Dsrna, use thereof, and method for preparing same

Abstract

The present invention relates to a modulator, such as a double-stranded RNA (dsRNA) active agent or an antisense polynucleotide formulation, that can modulate, e.g., inhibit, the expression and / or activity of cell death-inducing DFFA-like effector B (CIDEB). The present invention also relates to a method for inhibiting CIDEB expression and / or activity using such a modulator, and a method for preventing and treating a CIDEB-related disease (such as a chronic inflammatory disease) in a subject.

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 Application No. CN 202410935054.0, filed on July 12, 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, inhibit the expression and / or activity of cell death-induced DFFA-like effector B (CIDEB). The invention also relates to methods for inhibiting CIDEB expression and / or activity using such modulators, and methods for preventing and treating CIDEB-related diseases (such as chronic inflammatory diseases) in subjects.

[0004] Background of the Invention

[0005] CIDEB (Cell Death Inducing DFFA-Like Effector B) is a protein involved in apoptosis and lipid metabolism. As a member of the CIDE (Cell Death Inducing DFFA-Like Effector) family, CIDEB plays a crucial role in regulating cell death and lipid droplet fusion. It is primarily expressed in hepatocytes and participates in lipid droplet formation and fusion, particularly by promoting lipid transfer from small to large droplets, thereby functioning in lipid storage and metabolism.

[0006] The molecular functions of the CIDEB protein include acting as a hepatocyte-specific lipid transferase that promotes the formation of unilocular lipid droplets by mediating lipid droplet fusion. This fusion helps limit lipolysis and promote lipid storage. CIDEB localizes to the surface of lipid droplets and at contact points between them, and promotes the fusion of atypical lipid droplets by driving the directional transfer of net neutral lipids from smaller droplets to larger ones. Furthermore, CIDEB is involved in the biosynthesis and transport of cytoplasmic vesicles and is essential for the lipidation and maturation of very low-density lipoprotein (VLDL), thus being more closely associated with the VLDL pathway. Regarding apoptosis, CIDEB participates in upstream or internal regulation of apoptosis by activating cysteine-type endopeptidase activity, positively regulating cell death, and the release of cytochrome c from mitochondria. Mutations in the CIDEB gene are associated with certain genetic diseases, and it is also involved in the assembly of hepatitis C virus (HCV), which requires entry into hepatocytes.

[0007] CIDEB is a lipid droplet regulatory protein in liver cells that mediates the formation of large lipid droplets from small ones. The accumulation of these droplets in the liver leads to inflammation. Therefore, CIDEB could be an effective target for treating metabolic dysfunction-related steatohepatitis.

[0008] At present, there is still a need for inhibitors with better activity that can effectively regulate CIDEB expression and / or activity for the treatment of metabolic dysfunction-related steatohepatitis, etc. Summary of the Invention

[0009] This invention provides a specific RNAi activator that can effectively reduce the expression and / or activity of CIDEB.

[0010] This invention provides a double-stranded ribonucleic acid (dsRNA) activator for inhibiting the expression of CIDEB 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.

[0011] The present invention also provides a cell comprising the dsRNA activator described herein. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is located within an individual, such as a human body.

[0012] The present invention provides a pharmaceutical composition comprising the dsRNA activator described herein and optionally a pharmaceutically acceptable carrier.

[0013] The present invention provides a pharmaceutical combination comprising the above-mentioned dsRNA activator and one or more other therapeutic agents, said other therapeutic agents being any therapeutic agents effective, for example, in preventing or treating CIDEB-related diseases and / or conditions, covering a wide range of therapeutic agents for treating chronic inflammatory diseases.

[0014] 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 CIDEB gene expression.

[0015] This invention provides the use of the dsRNA activator described herein in the preparation of a drug that inhibits CIDEB gene expression in cells, preferably inhibiting CIDEB expression in liver tissue or hepatocytes.

[0016] The present invention provides a method for preventing or treating diseases and / or conditions caused by CIDEB 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.

[0017] On the other hand, the present invention provides a method for inhibiting CIDEB gene expression, the method comprising contacting an effective amount of the dsRNA activator and / or pharmaceutical composition and / or drug combination described herein with cells, preferably hepatocytes, and optionally maintaining the cells produced in this step for a period of time sufficient to degrade the mRNA transcript of the CIDEB gene, thereby inhibiting the expression of the CIDEB gene in cells such as hepatocytes. Attached Figure Description

[0018] Figure 1 shows the single-point inhibition of CIDEB mRNA after transfection with siRNA molecules in Hep3B;

[0019] Figure 2 shows the inhibition of CIDEB mRNA after transfection with siRNA molecules in Hep3B.

[0020] Figure 3 shows the inhibition of CIDEB mRNA after transfection with siRNA molecules in PHH.

[0021] Figure 4 shows the inhibition of CIDEB mRNA after transfection with siRNA molecules in PCH.

[0022] Figure 5 shows the inhibition of CIDEB mRNA in transgenic hCIDEB mice.

[0023] Figure 6 shows the inhibition of CIDEB mRNA in transgenic hCIDEB mice.

[0024] Invention Details

[0025] 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.

[0026] I. Definition

[0027] 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.

[0028] 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.

[0029] 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.

[0030] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0031] 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.

[0032] As used herein, "regulator" is a molecule that can reduce or increase CIDEB expression and / or activity. Preferably, the regulator is a molecule that can reduce or inhibit CIDEB expression and / or activity, such as an RNAi activator, such as a dsRNA activator.

[0033] Unless otherwise stated, the term "CIDEB," also known as "cell death-inducing DFFA-like effector b," "cell death activator CIDE-B," or "cell death-inducing DFF45-like effector B," and further referred to as cell death-inducing DFFA-like effector B235, cell death-inducing DFFA-like effector B24, cell death activator CIDE-B34, and lipid transferase CIDEB34, refers to a known gene encoding a CIDEB protein from any vertebrate or mammalian source, including but not limited to humans, bovines, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs. The term also refers to fragments and variants of natural CIDEB that maintain at least one in vivo or in vitro activity of natural CIDEB. CIDEB is required for HCV entry into hepatocytes and HCV assembly. CIDEB interacts with the HCV NS5A protein and regulates the association of HCV particles with ApoE. CIDEB also regulates the post-entry phase of the dengue virus (DENV) life cycle. As used herein, the term “CIDEB” also refers to a specific polypeptide expressed in cells through naturally occurring DNA sequence variations in the CIDEB gene, such as single nucleotide polymorphisms (SNPs) within the CIDEB gene. Many SNPs within the CIDEB gene have been identified and can be found, for example, at NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp).

