316] The sense strand consists of a modified nucleotide sequence with an L96 structural ligand attached to the 3' end, as shown in SEQ ID NO:663, and the antisense strand consists of a modified nucleotide sequence, as shown in SEQ ID NO:662.

[0317] III. Preparation of dsRNA activators

[0318] dsRNA can be synthesized using standard methods known in the art. The double-stranded RNA of the present invention can be prepared using a two-step procedure. First, a single strand of the double-stranded RNA molecule is prepared separately. Then, the component strands are annealed. In some embodiments, the single strand of dsRNA, such as siRNA, can be prepared using solution-phase or solid-phase organic synthesis, or both. Organic synthesis offers the advantage of readily preparing oligonucleotide chains comprising non-natural or modified nucleotides.

[0319] In some embodiments, the dsRNA of the present invention is prepared by RNA solid-phase synthesis. RNA solid-phase synthesis is a commonly used technique for synthesizing RNA molecules, which allows for the stepwise construction of RNA chains on a solid support. This method is characterized by high throughput, high efficiency, and automation, and is widely used in biotechnology and research fields. In some embodiments, RNA solid-phase synthesis comprises the following basic methods and steps:

[0320] 1. Template recognition:

[0321] In solid-phase synthesis, a template is first required, typically a single-stranded DNA sequence containing a sequence complementary to the desired RNA sequence. This template DNA is immobilized on a solid support, such as controlled-pore glass (CPG) or polystyrene beads.

[0322] 2. Transcription initiation:

[0323] RNA polymerase recognizes and binds to the promoter sequence on template DNA. In solid-phase synthesis, this process typically does not require primers because RNA polymerase can directly initiate RNA chain synthesis at the promoter region.

[0324] 3. Transcription elongation:

[0325] Once RNA polymerase binds to the promoter, it begins synthesizing an RNA chain on the template DNA. In this process, RNA polymerase moves along the DNA template, adding one nucleotide triphosphate (NTP) complementary to the template DNA bases one by one. Each time an NTP is added, the RNA chain extends by one nucleotide at its 3' end.

[0326] 4. Cyclic Synthesis:

[0327] A key feature of solid-phase synthesis is its ability to be performed in multiple cycles. Each cycle involves adding a new NTP, removing unreacted NTPs, and eluting and rebinding with RNA polymerase. This process can be automated, significantly improving synthetic efficiency.

[0328] 5. Transcription termination:

[0329] Once the RNA strand reaches the desired length, the transcription process needs to terminate. This is typically achieved by adding a specific termination signal or chemical substance.

[0330] 6. Post-processing:

[0331] After synthesis, the RNA strands need to be released from the solid support, and any unreacted NTPS, protecting groups, and other impurities need to be removed. This is typically achieved through chemical or enzymatic methods, such as using specific enzymes to cleave the links on the solid support.

[0332] 7. Purification and analysis:

[0333] Finally, the synthesized RNA needs to be purified using appropriate purification methods (such as gel electrophoresis, column chromatography, etc.). The purified RNA can then be verified for its length, purity, and sequence correctness using various analytical methods (such as capillary electrophoresis, mass spectrometry, etc.).

[0334] In some embodiments, the solid support is a blank solid support, such as a blank CPG solid support. In some embodiments, the solid support is a solid support containing ligands.

[0335] IV. Pharmaceutical Compositions

[0336] In some embodiments, the present invention provides compositions comprising the dsRNA active agent of the present invention or a pharmaceutically acceptable salt thereof, preferably pharmaceutical compositions or pharmaceutical formulations. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, for example a pharmaceutical composition, comprises the dsRNA active agent of the present invention, and a combination of one or more other therapeutic agents.

[0337] The present invention also includes compositions (including pharmaceutical compositions) comprising the dsRNA active agent of the present invention or a pharmaceutically acceptable salt thereof. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0338] As used herein, "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, or combinations thereof.

[0339] For information on the use and applications of pharmaceutical excipients, see "Handbook of Pharmaceutical Excipients", 8th edition, R.C. Rowe, P.J. Seskey and S.C. Swen, Pharmaceutical Press, London, Chicago.

[0340] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0341] A drug or pharmaceutical composition comprising the dsRNA active agent of the present invention can be prepared by mixing the dsRNA active agent of the present invention having the desired purity with one or more optional pharmaceutical excipients.

[0342] In some embodiments, the dsRNA activator is present in an unbuffered solution, such as saline or water. In some embodiments, the dsRNA activator is present in a buffered solution, such as an acetate, citrate, prolyl, carbonate, or phosphate, or any combination thereof, such as phosphate-buffered saline (PBS).

[0343] The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient, said active ingredient being required for the specific indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, such as insulin, glucagon-like peptide-1 agonists, sulfonylureas, sitaglinides, biguanides, thiazolidinediones, α-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and combination formulations of any of the above. The active ingredients are suitably combined in an amount effective for the intended use.

[0344] In some embodiments, the pharmaceutical composition or formulation of the present invention may be contained in a vial or in a syringe.

[0345] V. Drug combinations and pillboxes

[0346] In some embodiments, the present invention also provides pharmaceutical combinations or pharmaceutical combination products comprising the dsRNA activator of the present invention, and one or more other therapeutic agents.

[0347] Another object of the present invention is to provide a complete pillbox containing the drug combination of the present invention, preferably said pillbox in the form of drug dosage units. This allows dosage units to be provided according to a dosing regimen or drug administration interval.

[0348] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0349] - A first container containing a pharmaceutical composition comprising the dsRNA active agent of the present invention;

[0350] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0351] In some implementations, other therapeutic agents, such as any therapeutic agent effective in preventing or treating INHBE-related diseases and / or conditions, encompass a wide range of therapeutic agents used to treat metabolic disorders or obesity or cardiovascular diseases, such as insulin, glucagon-like peptide-1 agonists, sulfonylureas, sitaglinides, biguanides, thiazolidinediones, alpha-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and combination preparations of any of the above drugs.

[0352] VI. Uses and Methods

[0353] One aspect of the present invention provides a method for inhibiting the expression and / or activity of the INHBE gene in cells, comprising deactivating the cells from the dsRNA activator, pharmaceutical composition, or combination of drugs of the present invention, thereby inhibiting the expression of the INHBE gene in the cells. In some embodiments, the cells are in a subject. In some embodiments, the subject suffers from an INHBE-related disease and / or condition.

[0354] In some embodiments, contacting the cells with the dsRNA activator thereby inhibiting INHBE expression by at least about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, or about 94%. In some embodiments, inhibition of INHBE expression reduces the level of INHBE protein in the serum of the subject by at least about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, or about 94%. In some embodiments, administration of the dsRNA activator to the subject results in a decrease in INHBE concentration or a reduction in the accumulation of INHBE protein in the subject. In some embodiments, administration of the dsRNA activator to the subject results in a decrease in INHBE concentration or content, or a decrease in INHBE protein accumulation or content in the subject's body. In some embodiments, administration of the dsRNA activator to the subject results in a decrease in INHBE concentration or content in the subject's body fluids. In some embodiments, administration of the dsRNA formulation to the subject results in a decrease in INHBE expression or protein content in the subject's liver or hepatocytes.

[0355] This invention provides, in one aspect, a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of the dsRNA active agent, pharmaceutical composition, drug combination, or kit of the invention. In some embodiments, the disease is an INHBE-related disease and / or condition.

[0356] The present invention also provides a method for reducing weight in subjects, comprising administering to the subject an effective amount of the dsRNA activator, pharmaceutical composition, pharmaceutical combination or kit of the present invention.

[0357] In some embodiments, the present invention relates to the dsRNA activator, pharmaceutical composition, pharmaceutical combination or kit of the present invention for use in therapies, such as for treating INHBE-related diseases and / or conditions, and / or for weight loss.

[0358] In some embodiments, the present invention relates to methods for treating diseases, such as those mentioned herein, using the dsRNA active agent, pharmaceutical composition, pharmaceutical combination, or cassette of the present invention, or to uses for said treatment, or for weight loss, or to uses for preparing a medicament for said treatment or for weight loss.