[0034] Exemplary nucleotide and amino acid sequences of CIDEB can be found, for example, in Homo sapiens GenBank accession number NM_001393338.1 (SEQ ID NO: 175; anticomplement SEQ ID NO: 176). Further examples of CIDEB mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, and OMIM. Further information about CIDEB is provided, for example, in the NCBI gene database at http: / / www.ncbi.nlm.nih.gov / gene / 27141. In some embodiments, an iRNA substantially complementary to a region of mouse or rat CIDEB mRNA cross-reacts with human CIDEB mRNA and is a potential candidate for human targeting.

[0035] NM_001393338.1 (SEQ ID NO:175)

[0036] >NM_001393338.1 (Reverse complementary sequence SEQ ID NO: 176)

[0037] As used herein, a “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during CIDEB 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 CIDEB 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 length 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 length is preferably at least 18, 19, 20, or 21 nucleotides. In some embodiments of the invention, the target sequence length is about 19 to about 23 nucleotides. In some embodiments of the invention, the target sequence length is about 21 nucleotides.

[0038] “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.

[0039] When this article refers to a "nucleotide sequence", it means a continuous nucleotide sequence, which can be a natural nucleotide or a modified nucleotide.

[0040] 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 with “sense” and “antisense” orientations relative to the target RNA (i.e., the CIDEB 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.

[0041] 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.

[0042] 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., CIDEB mRNA).

[0043] As used herein, the terms “sense strand” or “lazy strand” or “sense strand” refer to an oligonucleotide strand containing a region substantially complementary to an antisense strand as defined herein, which can complement the antisense strand to form a dsRNA, and which can complement the antisense strand to form a double-stranded region of the dsRNA.

[0044] 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).

[0045] 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 bistrand formed by hybridization, 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 bistrand regions, it means perfect complementarity between two nucleotide chains of the same length 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 may still be considered “perfectly complementary” if the longer antisense oligonucleotide contains a 19-nucleotide sequence that is perfectly complementary to the shorter sense oligonucleotide.

[0046] 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 CIDEB 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.

[0047] 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.

[0048] "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.

[0049] 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.

[0050] 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 non-ribonucleotides or modified ribonucleotides, such as deoxyribonucleotides or chemically modified nucleotides. Additionally, 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. Therefore, 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] As used herein, the term “inhibition” is used interchangeably with “reduction,” “silence,” “downregulation,” and other similar terms, and includes any level of inhibition.

[0055] The expression "inhibit CIDEB" refers to the inhibition of the activity or expression of any CIDEB. The expression "inhibit CIDEB expression" refers to the inhibition of the expression of any CIDEB gene, as well as variants or mutants of the CIDEB gene. Therefore, the CIDEB gene can be a wild-type CIDEB gene, a mutant CIDEB gene, or a transgenic CIDEB gene in the case of genetically manipulated cells, cell groups, or organisms.

[0056] "Inhibition of CIDEB gene expression" includes inhibition of any level of the CIDEB gene, such as at least partial repression of CIDEB gene expression. CIDEB gene expression can be assessed based on the level or change in the level of any variable associated with CIDEB gene expression, such as CIDEB mRNA or CIDEB protein levels. 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 CIDEB 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 or inert control) individuals, cells, or samples.

[0057] As used herein, the term "CIDEB-related disease or condition" refers to a disease or condition caused by or associated with abnormal expression and / or activity of CIDEB. The term "CIDEB-related disease or condition" includes diseases, disorders, or conditions from which one may benefit from decreased CIDEB gene expression, replication, or protein activity. In some embodiments, a CIDEB-related disease or condition is a chronic inflammatory disease. "Chronic inflammatory disease" is any disease, condition, or symptom associated with chronic inflammation. Non-limiting examples of chronic inflammatory diseases include, for example, inflammation of the liver and / or other tissues.

[0058] 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".

[0059] "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 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0060] "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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] The term "drug combination" refers to a non-fixed or fixed combination, including but not limited to a pillbox or a pharmaceutical composition. The term "non-fixed combination" means that the 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 and other therapeutic agents of the present invention used in the drug combination are administered at levels not exceeding those achieved 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. It should be understood that the components in a drug combination are not limited to being packaged in the same pillbox or the same pharmaceutical composition; they may be in separate pharmaceutical compositions or pharmaceutical formulations.

[0066] 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.

[0067] The term "other therapeutic agents" encompasses any therapeutic agent effective for the prevention or treatment of CIDEB-related diseases or conditions (e.g., diseases and / or conditions caused by abnormal expression of the CIDEB gene), other than the dsRNA active agent of the present invention or pharmaceutical compositions comprising the present invention. It also encompasses therapeutic agents for the treatment of chronic inflammatory diseases, such as chronic inflammatory liver disease, such as those selected from various therapeutic agents for hepatic steatosis, hepatitis, liver fibrosis, fatty liver disease (steatohepatitis), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and cirrhosis.

[0068] 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.

[0069] 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.

[0070] 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."

[0071] "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 (amniotic fluid), peritoneal fluid (ascites), 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 contaminate with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. In some embodiments, the subject sample described herein is tissue, such as liver tissue homogenate, or hepatocytes.

[0072] In this document, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and the description includes both the possibility that the event or condition occurs and the possibility that it does not occur. For example, "alkyl" in "optionally substituted" includes "alkyl" and "substituted alkyl" as defined below. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is spatially impractical, synthetically infeasible, and / or inherently unstable.

[0073] In this document, "alkyl" refers to a straight-chain or branched chain having a specified number of carbon atoms, which can be from 1 to 30 carbon atoms, for example, 1 to 20 carbon atoms, 1 to 16 carbon atoms, or 12 to 16 carbon atoms. When referring to an alkyl residue having a specific number of carbons, it is intended to cover all branched and straight-chain forms having that number of carbons, and optionally substituted. When an alkyl group is substituted with one or more hydroxyl groups, it may be called a hydroxyalkyl group. In particular, the term "hydroxyethyl" refers to an ethyl group substituted with one hydroxyl group.