[0359] In some embodiments, the INHBE-related diseases and / or conditions are associated with abnormal expression (e.g., overexpression or upregulation) or abnormal activity (e.g., increased activity) of INHBE. In some embodiments, the disease or condition is an indication for which one benefits from reduced expression and / or activity of inhibin subunit βE (INHBE).

[0360] In some embodiments, a subject suffering from an INHBE-related disease and / or condition, or whose cells exhibit abnormal INHBE expression or activity. In some embodiments, the subject (particularly an adult subject) has INHBE overexpression. In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels or activity) INHBE (e.g., compared to a healthy subject). In some embodiments, the subject's biological sample (e.g., blood, serum, tissue such as liver tissue, or cells such as hepatocytes) has (e.g., elevated levels, such as nucleic acid or protein levels or activity) INHBE (e.g., compared to a biological sample from a healthy subject (e.g., the corresponding tissue or cells in a healthy subject)). In some embodiments, the subject has cells that overexpress INHBE, such as hepatocytes. In some embodiments, the individual's cells (e.g., hepatocytes) overexpress INHBE, for example, moderately or highly. In some embodiments, abnormal INHBE expression refers to higher INHBE expression in cells (e.g., hepatocytes) compared to INHBE expression in control cells (e.g., healthy cells in the corresponding tissue of a healthy individual, such as healthy hepatocytes). In some implementation schemes, abnormal expression of INHBE refers to higher INHBE expression in liver tissue or hepatocytes compared to control tissues or cells (e.g., corresponding tissues or corresponding healthy hepatocytes of a healthy individual).

[0361] In this article, “INHBE overexpression or upregulation” refers to an increase in the protein level (e.g., protein concentration or accumulation) of INHBE compared to the corresponding tissues or cells of healthy individuals.

[0362] In some implementations, the INHBE-related disease and / or condition is a metabolic disorder or obesity or cardiovascular disease, such as metabolic disorder being metabolic syndrome, or obesity being obesity.

[0363] The dsRNA active agent or composition or drug or formulation comprising the present invention may also be administered in combination with one or more other therapies, such as other treatment modalities and / or other therapeutic agents, for the purposes described herein, such as for the prevention and / or treatment of the related diseases or conditions mentioned herein. Therefore, the present invention also relates to combination therapies of the dsRNA active agent or composition or drug or formulation comprising the present invention with one or more other therapies.

[0364] In other respects, the present invention provides the use of the dsRNA active agent of the present invention or a composition or drug or formulation thereof in the manufacture or preparation of a medicament for the purposes described herein, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0365] In other respects, the present invention also provides the dsRNA activator of the present invention, or compositions, pharmaceuticals, formulations or combination products comprising the present invention, for use in therapies, such as for treating the related diseases or conditions mentioned herein.

[0366] Subjects may be mammals, such as primates, preferably higher primates, such as humans (e.g., individuals who have the disease described herein or are at risk of having the disease described herein).

[0367] In one implementation, the subject has the disease described herein or is at risk of having the disease described herein.

[0368] The combination therapy of the present invention covers combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations) and separate administration. In the case of separate administration, the dsRNA active agent or composition or drug or formulation of the present invention may be administered before, simultaneously with, and / or after the administration of other therapeutic agents and / or active agents.

[0369] In some embodiments, other therapeutic agents that may be combined or administered in combination with the dsRNA activator, drug, formulation, or composition of the present invention are selected from any therapeutic agent effective for, for example, the prevention or treatment of INHBE-related diseases and / or conditions, covering a variety of therapeutic agents for the treatment of metabolic disorders or obesity or cardiovascular diseases, such as insulin, glucagon-like peptide-1 agonists, sulfonylureas, sitaglinides, biguanides, thiazolidinediones, α-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and combination formulations of any of the above drugs. Example

[0370] Example 1: siRNA Synthesis

[0371] RNA solid-phase synthesis is a commonly used technique for synthesizing RNA molecules, allowing for the stepwise construction of RNA chains on a solid support. This method is characterized by high throughput, high efficiency, and automation, and is widely used in biotechnology and research fields.

[0372] The INHBE RNAi agent duplexes shown in Table 2 were synthesized according to the following method:

[0373] A. Synthesis. The sense and antisense strands of the INHBE RNAi agent were synthesized using the solid-phase phosphoramide technique employed in oligonucleotide synthesis. Mer Made 192 (Bioautomation) or OP Pilot 100 (GE Healthcare) were used, depending on the proportions. Synthesis was performed on a solid support made of controlled-pore glass (CPG, 500A or 600A, available from Prime Synthesis, Aston, PA, USA). All RNA and 2'-modified phosphoramide were purchased from Shanghai Zhaowei Technology Development Co., Ltd. Specifically, the following 2'-O-methylphosphoramides are used: (5'-O-dimethoxytriphenylmethyl-N6-(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramide, (5'-O-dimethoxytriphenylmethyl-N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramide, (5'-O-dimethoxytriphenylmethyl-N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramide, (5'-O-dimethoxytriphenylmethyl-N6 ... Triphenylmethyl-N2-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide and 5'-O-dimethoxytriphenylmethyl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide. 2'-Deoxy-2'-fluorophosphamide has the same protecting group as 2'-O-methylphosphamide. Debasing (3'-O-dimethoxytriphenyl) Methyl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide was purchased from Shanghai Zhaowei Technology Development Co., Ltd. The targeting ligand containing the phosphoramide was dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other phosphoramides were dissolved in anhydrous acetonitrile (50 mM), and a molecular sieve (3A) was added. 5-Benzylthio-1H-tetrazole (BTT, 250 mM, soluble in acetonitrile) or 5-ethylthio- -1H-tetrazole (ETT, 250 mM, soluble in acetonitrile) was used as the activator solution. Coupling times were 12 min (RNA), 15 min (targeting ligand), 90 s (2'OMe), and 60 s (2'F). To introduce the thiophosphate bond, 100 mM of 3-phenyl-1,2,4-dithiazolin-5-one (POS, available from PolyOrg, Inc., Leominster, MA, USA) dissolved in anhydrous acetonitrile was used.

[0374] B. Cleavage and deprotection of the support-bonded oligomers. After solid-phase synthesis, the dried solid support was treated with a 1:1 volume ratio of 40% by weight aqueous methylamine and 28% ammonium hydroxide solution (Aldrich) at 30°C for 1.5 hours. The solution was evaporated, and the solid residue was reconstituted in water (see below).

[0375] C. Purification. Crude oligomers were purified by anion-exchange HPLC using a TSKgel SuperQ-5PW 13μm column and a Shimadzu LC-8 system. Buffer A consisted of 20mM Tris, 5mM EDTA, and 20% acetonitrile at pH 9.0. Buffer B was identical to buffer A except for the addition of 1.5M sodium chloride. UV traces were recorded at 260 nm. Appropriate fractions were combined and then run on size exclusion HPLC using a GE Healthcare XK 26 / 40 column packed with Sephadex G-25 gel and a run buffer of filtered DI water or 100mM ammonium bicarbonate and 20% acetonitrile at pH 6.7.

[0376] D. Annealing. Complementary strands (sense and antisense strands) were mixed by combining equimolar RNA solutions in 1× phosphate-buffered saline (Corning, Cellgro) to form an RNAi agent. A portion of the RNAi agent was lyophilized and stored at -15°C to -25°C. The duplex concentration was determined by measuring the absorbance of the solution in 1× phosphate-buffered saline using a UV-Vis spectrometer. The absorbance at 260 nm was then multiplied by the conversion factor and dilution factor to determine the duplex concentration. Unless otherwise specified, all conversion factors were 0.037 mg / (mL·cm). For some experiments, the conversion factor was calculated from the extinction coefficient determined experimentally.

[0377] Table 1: Sensitive and antisense strands of unmodified INHBE double-stranded siRNA

[0378] Table 2: Sensitive and antisense strands of INHBE-modified double-stranded siRNA

[0379] The naked sequence (i.e., the unmodified nucleotide sequence) of the nucleotide sequence shown in SEQ ID NO is also listed in the sequence listing.

[0380] Preparation of GalNAc-siRNA conjugates:

[0381] Preparation of GalNAc-siRNA conjugates (Table 3): The synthesis method of GalNAc-siRNA conjugates can be referred to WO2023003922A1.

[0382] It should be understood that, in the context of this article, a siRNA ID without a decimal ".1" corresponds to a siRNA ID with a decimal ".1", for example, XD000010.1 corresponds to XD000010.

[0383] Table 3. Table of items containing GalNAc-siRNA

[0384] The naked sequence (i.e., the unmodified nucleotide sequence without L96 conjugation) of the nucleotide sequence shown in SEQ ID NO is also listed in the sequence listing.

[0385] Example 2: Inhibition of human INHBE in HepG2 cells by siRNA

[0386] HepG2 cells (Nanjing Kebai Biotechnology Co., Ltd., Cat#CBP60199) were cultured in EMEM medium (ATCC, Cat#30-2003) containing 10% fetal bovine serum at 37°C and 5% CO2.

[0387] siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat#13778150). The specific method is as follows: SiRNA was prepared into a 1 μM working solution using DEPC-treated water. Solution A was prepared, with each aliquot containing 1 μl of siRNA working solution and 9 μl of Opti-MEM medium (GIBCO, Cat#31985070). Solution B was prepared, with each aliquot containing 0.3 μl of Lipofectamine RNAiMAX and 9.7 μl of Opti-MEM medium. After mixing solutions A and B, the mixture was placed in a 96-well plate and incubated at room temperature for 20 min. Then, 80 μl of HepG2 cells were added, with 15,000 cells per well, resulting in a final siRNA concentration of 10 nM. Twenty-four hours after transfection, RNA was extracted from cells using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat#CL132), and reverse transcription and qPCR were performed according to the kit instructions to determine INHBE mRNA levels. INHBE mRNA levels were corrected for GAPDH internal control mRNA levels. INHBE mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of remaining INHBE mRNA expression in cells treated with negative siRNA, as follows: ΔCt = Ct (target gene) – Ct (internal control gene) ΔΔCt = ΔCt (drug-treated group) - ΔCt (negative siRNA control group) INHBE mRNA relative expression level = 2 -ΔΔCt ×100% (i.e., the remaining mRNA percentage in the table below)

[0388] Table 4 shows the inhibition of INHBE mRNA after transfection with the siRNA molecules in Table 2 in HepG2.

[0389] Table 4: Single-dose screening of dsRNA drugs targeting INHBE in HepG2 cells

[0390] Example 3: Inhibition of human INHBE in Hep3b cells by siRNA (dual concentration points)

[0391] In Hep3B cells, siRNA activity was screened at two concentration points: 1 nM and 0.1 nM.

[0392] Hep3B cells (CyberKang Biotechnology Co., Ltd., Cat#iCell-h091) were cultured in MEM medium (Gibco, Cat#11090-081) containing 10% fetal bovine serum at 37°C and 5% CO2.

[0393] siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat#13778150). The specific method is as follows: SiRNA was prepared into 0.1 μM and 0.01 μM working solutions using DEPC-treated water. Solution A was prepared, with each aliquot containing 1 μl of siRNA working solution and 9 μl of Opti-MEM medium (Gibco, Cat#31985070). Solution B was prepared, with each aliquot containing 0.3 μl of Lipofectamine RNAiMAX and 9.7 μl of Opti-MEM medium. After mixing solutions A and B, the mixture was placed in a 96-well plate and incubated at room temperature for 20 min. Then, 80 μl of Hep3B cells were added, with 15,000 cells per well. The final siRNA concentrations were 1 nM and 0.1 nM, respectively. Twenty-four hours after transfection, RNA was extracted from cells using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat#CL132), and reverse transcription and qPCR were performed according to the kit instructions to determine INHBE mRNA levels. INHBE mRNA levels were corrected for GAPDH internal control mRNA levels. INHBE mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of remaining INHBE mRNA expression in cells treated with negative siRNA, as shown in Table 5. The calculation formula is as follows: ΔCt = Ct (target gene) – Ct (internal control gene) ΔΔCt = ΔCt (drug-treated group) - ΔCt (negative siRNA control group) INHBE mRNA relative expression level = 2 -ΔΔCt ×100%

[0394] Table 5: Two-dose screening of dsRNA targeting INHBE in Hep3B cells

[0395] XD000202 is AD-1708473 in WO2023003922A1.

[0396] Example 4: Inhibition of human INHBE by siRNA in primary human hepatocytes

[0397] The activity of siRNA was screened at four concentration points in human primary hepatocytes (IPHASE, Cat#085A12.21).

[0398] Following the product instructions, siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat#13778150). The specific method was as follows: siRNA was diluted with DEPC-treated water to prepare four concentration gradients: 1 μM starting solution, 10-fold dilution, and so on. Solution A was prepared, with each aliquot containing 1 μl of siRNA working solution and 9 μl of Opti-MEM medium (Gibco, Cat#31985070). Solution B was prepared, with each aliquot containing 0.3 μl of Lipofectamine RNAiMAX and 9.7 μl of Opti-MEM medium. Equal volumes of solutions A and B were mixed and incubated in 96-well plates at room temperature for 20 min. Then, 80 μl of resuscitated human primary hepatocytes were added, with 40,000 cells per well. The final siRNA concentration was 10 nM starting solution, with four 10-fold dilutions. Twenty-four hours after transfection, RNA was extracted from cells using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat#CL132), and reverse transcription and qPCR were performed according to the kit instructions to determine INHBE mRNA levels. INHBE mRNA levels were corrected for GAPDH internal control mRNA levels. INHBE mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of remaining INHBE mRNA expression in cells treated with negative siRNA, as shown in the following formula (results are shown in Table 6): ΔCt = Ct (target gene) – Ct (internal control gene) ΔΔCt = ΔCt (drug-treated group) - ΔCt (negative siRNA control group) Relative expression level of INHBE mRNA = 2 -ΔΔCt ×100%

[0399] Table 6: Multi-dose screening of dsRNA targeting INHBE in human primary hepatocytes

[0400] Table 7: Multi-dose screening of siRNA targeting INHBE in human primary hepatocytes

[0401] The results are shown in Table 7, indicating that the siRNA disclosed herein has a high level of inhibitory activity against the INHBE gene in human primary hepatocytes.

[0402] Example 5. Inhibition of monkey INHBE in primary monkey hepatocytes by siRNA

[0403] The activity of siRNA was screened at four concentration points in primary monkey hepatocytes (IPHASE, Cat#01932B1.21).

[0404] Following the product instructions, siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat#13778150). The specific method was as follows: siRNA was diluted with DEPC-treated water to prepare working solutions of 1 μM starting concentration, with 10-fold serial dilutions thereafter, for a total of four concentrations. Solution A was prepared, with each aliquot containing 1 μl of siRNA working solution and 9 μl of Opti-MEM medium (Gibco, Cat#31985070). Solution B was prepared, with each aliquot containing 0.3 μl of Lipofectamine RNAiMAX and 9.7 μl of Opti-MEM medium. Equal volumes of solutions A and B were mixed and incubated in 96-well plates at room temperature for 20 min. Then, 80 μl of resuscitated primary monkey hepatocytes were added, with 40,000 cells per well. The final siRNA concentration was 10 nM starting concentration, with 10-fold serial dilutions for a total of four concentrations. Twenty-four hours after transfection, RNA was extracted from cells using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat#CL132), and reverse transcription and qPCR were performed according to the kit instructions to determine INHBE mRNA levels. INHBE mRNA levels were corrected for GAPDH internal control mRNA levels. INHBE mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of remaining INHBE mRNA expression in cells treated with negative siRNA, as follows: ΔCt = Ct (target gene) – Ct (internal control gene) ΔΔCt = ΔCt (drug-treated group) - ΔCt (negative siRNA control group) INHBE mRNA relative expression level = 2 -ΔΔCt ×100%

[0405] Table 8: Multi-dose screening of siRNA targeting INHBE in primary monkey hepatocytes

[0406] Example 6: psiCHECK-2 off-target activity test of AS and SS chains

[0407] Off-target plasmids were constructed using the psiCHECK-2 plasmid (Genewiz):

[0408] (1) PSCM is used to detect off-target effects of the sense strand. The target sequence is completely complementary to all 19 nucleotide sequences of the sense strand of the conjugate being detected.