[0074] Accordingly, the term "alkylene" refers to a divalent group derived from an alkane, such as a straight-chain or branched alkane having a specified number of carbon atoms, by removing two hydrogen atoms, for example, from 0 to 30 carbon atoms. It is understood that when the number of carbon atoms is 0, i.e., C0 alkylene, it indicates a valence bond. Examples include C... 0-20 Alkylene, C 0-12 Alkylene, C 0-8 Alkylene, C 0-6 Alkylenes, etc.

[0075] In this document, "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group having a specified number of carbon atoms, comprising at least one double bond, for example, from 2 to 30 carbon atoms. Specifically, the alkenyl group has 2 to 20, for example 2 to 16, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms, and is optionally substituted. For example, as used herein, the term "C 2-20 "Alkenyl" refers to alkenyl groups with straight or branched chains having 2 to 20 carbon atoms, such as vinyl, propenyl, allyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, etc.

[0076] In this document, "alkynyl" refers to a straight-chain or branched unsaturated hydrocarbon group having a specified number of carbon atoms, including at least one triple bond, such that the number of carbon atoms can be, for example, 2 to 30 carbon atoms. Specifically, the alkynyl group has 2 to 20, for example 2 to 16, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms, and is optionally substituted. For example, as used herein, the term "C2-C6 alkynyl" refers to a straight-chain or branched alkynyl group having 2 to 6 carbon atoms, such as ethynyl, propynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-methyl-1-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, etc.

[0077] The terms "alkoxy" and "alkyl-O-" are used interchangeably to refer to an alkyl group as defined above, linked by an oxygen atom. Preferably, the alkoxy group has 1-20 carbon atoms (C... 1-20 alkoxy group), 1-16 carbon atoms (C 1-16 alkoxy group), 1-6 carbon atoms (C 1-6 alkoxy group), 1-4 carbon atoms (C 1-4 alkoxy group or 1-3 carbon atoms (C 1-3 Alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, etc.), pentoxy (including n-pentoxy, isopentoxy, neopentoxy, etc.), hexoxy, heptoxy, octoxy, etc.

[0078] The terms "alkathio" and "alkyl-S-" are used interchangeably to refer to an alkyl group as defined above, linked by a sulfur atom. Preferably, the alkathio group has 1-20 carbon atoms (C2-C2). 1-20 Alkylthioyl), 1-16 carbon atoms (C 1-16 Alkylthioyl), 1-6 carbon atoms (C 1-6 Alkyl thioyl), 1-4 carbon atoms (C 1-4 (alkylthio) or 1-3 carbon atoms (C 1-3 Alkylthio (Alkylthio). Examples of alkylthio groups include, but are not limited to, methylthio, ethylthio, propanethio, pentylthio, hexylthio, heptylthio, octylthio, etc.

[0079] The term "cycloalkyl" refers to a fully or partially saturated non-aromatic monocyclic or bicyclic hydrocarbon group consisting of carbon and hydrogen atoms, preferably fully saturated. Preferably, the cycloalkyl group has 3-8 ring carbon atoms (C6H ... 3-8 cycloalkyl groups or 5-6 cyclic carbon atoms (C 5-6 Cycloalkyl groups. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. When a cycloalkyl group is replaced by one or more hydroxyl groups, it may be called a "hydroxycycloalkyl group," including C14 and C24. 3-8 Hydroxycycloalkyl or C 5-6 Hydroxycycloalkyl.

[0080] In this document, the term "aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6-20, for example, 6-12 carbon atoms in the ring moiety, which may be substituted or unsubstituted. Preferably, the aryl group is (C6-C6) 10Aryl. Non-limiting examples include phenyl, biphenyl, naphthyl, or tetrahydronaphthyl, each of which may optionally be substituted with 1 to 4 substituents, such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxyl, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, mercapto, alkyl-S-, aryl-S-, nitro, cyano, carboxyl, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamide, heterocyclic, etc. Preferably, the aryl group is an optionally substituted phenyl group.

[0081] The terms "-CO-" or "-C(O)-" represent carbonyl groups.

[0082] In this article, valence bond When present in a group or part, it indicates that the group or part is connected to the rest of the molecule via the wavy valence bond. When present in the molecule, it indicates that the chiral carbon atom connected by the wavy valence bond can have any chiral configuration, such as R and / or S. Additionally, when the wavy line passes through the valence bond originating from a group or part, for example in… In this context, it indicates that the group or part of it is connected to the rest of the molecule via the valence bond.

[0083] II. dsRNA activator

[0084] This invention provides RNAi activators for inhibiting CIDEB, 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 CIDEB gene in cells such as hepatocytes in a subject (e.g., mammals, such as individuals susceptible to CIDEB-related diseases or conditions).

[0085] 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 for the purposes of this invention). 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.

[0086] 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 CIDEB gene (e.g., the human CIDEB gene) by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, or about 93% 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, for example, concentrations of about 10 nM, about 1 nM, or about 0.1 nM of dsRNA, such as siRNA, via 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.

[0087] 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 CIDEB 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 at 100 nM or 10 nM) of dsRNA, such as siRNA, as provided herein by the qPCR method.

[0088] In some embodiments, the dsRNA active agents of this disclosure, such as siRNA (including siRNA having modified nucleotides and siRNA having modified nucleotides and ligands), inhibit the expression of the CIDEB gene (e.g., the human CIDEB 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%, about 90%, or about 95%, for example, by detection of a single subcutaneous administration to mice, as described in, for example, as in Examples 7 or 8.

[0089] In some embodiments, the dsRNA active agents of this disclosure, such as siRNAs (including siRNAs with modified nucleotides and siRNAs with modified nucleotides and ligands), exhibit prolonged inhibitory effects on the target gene CIDEB, particularly at low doses, such as 1 week, 2 weeks, or 3 weeks after subcutaneous administration of a low dose of 1 mpk. In some embodiments, the dsRNA active agents of this disclosure, such as siRNAs (including siRNAs with modified nucleotides and siRNAs with modified nucleotides and ligands), exhibit prolonged in vivo inhibition (knockdown) of the target gene CIDEB, such as siRNAs with modified nucleotides or 1 week, 2 weeks, or 3 weeks after administration, superior to control siRNAs, such as known CIDEB-targeting siRNAs, such as siRNA in WO2023034837A2, such as AD-1699964.1.