[0409] (2) GSSM is used to detect off-target effects in the seed region of the antisense strand. The target sequence is completely complementary to the nucleotide sequence at positions 1-8 of the 5' end of the antisense strand of the conjugate being detected. The nucleotide sequence at positions 9-21 of the 5' end of the antisense strand of the conjugate being detected is complementary to its corresponding target sequence but mismatched. The mismatch rule is that any G, C, A, or U nucleotide at any position 9-21 of the 5' end of the antisense strand of the conjugate being detected mismatches with the corresponding T, A, C, or G nucleotide in the target sequence. To improve detection sensitivity, the GSSM-5hits off-target plasmid was constructed, consisting of five identical GSSM sequences linked by TTCC.

[0410] The target sequence was embedded into the Xho I / Not I site of the psiCHECK-2 plasmid.

[0411] Off-target activity of siRNA was tested in Hepa1-6 cells using multiple concentration sites.

[0412] Hepa1-6 cells (Nanjing Kebai Biotechnology Co., Ltd., Cat#CBP60574) were cultured in DMEM medium (Gibco, Cat#11965-092) containing 10% fetal bovine serum at 37°C and 5% CO2.

[0413] After digesting the cells, resuspend them in complete culture medium and add them to a 96-well plate with 10,000 cells per well. The next day, when the cell confluence reaches 80-90%, discard the supernatant and add 90 μl of Opti-MEM to each well.

[0414] Refer to the product instruction manual for use. 2000Reagent(ThermoFisher,Cat#11668019)

[0415] Co-transfecting siRNA and plasmid was performed as follows: siRNA was diluted with DEPC-treated water to prepare a 2 μM starting solution, followed by 10-fold serial dilutions to obtain six working solutions. The plasmid was diluted to a working solution of 200 ng / μl. Solution A was prepared, with each aliquot containing 2 μl of siRNA working solution, 0.05 μl of plasmid working solution, and 2.95 μl of Opti-MEM medium. Solution B was prepared, with each aliquot containing 0.2 μl of siRNA working solution. 2000 Reagent and 4.8 μl Opti-MEM medium. Mix equal volumes of A and B, incubate at room temperature for 15-20 min, and add 10 μl to each well of a 96-well plate. The final siRNA concentration is 40 nM, serially diluted 10-fold downwards for a total of 6 concentrations, with 10 ng of plasmid per well. After 4 h, add 100 μl of 20% FBSDMEM complete medium and continue culturing for 24 h.

[0416] 24 hours later, dual fluorescence was detected using the Dual-Glo Luciferase Assay (Promega, Cat#E2940) according to the instructions. The fluorescence ratio per well was calculated as Ratio = Ren / Fir. Using the control group as a baseline, the Ratio for each test group was normalized to obtain R = Ratio(test) / Ratio(control), which represents the expression level of the Renilla reporter gene, i.e., residual activity. Dose-response curves were plotted using the activity results measured at different siRNA concentrations.

[0417] The results are shown in Tables 9 and 10. The off-target risk of the siRNA of this disclosure is low for the sense strand, and the off-target risk of the antisense strand seed region of the siRNA of this disclosure is lower than that of XD000202. In summary, the siRNA of this disclosure has a low off-target risk and high potential safety.

[0418] Table 9: Off-target activity results of siRNA targeting INHBE (GSSM)

[0419] Table 10: Off-target activity results of siRNA targeting INHBE (PSCM)

[0420] Example 7: Inhibition of human INHBE in Hep3B cells by fluorinated siRNA

[0421] The activity of fluorinated siRNA was screened in Hep3B cells at three concentration points: 10 nM, 1 nM, and 0.1 nM.

[0422] Hep3B cells (CyberKang Biotechnology Co., Ltd., Cat#iCell-h091) were cultured in MEM medium (Gibco, Cat#11090-081) containing 10% fetal bovine serum at 37°C and 5% CO2.

[0423] siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat#13778150). The specific method is as follows: siRNA was prepared into 1 μM, 0.1 μM, and 0.01 μM working solutions using DEPC-treated water. Solution A was prepared, with each aliquot containing 1 μl of siRNA working solution and 9 μl of Opti-MEM medium (Gibco, Cat#31985070). Solution B was prepared, with each aliquot containing 0.3 μl of Lipofectamine RNAiMAX and 9.7 μl of Opti-MEM medium. After mixing solutions A and B, the mixture was placed in a 96-well plate and incubated at room temperature for 20 min. Then, 80 μl of Hep3B cells were added, with 15,000 cells per well. The final siRNA concentrations were 10 nM, 1 nM, and 0.1 nM, respectively. Twenty-four hours after transfection, RNA was extracted from cells using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat#CL132), and reverse transcription and qPCR were performed according to the kit instructions to determine INHBE mRNA levels. INHBE mRNA levels were corrected for GAPDH internal control mRNA levels. INHBE mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of remaining INHBE mRNA expression in cells treated with negative siRNA, as follows: ΔCt = Ct (target gene) – Ct (internal control gene) ΔΔCt = ΔCt (drug-treated group) - ΔCt (negative siRNA control group) INHBE mRNA relative expression level = 2 -ΔΔCt ×100%

[0424] The results are shown in Table 11. The results indicate that the fluorinated siRNA disclosed herein has similar or better inhibitory activity against the INHBE gene in Hep3B cells as the maternal parent.

[0425] Table 11: Three-dose screening of fluorinated dsRNA targeting INHBE in Hep3B cells

[0426] Example 8: Inhibition of human INHBE in primary human hepatocytes (free uptake) by siRNA

[0427] The compound enters human primary hepatocytes via free uptake, as described below:

[0428] 1) Dilute the compound to 10 times the final concentration with PBS, and transfer 10 μL of the compound into a 96-well plate.

[0429] 2) Resuscitated human primary hepatocytes (IPHASE, Cat#085A12.21) were added to 96-well plates, 90 μL per well, for a total of 40,000 cells. The final concentration of siRNA was 500 nM, with 7 concentration gradients of 4-fold dilution.

[0430] After 48 hours of culture, each well was washed once with PBS, and cellular RNA was extracted using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat#CL132). Reverse transcription and qPCR were performed according to the kit instructions to determine INHBE mRNA levels. INHBE mRNA levels were corrected for GAPDH internal control mRNA levels. INHBE mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of remaining INHBE mRNA expression in cells treated with negative siRNA, as shown in the following formula: ΔCt = Ct (target gene) – Ct (internal control gene) ΔΔCt = ΔCt (drug-treated group) - ΔCt (negative siRNA control group) INHBE mRNA relative expression level = 2 -ΔΔCt ×100%

[0431] The results are shown in Table 12, indicating that the siRNA disclosed herein has a high level of inhibitory activity against the INHBE gene in human primary hepatocytes.

[0432] Example 9: Inhibition of monkey INHBE in primary monkey hepatocytes (free uptake) by siRNA

[0433] The compound (siRNA) enters primary monkey hepatocytes via free uptake, as described below:

[0434] Dilute the compound to 10 times its final concentration with PBS, and transfer 10 μL of the compound into a 96-well plate.

[0435] Resuscitated primary monkey hepatocytes (IPHASE, Cat#01932B1.21) were added to 96-well plates, 90 μL per well, for a total of 50,000 cells. The final concentration of siRNA was 100 nM, with four 10-fold dilutions.

[0436] After 48 hours of culture, each well was washed once with PBS, and cellular RNA was extracted using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat#CL132). Reverse transcription and qPCR were performed according to the kit instructions to determine INHBE mRNA levels. INHBE mRNA levels were corrected for GAPDH internal control mRNA levels. INHBE mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of remaining INHBE mRNA expression in cells treated with negative siRNA, as shown in the following formula: ΔCt = Ct (target gene) – Ct (internal control gene) ΔΔCt = ΔCt (drug-treated group) - ΔCt (negative siRNA control group) INHBE mRNA relative expression level = 2 -ΔΔCt ×100%

[0437] The results are shown in Table 13, indicating that the siRNA disclosed herein has a high level of inhibitory activity against the INHBE gene in primary monkey hepatocytes.

[0438] Table 13: Multi-dose screening of GalNAc-siRNA drugs targeting INHBE in primary monkey hepatocytes

[0439] Example 10: Inhibitory effect of siRNA on human INHBE in humanized INHBE mice.

[0440] Humanized INHBE mice (all male) were randomly divided into groups of 15 each. The experimental groups received XD000155.1, XD000202.1, XD000275.1, and XD000275.36, while the control group received PBS. All animals were administered a single subcutaneous dose of 9 mg / kg, calculated based on their body weight. Animals (n=5 per group) were sacrificed on days 7, 21, and 35 post-administration, and their livers were collected. Liver tissue was homogenized using a tissue homogenizer, and total RNA was extracted using an RNA extraction kit (Vazyme, Cat#RC113). The RNA was then reverse transcribed into cDNA using a reverse transcription kit (Vazyme, Cat#R433), and the expression level of human INHBE mRNA in liver tissue was detected using real-time quantitative PCR (QPCR) using a qPCR kit (Vazyme, Cat#Q712). Primer sequences are shown in Table 14. INHBE mRNA levels were corrected for GAPDH internal control mRNA levels. INHBE mRNA expression level was calculated using the ΔΔCt relative quantification method, expressed as the percentage of remaining INHBE mRNA expression relative to the PBS control group. The calculation formula is as follows: ΔCt = Ct (target gene) – Ct (internal reference gene) ΔΔCt = ΔCt (drug-treated group) - ΔCt (PBS control group) INHBE mRNA relative expression level = 2 -ΔΔCt ×100%

[0441] The results are shown in Table 15. The results indicate that the disclosed siRNA exhibits high levels of inhibitory activity against the INHBE gene in humanized INHBE mice. Compared to the control XD000202.1, XD000275.1 and XD000275.36 showed better knockdown of the INHBE gene in mice. XD000275.36 maintained a good knockdown effect even on day 35, demonstrating good long-term efficacy.

[0442] Table 14: Sequences of Detection Primers

[0443] Table 15: Inhibitory effect of human INHBE mRNA in humanized INHBE mice

[0444] XD000202.1 is AD-1708473.1 in WO2023003922A1.

[0445] Example 11: Pharmacological effects of INHBE siRNA in high-fat diet-induced obese humanized INHBE mice

[0446] This experiment used high-fat diet-induced obese humanized INHBE mice to determine the efficacy of INHBE siRNA drugs.

[0447] Obese humanized INHBE mice induced by a high-fat diet (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were divided into five groups of seven mice each, based on body weight and body fat percentage. The control group received PBS, while the experimental groups received XD000202.1, XD000155.1, XD000275.1, and XD000275.36, respectively, at a dose of 9 mg / kg, administered subcutaneously once a week for a total of four weeks. Body weight was measured twice a week during the treatment period and the observation period after treatment, and body fat percentage was measured weekly. The results are shown in Figures 1 and 2. Compared with the control group, all INHBE siRNA experimental groups significantly reduced the weight and body fat of obese humanized INHBE mice. Compared with XD000202.1, XD000275.1 was more effective in reducing mouse weight and body fat than XD000202.1, while XD000275.36 was comparable to XD000202.1 in reducing mouse weight and body fat.

[0448] The siRNAs of the present invention, particularly the modified siRNAs, exhibit superior in vivo activity, exceeding expectations based on their activity in in vitro screening, and surpassing or being comparable to siRNAs known in the prior art.

Claims

1. A dsRNA activator, wherein the dsRNA activator comprises a sense strand and an antisense strand capable of forming a double-stranded region, wherein the antisense strand comprises a complementary region complementary to at least 15 consecutive nucleotides of a target sequence, and the complementary region and the at least 15 consecutive nucleotides of the target sequence contain a mismatch of no more than 3, 2, or 1 nucleotide, wherein the target sequence is selected from... (i) The nucleotide sequence shown in SEQ ID NO: 280 or 321; (ii) The nucleotide sequence corresponding to positions 2164-2184 or positions 1353-1373 in the sequence shown in SEQ ID NO:664; (iii) The nucleotide sequence shown in any one of SEQ ID NO: 221-279, 281-320, or 322-440; or (iv) The target sequences corresponding to the positions in the INHBE mRNA (e.g., NM_031479.5) shown in Table 1; Optionally, the dsRNA activator is used to inhibit the expression of the gene encoding INHBE.

2. A dsRNA activator, wherein the dsRNA activator comprises a sense strand and an antisense strand capable of forming a double-stranded region, wherein the antisense strand comprises a sequence (complementary region) complementary to a target sequence encoding an mRNA of INHBE, and comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differing from a nucleotide sequence selected from the following: (i) The nucleotide sequence shown in SEQ ID NO:170; (ii) The nucleotide sequence shown in SEQ ID NO:211; or (iii) The nucleotide sequence shown in any one of SEQ ID NO: 110-169, 171-210 and 212-220; Optionally, the antisense strand contains a sequence (complementary region) that is complementary to the target sequence and is between 15 and 30 nucleotides, 18 and 30 nucleotides, or 18 and 23 nucleotides in length, for example, the length of the complementary region is 18, 19, 20, or 21 nucleotides. Optionally, the target sequence of the mRNA is as defined in claim 1; Optionally, the complementary region includes at least nucleotides 2-17, 2-18, 2-19, 2-20, or 20-21, for example, nucleotides 2-19, starting from the 5' end of the antisense strand. Optionally, the dsRNA activator is used to inhibit the expression of the gene encoding INHBE.

3. The dsRNA activator according to claim 1 or 2, wherein the antisense strand has the same number of nucleotides as the mRNA target sequence and is mismatched with the mRNA target sequence at the first nucleotide at the 5' end, for example, the entire nucleotide sequence except for the first nucleotide at the 5' end is completely complementary to the corresponding part of the mRNA target sequence; Optionally, the first nucleotide at the 5' end of the antisense strand is A or U, for example, U.

4. The dsRNA activator according to any one of claims 1 to 3, wherein the dsRNA activator comprises a sense strand and an antisense strand, the sense strand comprising at least 15, 16, 17, 18, or 19 consecutive nucleotides differing from a nucleotide sequence selected from the group consisting of: (i) The nucleotide sequence shown in SEQ ID NO:60; (ii) The nucleotide sequence shown in SEQ ID NO:101; or (iii) The nucleotide sequence shown in any one of SEQ ID NO: 1-59, 61-100 and 102-110; Optionally, the double-stranded region formed by the sense chain and the antisense chain is completely complementary or may contain 1, 2, 3, 4 or 5 non-complementary sites; Optionally, the length of the fully complementary double-stranded region is between 15 and 25 nucleotide pairs, 16 and 25 nucleotide pairs, 16 and 24 nucleotide pairs, 16 and 22 nucleotides, 17 and 24 nucleotide pairs, 17 and 23 nucleotides, 18 and 23 nucleotide pairs, or 19 and 22 nucleotides, for example, the length of the fully complementary double-stranded region is 16, 17, 18, or 19 nucleotides, for example, 19 nucleotides; Optionally, the lengths of the sense strand and the antisense strand are each independently 15-30 nucleotides, such as 17-27 nucleotides, 19-25 nucleotides, 19-23 nucleotides, or 19-21 nucleotides, for example, the length of the sense strand is 19 nucleotides and the length of the antisense strand is 21 nucleotides.

5. The dsRNA activator according to any one of claims 1 to 4, wherein the sense strand and / or antisense strand comprises a 3' or 5' overhang of at least one, two, or three nucleotides, for example, only the antisense strand comprises a 3' overhang of two nucleotides.

6. The dsRNA activator according to any one of claims 1 to 5, comprising a sense strand and an antisense strand forming a double-stranded region, wherein (i) The positive strand contains or is 19 nucleotides. (ii) The antisense strand comprises or is 21 nucleotides and is completely complementary to the mRNA target sequence encoding INHBE in the region except for the first nucleotide at the 5' end, wherein the first nucleotide at the 5' end of the antisense strand is A or U, for example, U; and (iii) The antisense strand contains a 3' overhang of 2 nucleotides compared to the sense strand, and the sense strand and the antisense strand are completely complementary at 19 nucleotides, for example, completely complementary at 19 consecutive nucleotides.