[0090] 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 the expression of the CIDEB gene. 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 18, 19, 20, or 21 nucleotides. In some embodiments, the length of the complementary region is at least 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 the following nucleotides from the 5' end: nucleotides 1-16, nucleotides 2-16, nucleotides 1-17, nucleotides 2-17, nucleotides 1-18, nucleotides 2-18, nucleotides 1-19, nucleotides 2-19, nucleotides 1-20, nucleotides 2-20, nucleotides 1-21, or nucleotides 2-21. In some embodiments, the complementary region of the antisense strand contains at least 2-19 consecutive nucleotides starting from the 5' end of the antisense strand. In some embodiments, the complementary region of the antisense strand contains, or is composed of, consecutive nucleotides starting from the 5' end of the antisense strand, starting from positions 2-19, 2-20, or 2-21.

[0091] 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.

[0092] 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 the mRNA formed during CIDEB 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' end and / or the 3' end, 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.

[0093] The other strand of dsRNA (the sense strand) contains a region complementary to the antisense strand, allowing the two strands to hybridize and form a double-stranded structure (double-stranded region) when combined under appropriate conditions.

[0094] In some embodiments, 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, the antisense strand of the dsRNA, from the second nucleotide from the 5' end to the first, second, or third nucleotide from the 3' end, is completely complementary to the corresponding portion of the target sequence. 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.

[0095] 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.

[0096] In some implementations, the dsRNA described herein targets the sequence of the CIDEB gene at or near the location shown in Table 1 of the CIDEB genome (e.g., NM_001393338.1).

[0097] 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, of the sequence shown in SEQ ID NO:175 or its complementary sequence, in the CIDEB genome (e.g., NM_001393338.1). The nucleic acid sequence shown in NO:175 consists of sequences 1378-1398, 1379-1399, 1380-1400, 1381-1401, 1411-1431, 1418-1438, 1419-1439, 1420-1440, 1557-1577, 1558-1578, 1559-1579, 1560-1580, 1566-1586, and 1577- 1597, 1578-1598, 1600-1620, 1603-1623, 1606-1626, 1607-1627, 1612-1632, 1632-1652, 1775-1795, 1779-1799, 1781-1801, 1782-1802, 1796-1816, 1808-1828, 1809-1829, 1822-1 842, 1823-1843, 1893-1913, 1894-1914, 1945-1965, 1949-1969, 1952-1972, 2046-2066, 2047-2067, 2048-2068, 2049-2069, 2050-2070, 2051-2071, 2052-2072, 2079-2099, 2080-2 The sequence corresponding to bits 100, 2081-2101, 2082-2102, 2083-2103, 2086-2106, 2099-2119, 2101-2121, 2102-2122, 2103-2123, 2106-2126, 2218-2238, 2240-2260, 2292-2312, 2344-2364, or 2346-2366.

[0098] In some implementations, the dsRNA described herein targets a nucleotide sequence (target sequence) of the mRNA selected from the CIDEB gene:

[0099] (i) A continuous sequence of CIDEB mRNA at or near the CIDEB genome (e.g., within 10 nucleotides before and after) 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 said position;

[0100] (ii) The CIDEB genome (e.g., NM_001393338.1) mRNA or the nucleic acid sequence shown in SEQ ID NO:175 or its complementary sequence at positions 1378, 1379, 1380, 1381, 1411, 1418, 1419, 1420, 1557, 1558, 1559, 1560, 1566, 1577, 1578, 1600, 1603, 1606, 1607, 1612, 1632, 1775, 1779, 1781, 1782, 1796, 1808, 1809, 1822, 1823, 189 3. At least 15-35 consecutive nucleotides starting at position 1894, 1945, 1949, 1952, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2079, 2080, 2081, 2082, 2083, 2086, 2099, 2101, 2102, 2103, 2106, 2218, 2240, 2292, 2344, or 2346, for example, 18, 19, 20, or 21 consecutive nucleotides;

[0101] (iii) CIDEB genomic (e.g., NM_001393338.1) mRNA or the nucleic acid sequence shown in SEQ ID NO: 175 or its complementary sequence containing digits 1378-1398, 1379-1399, 1380-1400, 1381-1401, 1411-1431, 1418-1438, 1419-1439, 1420-1440, 1557-1577, 1558-1578, 1559-1579, 1560-1580, 1566-1586, 1577-1597, 1 578-1598, 1600-1620, 1603-1623, 1606-1626, 1607-1627, 1612-1632, 1632-1652, 1775-1795, 1779-1799, 1781-1801, 1782-1802, 1796-1816, 1808-1828, 1809-1829, 1822-1842, 1823-1843, 1893-1 913, 1894-1914, 1945-1965, 1949-1969, 1952-1972, 2046-2066, 2047-2067, 2048-2068, 2049-2069, 2050-2070, 2051-2071, 2052-2072, 2079-2099, 2080-2100, 2081-2101, 2082-2102, 2083-2103, 2 The 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, or 35th consecutive nucleotides of the sequence corresponding to positions 086-2106, 2099-2119, 2101-2121, 2102-2122, 2103-2123, 2106-2126, 2218-2238, 2240-2260, 2292-2312, 2344-2364, or 2346-2366;

[0102] (iv) CIDEB genomic (e.g., NM_001393338.1) mRNA or nucleic acid sequence of SEQ ID NO:175, numbers 1378-1398, 1379-1399, 1380-1400, 1381-1401, 1411-1431, 1418-1438, 1419-1439, 1420-1440, 1557-1577, 1558-1578, 1559-1579, 1560-1580, 1566-1586, 1577-1597, 1578-1 598, 1600-1620, 1603-1623, 1606-1626, 1607-1627, 1612-1632, 1632-1652, 1775-1795, 1779-1799, 1781-1801, 1782-1802, 1796-1816, 1808-1828, 1809-1829, 1822-1842, 1823-1843, 1893-1913 1894-1914, 1945-1965, 1949-1969, 1952-1972, 2046-2066, 2047-2067, 2048-2068, 2049-2069, 2050-2070, 2051-2071, 2052-2072, 2079-2099, 2080-2100, 2081-2101, 2082-2102, 2083-2103, 2 The sequence corresponding to positions 086-2106, 2099-2119, 2101-2121, 2102-2122, 2103-2123, 2106-2126, 2218-2238, 2240-2260, 2292-2312, 2344-2364, or 2346-2366, consisting of any 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides, preferably the nucleotide sequence corresponding to the positions mentioned above; or

[0103] (v) A nucleotide sequence that contains or consists of the nucleotide sequences shown in any of SEQ ID NO:117-174.

[0104] 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 CIDEB gene shown in Table 1.

[0105] 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 CIDEB genome (e.g., NM_001393338.1) 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 CIDEB mRNA target sequence targeted by the dsRNA described herein comprises or is composed of the nucleotide sequence shown in any of SEQ ID NO: 117-174. In some embodiments, the target sequence comprises a continuous sequence of CIDEB mRNA at or near the CIDEB genome (e.g., NM_001393338.1) mRNA location shown in Table 1. In some embodiments, the antisense strand of the dsRNA has the same number of nucleotides as the CIDEB target sequence (e.g., the target sequence shown in Table 2).

[0106] In some embodiments, the antisense strand of the dsRNA is fully complementary, substantially complementary, or at least partially complementary to the CIDEB 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 CIDEB 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 CIDEB 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.

[0107] In some embodiments, the target sequence comprises a continuous sequence of CIDEB mRNA located at or near the CIDEB genome position (e.g., NM_001393338.1) as shown in Table 1. In some embodiments, the mRNA sequence targeted by the dsRNA described herein (i.e., the CIDEB gene target sequence) comprises, or is composed of, the nucleotide sequence shown in any of SEQ ID NO: 117-174.

[0108] In some embodiments, the antisense strand of the dsRNA disclosed herein contains complementary regions that are completely, substantially, or at least partially complementary to the target sequences disclosed in Table 2. In some embodiments, the antisense strand of the dsRNA has the same number of nucleotides as or differs from the CIDEB gene target sequence (e.g., the target sequence corresponding to the position shown in Table 1 or the target sequence shown in Table 2) by 1, 2, or 3 nucleotides. In some embodiments, the antisense strand of the dsRNA is completely complementary to the CIDEB gene target sequence (e.g., the target sequence corresponding to the position shown in Table 1 or the 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, mismatches at 1-3 nucleotides. In some embodiments, the antisense strand of the dsRNA is completely complementary to the CIDEB gene target sequence (e.g., the target sequence corresponding to the position shown in Table 1 or the target sequence shown in Table 2) except for 1, 2, or 3 nucleotides. In some embodiments, the antisense strand of the dsRNA is completely complementary to the CIDEB gene target sequence (e.g., the target sequence corresponding to the position shown in Table 1 or the 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.

[0109] 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.

[0110] In some embodiments, the antisense and sense strands hybridize to form a double-stranded region. 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 embodiments, the length of the double-stranded region is 18, 19, 20, or 21 nucleotide pairs, for example, 19 nucleotide pairs.

[0111] 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 nucleotide pairs, 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.

[0112] 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 compared 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] In some embodiments, the present invention relates to a double-stranded RNA (dsRNA) activator for inhibiting the expression of cell death-induced DFFA-like effector B (CIDEB), 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 CIDEB 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.

[0118] In some embodiments, the present invention relates to a double-stranded RNA (dsRNA) activator for inhibiting cell death-induced DFFA-like effector B (CIDEB) expression, 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 CIDEB. In some embodiments, the antisense strand is completely complementary to the target sequence of the CIDEB 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.

[0119] 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).

[0120] In some embodiments, the dsRNA of the present invention comprises a sense strand and an antisense strand forming a double-stranded region, wherein

[0121] (i) The positive strand contains or is 19 nucleotides.

[0122] (ii) The antisense strand comprises or is 21 nucleotides and is completely complementary to the target sequence of the CIDEB 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;

[0123] (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, the sense strand and the antisense strand are completely complementary over 1-19 consecutive nucleotides starting from the 5' end.

[0124] In one aspect of the invention, the invention relates to a double-stranded RNA (dsRNA) activator for inhibiting the expression of cell death-induced DFFA-like effector B (CIDEB), 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.

[0125] 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.

[0126] 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 comprising at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differing by no more than 2 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.

[0127] 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 comprising at least 15, 16, 17, 18, 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.

[0128] 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:1-58. In some specific embodiments, the antisense strand differs from the nucleotide sequence shown in any one of SEQ ID NO:1-58 by no more than 1, 2, or 3 nucleotides. In some specific embodiments, the antisense and sense strands comprise or consist of the nucleotide sequences shown in any one of SEQ ID NO:1-58.

[0129] 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:59-116. In some specific embodiments, the positive strand differs from the nucleotide sequence shown in any one of SEQ ID NO:59-116 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 any one of SEQ ID NO:59-116.

[0130] 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)).

[0131] In some embodiments, the sense and antisense strands of the dsRNA activator respectively comprise, or are composed of, either the nucleotide sequences of the sense and antisense strands selected from any combination of sense and antisense strands in Table 1, or are composed of the nucleotide sequences respectively.

[0132] 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.

[0133] In some specific implementations, the dsRNA activator comprises an antisense strand and a sense strand.The antisense chain and the justice chain respectively include SEQ ID NO:1 / 59, SEQ ID NO:2 / 60, SEQ ID NO:3 / 61, SEQ ID NO:4 / 62, SEQ ID NO:5 / 63, SEQ ID NO:6 / 64, SEQ ID NO:7 / 65, SEQ ID NO:8 / 66, SEQ ID NO:9 / 67, SEQ ID NO:10 / 68, SEQ ID NO:11 / 69, SEQ ID NO:12 / 70, SEQ ID NO:13 / 71, SEQ ID NO:14 / 72, SEQ ID NO:15 / 73, SEQ ID NO:16 / 74, SEQ ID NO:17 / 75, SEQ ID NO:18 / 76, SEQ ID NO:19 / 77, SEQ ID NO:20 / 78, SEQ ID NO:21 / 79, SEQ ID NO:22 / 80, SEQ ID NO:23 / 81, SEQ ID NO:24 / 82, SEQ ID NO:25 / 83, SEQ ID NO:26 / 84, SEQ ID NO:27 / 85, SEQ ID NO:28 / 86, SEQ ID NO:29 / 87, SEQ ID NO:30 / 88, SEQ ID NO:31 / 89, SEQ ID NO:32 / 90, SEQ ID NO:33 / 91, SEQ ID NO:34 / 92, SEQ ID NO:35 / 93, SEQ ID NO:36 / 94, SEQ ID NO:37 / 95, SEQ ID NO:38 / 96, SEQ ID NO:39 / 97, SEQ ID NO:40 / 98, SEQ ID NO:41 / 99, SEQ ID NO:42 / 100, SEQ ID NO:43 / 101, SEQ ID NO:44 / 102, SEQ ID NO:45 / 103, SEQ ID NO:46 / 104, SEQ ID NO:47 / 105, SEQ ID NO:48 / 106, SEQ At least 15, 16, 17, 18, or 19 consecutive nucleotides in the nucleotide sequences shown in SEQ ID NO:49 / 107, SEQ ID NO:50 / 108, SEQ ID NO:51 / 109, SEQ ID NO:52 / 110, SEQ ID NO:53 / 111, SEQ ID NO:54 / 112, SEQ ID NO:55 / 113, SEQ ID NO:56 / 114, SEQ ID NO:57 / 115, or SEQ ID NO:58 / 116.Optionally, the antisense strand also has a 3' overhang of one or two nucleotides, and optionally, the first nucleotide at the 5' end of the antisense strand is A or U, for example, U.