7. The dsRNA activator according to any one of claims 1 to 6, wherein the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand respectively comprise SEQ ID NO:60 / 170, SEQ ID NO:101 / 211, SEQ ID NO:1 / 111, SEQ ID NO:2 / 112, SEQ ID NO:3 / 113, SEQ ID NO:4 / 114, SEQ ID NO:5 / 115, SEQ ID NO:6 / 116, SEQ ID NO:7 / 117, SEQ ID NO:8 / 118, SEQ ID NO:9 / 119, SEQ ID NO:10 / 120, SEQ ID NO:11 / 121, SEQ ID NO:12 / 122, SEQ ID NO:13 / 123, SEQ ID NO:14 / 124, SEQ ID NO:15 / 125, SEQ ID NO:16 / 126, SEQ ID NO:17 / 127, SEQ ID NO:60 / 170, SEQ ID NO:101 / 211, SEQ ID NO:1 / 111, SEQ ID NO:9 / 119, SEQ ID NO:10 / 120, SEQ ID NO:11 / 121, SEQ ID NO:12 / 122, SEQ ID NO:13 / 123, SEQ ID NO:14 / 124, SEQ ID NO:15 / 125, SEQ ID NO:16 / 126, SEQ ID NO:17 / 127, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:10 / 120, SEQ ID NO:101 / 21 ... NO:18 / 128, SEQ ID NO:19 / 129, SEQ ID NO:20 / 130, SEQ ID NO:21 / 131, SEQ ID NO:22 / 132, SEQ ID NO:23 / 133, SEQ ID NO:24 / 134, SEQ ID NO:25 / 135, SEQ ID NO:26 / 136, SEQ ID NO:27 / 137, SEQ ID NO:28 / 138, SEQ ID NO:29 / 139, SEQ ID NO:30 / 140, SEQ ID NO:31 / 141, SEQ ID NO:32 / 142, SEQ ID NO:33 / 143, SEQ ID NO:34 / 144, SEQ ID NO:35 / 145, SEQ ID NO:36 / 146, SEQ ID NO:37 / 147, SEQ ID NO:38 / 148, SEQ ID NO:39 / 149, SEQ ID NO:40 / 150, SEQ ID NO:41 / 151, SEQ ID NO:42 / 152, SEQ ID NO:43 / 153, SEQ ID NO:44 / 154, SEQ ID NO:45 / 155, SEQ ID NO:46 / 156, SEQ ID NO:47 / 157, SEQ ID NO:48 / 158, SEQ ID NO:49 / 159, SEQ ID NO:50 / 160, SEQ ID NO:51 / 161, SEQ ID NO:52 / 162, SEQ ID NO:53 / 163, SEQ IDThe nucleotide sequences shown in SEQ ID NO:54 / 164, SEQ ID NO:55 / 165, SEQ ID NO:56 / 166, SEQ ID NO:57 / 167, SEQ ID NO:58 / 168, SEQ ID NO:59 / 169, SEQ ID NO:61 / 171, SEQ ID NO:62 / 172, SEQ ID NO:63 / 173, SEQ ID NO:64 / 174, SEQ ID NO:65 / 175, SEQ ID NO:66 / 176, SEQ ID NO:67 / 177, SEQ ID NO:68 / 178, SEQ ID NO:69 / 179, SEQ ID NO:70 / 180, SEQ ID NO:71 / 181, SEQ ID NO:72 / 182, SEQ ID NO:73 / 183, SEQ ID NO:74 / 184, SEQ ID NO:75 / 185, SEQ ID NO:76 / 186, SEQ ID NO:77 / 187, SEQ ID NO:78 / 188, SEQ ID NO:79 / 189, SEQ ID NO:80 / 190, SEQ ID NO:81 / 191, SEQ ID NO:82 / 192, SEQ ID NO:83 / 193, SEQ ID NO:84 / 194, SEQ ID NO:85 / 195, SEQ ID NO:86 / 196, SEQ ID NO:87 / 197, SEQ ID NO:88 / 198, SEQ ID NO:89 / 199, SEQ ID NO:90 / 200, SEQ ID NO:91 / 201, SEQ ID NO:92 / 202, SEQ ID NO:93 / 203, SEQ ID NO:94 / 204, SEQ ID NO:95 / 205, SEQ ID NO:96 / 206, SEQ ID NO:97 / 207, SEQ ID NO:98 / 208, SEQ ID NO:99 / 209, SEQ ID NO:100 / 210, SEQ ID NO:102 / 212, SEQ ID NO:103 / 213, SEQ ID NO:104 / 214, SEQ ID NO:105 / 215, SEQ ID NO:106 / 216, SEQ ID NO:107 / 217, SEQ ID NO:108 / 218, SEQ ID NO:109 / 219, SEQ ID NO:110 / 220.

8. The dsRNA activator according to any one of claims 1-7, wherein at least one nucleotide in the dsRNA activator is a modified nucleotide, for example, substantially all nucleotides of the sense strand are modified nucleotides; substantially all nucleotides of the antisense strand are modified nucleotides; or substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides; Optionally, all nucleotides in the sense strand are modified nucleotides; all nucleotides in the antisense strand are modified nucleotides; or all nucleotides in both the sense strand and the antisense strand are modified nucleotides. Optionally, at least one of the modified nucleotides is selected from the group consisting of: LNA, HNA, TNA, CeNA, deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, 2'-5'-linked ribonucleotides (3'-RNA), unlocked nucleotides, conformation-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, etc. 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholinonucleotides, aminophosphates, nucleotides including non-natural bases, tetrahydropyran modified nucleotides, 1,5-dehydrohexyl modified nucleotides, cyclohexenyl modified nucleotides, nucleotides including thiophosphate groups, nucleotides including methylphosphonate groups, nucleotides including 5'-phosphates, nucleotides including 5'-phosphate mimics, vinyl-phosphonate nucleotides, heat-labile nucleotides, ethylene glycol modified nucleotides (GNA), nucleotides containing 2'-phosphates and nucleotides modified with 2-O-(N-methylacetamide); and combinations of one or more thereof; Optionally, at least one of the modified nucleotides is selected from the group consisting of: LNA, HNA, CeNA, 2′-methoxyethyl modified nucleotides, 2′-O-alkyl modified nucleotides, 2′-O-allyl modified nucleotides, 2′-C-allyl modified nucleotides, 2′-fluorine modified nucleotides, 2′-deoxy modified nucleotides, 2′-O-methyl modified nucleotides, 2′-fluorine modified nucleotides, 2′-deoxy modified nucleotides, nucleotides comprising 2′-phosphate esters and nucleotides comprising thiophosphate ester groups; and combinations of one or more thereof; Optionally, the antisense strand contains a thiophosphate bond, and / or the sense strand contains a thiophosphate bond, wherein the thiophosphate nucleotide bond is located between 1-3 nucleotides from the 5' end of the sense strand, and the thiophosphate nucleotide bond is located between 1-3 nucleotides from the 5' end and between 1-3 nucleotides from the 3' end of the antisense strand. Optionally, the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are 2'-fluorinated nucleotides, and the nucleotides at positions 7-9 from the 5' end of the sense strand are 2'-fluorinated nucleotides; or the nucleotides at positions 2, 7, 12, 14, and 16 from the 5' end of the antisense strand are 2'-fluorinated nucleotides, and the nucleotides at positions 7-10 from the 5' end of the sense strand are 2'-fluorinated nucleotides. Optionally, the modified nucleotides in the antisense and sense strands have the following modification patterns: antisense chain: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm, and Chain of Justice: NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm; or, antisense chain: NmsNfsNmNmNmNmNfNmNmNmNmNfNmNfNmNfNmNmNmsNmsNm, and Chain of Justice: NmsNmsNmNmNmNmNfNfNfNfNmNmNmNmNmNmNmNmNm; in Nf = any nucleotide modified with 2'-fluorine; Nfs = any 2'-fluorine modified nucleoside-3' thiophosphate; Nm = any 2'-methoxynucleotide; Nms = any 2'-methoxynucleoside-3'-thiophosphate; 's' indicates that the two nucleotides are linked by a phosphate thioester bond.