[0134] In some specific implementations, the dsRNA activator comprises an antisense strand and a sense strand.The antisense chain and the justice chain respectively include SEQ ID NO:1 / 59, SEQ ID NO:2 / 60, SEQ ID NO:3 / 61, SEQ ID NO:4 / 62, SEQ ID NO:5 / 63, SEQ ID NO:6 / 64, SEQ ID NO:7 / 65, SEQ ID NO:8 / 66, SEQ ID NO:9 / 67, SEQ ID NO:10 / 68, SEQ ID NO:11 / 69, SEQ ID NO:12 / 70, SEQ ID NO:13 / 71, SEQ ID NO:14 / 72, SEQ ID NO:15 / 73, SEQ ID NO:16 / 74, SEQ ID NO:17 / 75, SEQ ID NO:18 / 76, SEQ ID NO:19 / 77, SEQ ID NO:20 / 78, SEQ ID NO:21 / 79, SEQ ID NO:22 / 80, SEQ ID NO:23 / 81, SEQ ID NO:24 / 82, SEQ ID NO:25 / 83, SEQ ID NO:26 / 84, SEQ ID NO:27 / 85, SEQ ID NO:28 / 86, SEQ ID NO:29 / 87, SEQ ID NO:30 / 88, SEQ ID NO:31 / 89, SEQ ID NO:32 / 90, SEQ ID NO:33 / 91, SEQ ID NO:34 / 92, SEQ ID NO:35 / 93, SEQ ID NO:36 / 94, SEQ ID NO:37 / 95, SEQ ID NO:38 / 96, SEQ ID NO:39 / 97, SEQ ID NO:40 / 98, SEQ ID NO:41 / 99, SEQ ID NO:42 / 100, SEQ ID NO:43 / 101, SEQ ID NO:44 / 102, SEQ ID NO:45 / 103, SEQ ID NO:46 / 104, SEQ ID NO:47 / 105, SEQ ID NO:48 / 106, SEQ The nucleotide sequences shown in SEQ ID NO:49 / 107, SEQ ID NO:50 / 108, SEQ ID NO:51 / 109, SEQ ID NO:52 / 110, SEQ ID NO:53 / 111, SEQ ID NO:54 / 112, SEQ ID NO:55 / 113, SEQ ID NO:56 / 114, SEQ ID NO:57 / 115, or SEQ ID NO:58 / 116, or each of the nucleotide sequences shown, are composed of the nucleotide sequences shown.

[0135] In some embodiments, the dsRNA activator comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand comprise the nucleotide sequence shown in SEQ ID NO:8 and the nucleotide sequence shown in SEQ ID NO:66, respectively; or the antisense strand and the sense strand comprise the nucleotide sequence shown in SEQ ID NO:42 and the nucleotide sequence shown in SEQ ID NO:100, respectively.

[0136] 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 sequence expression, as those skilled in the art will understand, the antisense strand serving as a guide RNAi for specific binding to the target sequence 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 nucleotides 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.

[0137] 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.

[0138] 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).

[0139] In some embodiments, the dsRNA activator of the present invention comprises at least one 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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).

[0149] 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-anhydrohexyl-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 nucleotides modified with 2-O-(N-methylacetamide); and combinations thereof.

[0150] In some embodiments, at least one of the modified nucleotides 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 modified nucleotides of 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.

[0151] 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, 17 nucleotides are 2'-methoxy modified nucleotides. In some embodiments, the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand of the dsRNA activator of the present invention are 2'-methoxy modified nucleotides. In some embodiments, the sense strand of the dsRNA activator of the present invention comprises 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, 16 nucleotides are 2'-methoxy modified nucleotides. In some embodiments, the nucleotides at positions 1-6 and 10-19 of the sense strand of the dsRNA activator of the present invention are 2'-methoxy modified nucleotides.

[0152] 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 nucleotides are 2'-fluorinated nucleotides. In some embodiments, the nucleotides at positions 2, 6, 14, and 16 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 nucleotides are 2'-fluorinated nucleotides. In some embodiments, the nucleotides at positions 7-9 of the sense strand of the dsRNA activator of the present invention are 2'-fluorinated nucleotides.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] In some implementations, the modified nucleotides in the sense and antisense strands of the dsRNA activator have the following modification patterns:

[0158] antisense chain:

[0159] NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm; and / or

[0160] Chain of Justice:

[0161] NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm

[0162] in,

[0163] Nf = any nucleotide modified with 2'-fluorine;

[0164] Nm = any 2'-methoxynucleotide;

[0165] 's' indicates that the two nucleotides are linked by a phosphate thioester bond.

[0166] 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.

[0167] In some embodiments, the sense and antisense strands of the dsRNA activator respectively comprise modified nucleotide sequences of the sense and antisense strands selected from any combination of sense and antisense strands in Table 2, or are composed of the modified nucleotide sequences respectively.

[0168] In some embodiments, 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.

[0169] In some embodiments, the dsRNA activator is any of the dsRNA activators shown in Table 2.

[0170] In some specific embodiments, the dsRNA activator comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand respectively comprise SEQ ID NO:177 / 235, SEQ ID NO:178 / 236, SEQ ID NO:179 / 237, SEQ ID NO:180 / 238, SEQ ID NO:181 / 239, SEQ ID NO:182 / 240, SEQ ID NO:183 / 241, SEQ ID NO:184 / 242, SEQ ID NO:185 / 243, SEQ ID NO:186 / 244, SEQ ID NO:187 / 245, SEQ ID NO:188 / 246, SEQ ID NO:189 / 247, SEQ ID NO:190 / 248, SEQ ID NO:191 / 249, SEQ ID NO:192 / 250, SEQ ID NO:193 / 251, SEQ ID NO:194 / 252, SEQ ID NO:19 ... NO:195 / 253, SEQ ID NO:196 / 254, SEQ ID NO:197 / 255, SEQ ID NO:198 / 256, SEQ ID NO:199 / 257, SEQ ID NO:200 / 258, SEQ ID NO:201 / 259, SEQ ID NO:202 / 260, SEQ ID NO:203 / 261, SEQ ID NO:204 / 262, SEQ ID NO:205 / 263, SEQ ID NO:206 / 264, SEQ ID NO:207 / 265, SEQ ID NO:208 / 266, SEQ ID NO:209 / 267, SEQ ID NO:210 / 268, SEQ ID NO:211 / 269, SEQ ID NO:212 / 270, SEQ ID NO:213 / 271、SEQ ID NO:214 / 272、SEQ ID NO:215 / 273, SEQ ID NO:216 / 274, SEQ ID NO:217 / 275, SEQ ID NO:218 / 276, SEQ ID NO:219 / 277, SEQ ID NO:220 / 278, SEQ ID NO:221 / 279, SEQ ID NO:222 / 280, SEQ ID NO:223 / 281, SEQ ID NO:224 / 282, SEQ ID NO:225 / 283, SEQ ID NO:226 / 284, SEQ ID NO:227 / 285, SEQ ID NO:228 / 286, SEQ IDAt least 15, 16, 17, 18, or 19 consecutive modified nucleotides in the nucleotide sequences shown in NO:229 / 287, SEQ ID NO:230 / 288, SEQ ID NO:231 / 289, SEQ ID NO:232 / 290, SEQ ID NO:233 / 291, or SEQ ID NO:234 / 292, 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.

[0171] In some specific embodiments, the dsRNA activator comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand respectively comprise SEQ ID NO:177 / 235, SEQ ID NO:178 / 236, SEQ ID NO:179 / 237, SEQ ID NO:180 / 238, SEQ ID NO:181 / 239, SEQ ID NO:182 / 240, SEQ ID NO:183 / 241, SEQ ID NO:184 / 242, SEQ ID NO:185 / 243, SEQ ID NO:186 / 244, SEQ ID NO:187 / 245, SEQ ID NO:188 / 246, SEQ ID NO:189 / 247, SEQ ID NO:190 / 248, SEQ ID NO:191 / 249, SEQ ID NO:192 / 250, SEQ ID NO:193 / 251, SEQ ID NO:194 / 252, SEQ ID NO:19 ... NO:195 / 253, SEQ ID NO:196 / 254, SEQ ID NO:197 / 255, SEQ ID NO:198 / 256, SEQ ID NO:199 / 257, SEQ ID NO:200 / 258, SEQ ID NO:201 / 259, SEQ ID NO:202 / 260, SEQ ID NO:203 / 261, SEQ ID NO:204 / 262, SEQ ID NO:205 / 263, SEQ ID NO:206 / 264, SEQ ID NO:207 / 265, SEQ ID NO:208 / 266, SEQ ID NO:209 / 267, SEQ ID NO:210 / 268, SEQ ID NO:211 / 269, SEQ ID NO:212 / 270, SEQ ID NO:213 / 271、SEQ ID NO:214 / 272、SEQ ID NO:215 / 273, SEQ ID NO:216 / 274, SEQ ID NO:217 / 275, SEQ ID NO:218 / 276, SEQ ID NO:219 / 277, SEQ ID NO:220 / 278, SEQ ID NO:221 / 279, SEQ ID NO:222 / 280, SEQ ID NO:223 / 281, SEQ ID NO:224 / 282, SEQ ID NO:225 / 283, SEQ ID NO:226 / 284, SEQ ID NO:227 / 285, SEQ ID NO:228 / 286, SEQ IDThe modified nucleotide sequences shown in NO:229 / 287, SEQ ID NO:230 / 288, SEQ ID NO:231 / 289, SEQ ID NO:232 / 290, SEQ ID NO:233 / 291 or SEQ ID NO:234 / 292, or each of the modified nucleotide sequences shown.

[0172] In some specific embodiments, the dsRNA activator comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand comprise the nucleotide sequence shown in SEQ ID NO:184 and the nucleotide sequence shown in SEQ ID NO:242, respectively; or the antisense strand and the sense strand comprise the nucleotide sequence shown in SEQ ID NO:218 and the nucleotide sequence shown in SEQ ID NO:276, respectively.

[0173] 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, as will be readily understood by those skilled in the art, 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)):

[0174] T = 5'-methyluridine-3'-phosphate

[0175] Nf = any 2'-fluorine modified nucleotide

[0176] Nfs = any 2'-fluorine modified nucleoside-3'-thiophosphate ester

[0177] Af = 2'-Fluoroadenosine-3'-phosphate

[0178] Afs = 2'-Fluoroadenosine-3'-Thiophosphate

[0179] Cf = 2'-Fluorocytidine-3'-phosphate

[0180] Cfs = 2'-Fluorocytidine-3'-Thiophosphate

[0181] Gf = 2'-Fluoroguanosine-3'-phosphate

[0182] Gfs = 2'-Fluoroguanosine-3'-Thiophosphate

[0183] Tf = 2'-Fluoro-5'-methyluridine-3'-phosphate

[0184] Tfs = 2'-Fluoro-5'-methyluridine-3'-thiophosphate

[0185] Uf = 2'-fluorouridine-3'-phosphate

[0186] Ufs = 2'-fluorouridine-3'-thiophosphate

[0187] Nm = any 2'-methoxynucleotide

[0188] Nms = any 2'-methoxynucleoside-3'-thiophosphate

[0189] Am = 2'-methoxyadenosine-3'-phosphate

[0190] Ams = 2'-methoxyadenosine-3'-thiophosphate

[0191] Tm = 2'-methoxythymidine-3'-phosphate

[0192] Tms = 2'-methoxythymidine-3'-thiophosphate

[0193] Um = 2'-methoxyuridine-3'-phosphate

[0194] Ums = 2'-methoxyuridine-3'-thiophosphate

[0195] Gm = 2'-methoxyguanosine-3'-phosphate

[0196] Gms = 2'-methoxyguanosine-3'-thiophosphate

[0197] Cm = 2'-methoxycytidine-3'-phosphate

[0198] Cms = 2'-methoxycytidine-3'-thiophosphate

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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. In some embodiments, the ligands are monosaccharides.