9. The dsRNA activator according to claim 8, wherein... The antisense strand comprises any modified nucleotide sequence of the antisense strand in Table 2 of the specification, and / or the sense strand comprises any modified nucleotide sequence of the sense strand in Table 2 of the specification; optionally, the dsRNA activator comprises a combination of modified antisense strands and sense strands as shown in Table 2. Optionally, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand respectively comprise SEQ ID NO:390 / 500, SEQ ID NO:431 / 541, SEQ ID NO:661 / 662, SEQ ID NO:331 / 441, SEQ ID NO:332 / 442, SEQ ID NO:333 / 443, SEQ ID NO:334 / 444, SEQ ID NO:335 / 445, SEQ ID NO:336 / 446, SEQ ID NO:337 / 447, SEQ ID NO:338 / 448, SEQ ID NO:339 / 449, SEQ ID NO:340 / 450, SEQ ID NO:341 / 451, SEQ ID NO:342 / 452, SEQ ID NO:343 / 453, SEQ ID NO:344 / 454, SEQ ID NO:345 / 45 ...500, SEQ ID NO:431 / 541, SEQ ID NO:661 / 662, SEQ ID NO:331 / 441, SEQ ID NO:332 / 442, SEQ ID NO:343 / 453, SEQ ID NO:344 / 4 NO:346 / 456, SEQ ID NO:347 / 457, SEQ ID NO:348 / 458, SEQ ID NO:349 / 459, SEQ ID NO:350 / 460, SEQ ID NO:351 / 461, SEQ ID NO:352 / 462, SEQ ID NO:353 / 463, SEQ ID NO:354 / 464, SEQ ID NO:355 / 465, SEQ ID NO:356 / 466, SEQ ID NO:357 / 467, SEQ ID NO:358 / 468, SEQ ID NO:359 / 469, SEQ ID NO:360 / 470, SEQ ID NO:361 / 471, SEQ ID NO:362 / 472, SEQ ID NO:363 / 473, SEQ ID NO:364 / 474、SEQ ID NO:365 / 475、SEQ ID NO:366 / 476, SEQ ID NO:367 / 477, SEQ ID NO:368 / 478, SEQ ID NO:369 / 479, SEQ ID NO:370 / 480, SEQ ID NO:371 / 481, SEQ ID NO:372 / 482, SEQ ID NO:373 / 483, SEQ ID NO:374 / 484, SEQ ID NO:375 / 485, SEQ ID NO:376 / 486, SEQ ID NO:377 / 487, SEQ ID NO:378 / 488, SEQ ID NO:379 / 489, SEQ ID NO:380 / 490, SEQ IDNO:381 / 491、SEQ ID NO:382 / 492、SEQ ID NO:383 / 493、SEQ ID NO:384 / 494、SEQ ID NO:385 / 495、SEQ ID NO:386 / 496、SEQ ID NO:387 / 497、SEQ ID NO:388 / 498、SEQ ID NO:389 / 499、SEQ ID NO:391 / 501、SEQ ID NO:392 / 502、SEQ ID NO:393 / 503、SEQ ID NO:394 / 504、SEQ ID NO:395 / 505、SEQ ID NO:396 / 506、SEQ ID NO:397 / 507、SEQ ID NO:398 / 508、SEQ ID NO:399 / 509、SEQ ID NO:400 / 510、SEQ ID NO:401 / 511、SEQ ID NO:402 / 512、SEQ ID NO:403 / 513、SEQ ID NO:404 / 514、SEQ ID NO:405 / 515、SEQ ID NO:406 / 516、SEQ ID NO:407 / 517、SEQ ID NO:408 / 518、SEQ ID NO:409 / 519、SEQ ID NO:410 / 520、SEQ ID NO:411 / 521、SEQ ID NO:412 / 522、SEQ ID NO:413 / 523、SEQ ID NO:414 / 524、SEQ ID NO:415 / 525、SEQ ID NO:416 / 526、SEQ ID NO:417 / 527、SEQ ID NO:418 / 528、SEQ ID NO:419 / 529、SEQ ID NO:420 / 530、SEQ ID NO:421 / 531、SEQ ID NO:422 / 532、SEQ ID NO:423 / 533、SEQ ID NO:424 / 534、SEQ ID NO:425 / 535、SEQ ID NO:426 / 536、SEQ ID NO:427 / 537、SEQ ID NO:428 / 538、SEQ ID NO:429 / 539、SEQ ID NO:430 / 540、SEQ ID NO:432 / 542、SEQ ID NO:433 / 543、SEQ ID NO:434 / 544、SEQ ID NO:435 / 545、SEQ ID NO:436 / 546、SEQ ID NO:437 / 547、SEQ IDThe modified nucleotide sequences shown in NO:438 / 548, SEQ ID NO:439 / 549 or SEQ ID NO:440 / 550.

10. The dsRNA activator according to any one of claims 1 to 9, further comprising a ligand capable of delivering dsRNA molecules to liver tissue or hepatocytes as an asialic acid glycoprotein receptor (ASGPR) ligand, for example comprising a portion selected from galactose or galactose derivatives (e.g., galactosamine, N-formylgalactosamine, N-acetylgalactosamine (GalNAc), N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, etc.); preferably, the ligand comprises one or more GalNAc or GalNAc derivatives; Optionally, the oligonucleotide of the dsRNA activator is conjugated to one or more (e.g., 1) ligands, each of which independently has the structure of formula (I): in, Gal represents terminal galactose derivatives independently; L indicates a connector; n is an integer selected from 1, 2, 3, and 4; and The wavy line indicates the oligonucleotide linked to the dsRNA via this valence bond. Preferably, the ligand is linked to the 5' and / or 3' ends of the sense and / or antisense strands of the oligonucleotide of the dsRNA, preferably via a phosphate ester bond or a thiophosphate ester bond. Optionally, each of the ligands independently has the structure of formula (Ia-i): The wavy line indicates the oligonucleotide linked to the dsRNA via this valence bond. Preferably, the ligand is linked to the 5' and / or 3' ends of the sense and / or antisense strands of the dsRNA oligonucleotide, preferably via a phosphate ester bond or a thiophosphate ester bond. R1 is H independently; m1 and m2 are each independently 1, 2, 3, 4, 5, 6, 7 or 8, preferably 3 or 4; q is an integer selected from 1 to 16, preferably an integer selected from 1 to 12, more preferably an integer selected from 8 to 12, such as 8, 9, 10, 11 or 12. Optionally, each of the ligands is an L96 portion having the following structure: The wavy line represents the oligonucleotide linking to dsRNA via this valence bond; Preferably, the ligand is attached to the 3' end of the sense strand of the oligonucleotide of the dsRNA; Preferably, they are linked by phosphate ester bonds or thiophosphate ester bonds.

11. The dsRNA activator according to any one of claims 1 to 10, wherein the antisense strand of the dsRNA activator comprises any modified nucleotide sequence of any one of the antisense strands in Table 3 of the specification, and / or the sense strand comprises any one of the sense strands in Table 3 of the specification having a modified nucleotide sequence of any one of the sense strands at the 3' end conjugated to an L96 ligand structure via a phosphate ester bond. Optionally, the combination of antisense and sense strands in the dsRNA activator is as shown in Table 3 for any combination of antisense and sense strands; Optionally, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand respectively comprise SEQ ID NO:610 / 500, SEQ ID NO:651 / 541, SEQ ID NO:663 / 662, SEQ ID NO:551 / 441, SEQ ID NO:552 / 442, SEQ ID NO:553 / 443, SEQ ID NO:554 / 444, SEQ ID NO:555 / 445, SEQ ID NO:556 / 446, SEQ ID NO:557 / 447, SEQ ID NO:558 / 448, SEQ ID NO:559 / 449, SEQ ID NO:560 / 450, SEQ ID NO:561 / 451, SEQ ID NO:562 / 452, SEQ ID NO:563 / 453, SEQ ID NO:564 / 454, SEQ ID NO:565 / 455, SEQ ID NO:610 / 500, SEQ ID NO:651 / 54 ...663 / 662, SEQ ID NO:551 / 441, SEQ ID NO:552 / 442, SEQ ID NO:553 / 453, SEQ ID NO:564 / 454, SEQ ID NO:565 / 455, SEQ ID NO:610 / 500, SEQ ID NO:651 / 541, SEQ ID NO:562 / 452, SEQ ID NO:563 / 453, SEQ ID NO:564 / 4 NO:566 / 456, SEQ ID NO:567 / 457, SEQ ID NO:568 / 458, SEQ ID NO:569 / 459, SEQ ID NO:570 / 460, SEQ ID NO:571 / 461, SEQ ID NO:572 / 462, SEQ ID NO:573 / 463, SEQ ID NO:574 / 464, SEQ ID NO:575 / 465, SEQ ID NO:576 / 466, SEQ ID NO:577 / 467, SEQ ID NO:578 / 468, SEQ ID NO:579 / 469, SEQ ID NO:580 / 470, SEQ ID NO:581 / 471, SEQ ID NO:582 / 472, SEQ ID NO:583 / 473, SEQ ID NO:584 / 474、SEQ ID NO:585 / 475、SEQ ID NO:586 / 476, SEQ ID NO:587 / 477, SEQ ID NO:588 / 478, SEQ ID NO:589 / 479, SEQ ID NO:590 / 480, SEQ ID NO:591 / 481, SEQ ID NO:592 / 482, SEQ ID NO:593 / 483, SEQ ID NO:594 / 484, SEQ ID NO:595 / 485, SEQ ID NO:596 / 486, SEQ ID NO:597 / 487, SEQ ID NO:598 / 488, SEQ ID NO:599 / 489, SEQ ID NO:600 / 490, SEQ IDNO:601 / 491、SEQ ID NO:602 / 492、SEQ ID NO:603 / 493、SEQ ID NO:604 / 494、SEQ ID NO:605 / 495、SEQ ID NO:606 / 496、SEQ ID NO:607 / 497、SEQ ID NO:608 / 498、SEQ ID NO:609 / 499、SEQ ID NO:611 / 501、SEQ ID NO:612 / 502、SEQ ID NO:613 / 503、SEQ ID NO:614 / 504、SEQ ID NO:615 / 505、SEQ ID NO:616 / 506、SEQ ID NO:617 / 507、SEQ ID NO:618 / 508、SEQ ID NO:619 / 509、SEQ ID NO:620 / 510、SEQ ID NO:621 / 511、SEQ ID NO:622 / 512、SEQ ID NO:623 / 513、SEQ ID NO:624 / 514、SEQ ID NO:625 / 515、SEQ ID NO:626 / 516、SEQ ID NO:627 / 517、SEQ ID NO:628 / 518、SEQ ID NO:629 / 519、SEQ ID NO:630 / 520、SEQ ID NO:631 / 521、SEQ ID NO:632 / 522、SEQ ID NO:633 / 523、SEQ ID NO:634 / 524、SEQ ID NO:635 / 525、SEQ ID NO:636 / 526、SEQ ID NO:637 / 527、SEQ ID NO:638 / 528、SEQ ID NO:639 / 529、SEQ ID NO:640 / 530、SEQ ID NO:641 / 531、SEQ ID NO:642 / 532、SEQ ID NO:643 / 533、SEQ ID NO:644 / 534、SEQ ID NO:645 / 535、SEQ ID NO:646 / 536、SEQ ID NO:647 / 537、SEQ ID NO:648 / 538、SEQ ID NO:649 / 539、SEQ ID NO:650 / 540、SEQ ID NO:652 / 542、SEQ ID NO:653 / 543、SEQ ID NO:654 / 544、SEQ ID NO:655 / 545、SEQ ID NO:656 / 546、SEQ ID NO:657 / 547、SEQ IDThe nucleotide sequences shown in NO:658 / 548, SEQ ID NO:659 / 549 or SEQ ID NO:660 / 550; Optionally, the dsRNA activator comprises a sense strand and an antisense strand, wherein The sense strand consists of a modified nucleotide sequence with an L96 ligand structure attached to the 3' end, as shown in SEQ ID NO:610, and the antisense strand consists of a modified nucleotide sequence, as shown in SEQ ID NO:

500. The sense strand consists of a modified nucleotide sequence, as shown in SEQ ID NO:651, with an L96 ligand structure conjugated to its 3' end, and the antisense strand consists of a modified nucleotide sequence, as shown in SEQ ID NO:541; or The sense strand consists of a modified nucleotide sequence with an L96 ligand structure attached to the 3' end, as shown in SEQ ID NO:663, and the antisense strand consists of a modified nucleotide sequence, as shown in SEQ ID NO:

662.

12. A cell containing a dsRNA activator according to any one of claims 1 to 11.

13. A pharmaceutical composition wherein The pharmaceutical composition comprises a dsRNA activator according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier; Optionally, the dsRNA activator is in an unbuffered solution, such as saline or water; or the dsRNA activator is in a buffered solution, such as an acetate, citrate, prolyl, carbonate, or phosphate, or any combination thereof, such as phosphate buffered solution (PBS).

14. A pharmaceutical combination comprising a dsRNA activator according to any one of claims 1 to 11 and one or more other therapeutic agents, said other therapeutic agents being any therapeutic agent effective, for example, in preventing or treating INHBE-related diseases and / or conditions, covering a variety of therapeutic agents for treating metabolic disorders or obesity or cardiovascular diseases.

15. A method for inhibiting the expression of the INHBE gene in cells, the method comprising contacting the cells with a dsRNA activator according to any one of claims 1 to 11, or a pharmaceutical composition according to claim 13, or a pharmaceutical composition according to claim 14, thereby inhibiting the expression of the INHBE gene in the cells; optionally, the cells are in a subject, optionally the subject is a human being, optionally the subject suffers from an INHBE-related disease and / or condition; optionally, INHBE-related diseases are metabolic disorders, obesity, or cardiovascular diseases. For example, metabolic disorders are metabolic syndrome, and obesity is obesity. Optionally, the method includes contacting the cells with the dsRNA activator to inhibit INHBE expression by at least about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, or about 94%, or inhibiting INHBE expression to reduce INHBE protein levels in the serum or hepatocytes of the subject by at least about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, or about 94%.

16. A method for preventing or treating in a subject an indication or INHBE-related disease and / or condition that would benefit from a reduction in the expression and / or activity of inhibin subunit βE (INHBE), or for weight loss, comprising administering to the subject the dsRNA active agent of any one of claims 1-11 or the pharmaceutical composition of claim 13 or the pharmaceutical composition of claim 14; Optionally, the INHBE-related condition is a metabolic disorder or obesity or cardiovascular disease, such as metabolic syndrome, and obesity, such as obesity. Optionally, administration of the dsRNA activator to the subject results in a decrease in INHBE concentration or content, or a decrease in INHBE protein accumulation or protein content in the subject. Optionally, the method further includes administering one or more other therapeutic agents to the subject, said other therapeutic agents being any therapeutic agent effective, for example, in preventing or treating INHBE-related diseases and / or conditions, covering a variety of therapeutic agents used to treat metabolic disorders or obesity or cardiovascular diseases; Optionally, the method further includes determining the level of INHBE in a sample from the subject.

17. Use of the dsRNA activator of any one of claims 1-11, or the pharmaceutical composition of claim 13, or the pharmaceutical composition of claim 14, in the preparation of a medicament for the prevention or treatment in a subject of an indication having an INHBE-related disease and / or condition that would benefit from a reduction in the expression and / or activity of inhibin subunit βE (INHBE), or for weight loss. Optionally, the INHBE-related condition is either obesity or cardiovascular disease, such as metabolic disorder being metabolic syndrome, and the obesity being, for example, obesity. Optionally, administration of the drug results in a decrease in INHBE concentration or content, or a decrease in INHBE protein accumulation or protein content in the subject. Optionally, the subject's tissues or cells have overexpression or upregulated expression of INHBE.

18. A kit, vial, or syringe containing the dsRNA activator of any one of claims 1-11, the pharmaceutical composition of claim 13, or the pharmaceutical composition of claim 14.

19. An RNA-induced silencing complex (RISC) comprising the antisense strand of any one of the dsRNA activators according to any one of claims 1 to 11.

Images