[0204] 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-Plus TM (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.

[0205] 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.

[0206] 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").

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] In some embodiments, the ligand is L96, see, for example, WO2009073809 and WO2009082607, which are incorporated herein by reference in their entirety.

[0214] In some implementations, the ligand is

[0215] In some embodiments, the dsRNA activator comprises the ligand-conjugated nucleotide, as illustrated in the following schematic diagram.

[0216] And X is O or S, and the double-stranded structure is a nucleotide chain of dsRNA.

[0217] 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.

[0218] In some implementations, antibodies can also be used as ligands for the delivery of dsRNA, such as antibody-oligonucleotide conjugates (AOCs).

[0219] In some implementations, polymer matrix copolymerization can also be used to deliver dsRNA, such as the LODER (Local Drug EluteR) delivery platform.

[0220] 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.

[0221] 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.

[0222] 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 linker or linker group, such as a linker group contained in the ligand). In some embodiments of the present invention, the dsRNA of the present invention may optionally include a linker located between the nucleotide chain of the dsRNA and the ligand. In some embodiments, the linker is a biocleavable linker. In some embodiments, the linker does not necessarily have to be biocleavable. In some embodiments, the linker 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, a linker having a branching region can be used to conjugate multiple entities, such as N-acetylgalactosamine moieties, to the oligonucleotide of the dsRNA of the present invention. Linkers 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 invention, when referring to "ligand" or "ligand portion," it may also refer to a ligand or ligand portion that includes a connector.

[0223] 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):

[0224] in,

[0225] Gal represents terminal galactose derivatives independently;

[0226] L indicates a connector;

[0227] n is an integer selected from 1, 2, 3, and 4; and

[0228] 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.

[0229] 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 branches, three branches, or other multi-branched shapes.

[0230] 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.

[0231] Therefore, in some embodiments, the ligands of formula (I) of the present invention each independently have the structure of formula (Ia):

[0232] in,

[0233] L A1 This indicates the connection used to link the Gal section to L. A2 Partial linker;

[0234] 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;

[0235] Gal independently represents terminal galactose derivatives; and

[0236] n is an integer selected from 1, 2, 3, and 4; and

[0237] 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.

[0238] 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:

[0239] in,

[0240] R1 is an H or hydroxyl protecting group,

[0241] 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-C0-8 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

[0242] A wavy valence bond indicates that the bond is connected to the rest of the molecule.

[0243] 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.

[0244] 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.

[0245] 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. 1-16Alkoxy groups, such as C 1-6 Alkyl group.

[0246] In some implementations, R2 is independently H, OH,

[0247] Preferably, R2 is OH, More preferably, R2 is independently OH or in particular

[0248] 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.

[0249] 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.

[0250] In some implementations, Gal is represented independently. The wavy valence bond indicates that the valence bond is connected to the rest of the molecule.

[0251] In some implementations, in formula (Ia), L A1 Each can be independently represented as a linking base having the following structure:

[0252] -(CH2) m1 -C(O)-NH-(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0253] -(CH2) m1 -C(O)-NH-(CH2) m2 -C(O)-NH-(CH2) m3 -;

[0254] -(CH2) m1 -NH-C(O)-(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0255] -(CH2) m1 -NH-C(O)-(CH2) m2 -C(O)-NH-(CH2) m3 -;

[0256] -(CH2) m1 -O-(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0257] -(CH2CH2O) m4 -(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0258] 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.

[0259] In some implementations, m1 is preferably 3, 4, 5, 6, 7 or 8, more preferably 3, 4, 5 or 6, and most preferably 4.

[0260] In some implementations, m2 is preferably 1, 2, 3, 4, 5 or 6, more preferably 1, 2 or 3.

[0261] In some implementations, m3 is preferably 1, 2, 3, 4, 5 or 6, more preferably 1, 2 or 3, and most preferably 2.

[0262] 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.

[0263] In some implementations, L A1 Each of these independently represents a linker base selected from the following:

[0264] 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.

[0265] In some implementations, L A1 Each of these independently represents a linker base selected from the following:

[0266] Preferred is

[0267] The group is connected to Gal at position 1 and to the rest of the molecule at position 2.

[0268] In some implementations, L A2 This refers to a divalent, trivalent, or tetravalent connector comprising monohydroxymethylmethane, dihydroxymethylmethane, or trihydroxymethylmethane components, wherein the L... A2via 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.

[0269] In some implementations, L A2 This indicates a joint having a structure selected from the following:

[0270] 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.

[0271] In some implementations, L A3 Represents spacer bases with the following structure:

[0272] Preferred

[0273] 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 ester bond.

[0274] In some implementations, L A3 Represents spacer bases with the following structure:

[0275] Preferred

[0276] 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 ester bond.

[0277] In some implementations, the ligands of formula (I) or (Ia) each independently have the structure of formula (Ia-i):

[0278] 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.

[0279] In particular, the ligands of formula (I) or (Ia) are each independently an L96 moiety having the following structure:

[0280] 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' 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 some embodiments, the ligand is linked to the 3' end of the sense strand of the oligonucleotide of the dsRNA of the present invention.

[0281] In some embodiments, the dsRNA activators of the present invention having ligands of formula (Ia-i) are shown below:

[0282] 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.

[0283] In particular, the dsRNA activator of the present invention having a ligand with an L96 structure is shown below:

[0284] 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.

[0285] 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.

[0286] 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.

[0287] In some embodiments, the ligand is L96, see, for example, WO2009073809 and WO2009082607, which are incorporated herein by reference in their entirety.

[0288] 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).

[0289] 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.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] In some embodiments, the sense and antisense strands of the dsRNA activator comprise, respectively, a modified nucleotide sequence of the sense strand conjugated to the L96 ligand structure at the 3' end via a phosphate ester bond and a modified nucleotide sequence of the antisense strand, selected from any combination of sense and antisense strands in Table 3, or are composed of said modified nucleotide sequences.

[0294] 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.

[0295] In some embodiments, the dsRNA activator is any of the dsRNA activators shown in Table 3.

[0296] In some specific embodiments, the dsRNA activator comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise at least 15, 16, 17, 18, or 19 consecutive modified nucleotides from the nucleotide sequences shown in SEQ ID NO:293 / 299, SEQ ID NO:294 / 300, SEQ ID NO:295 / 301, SEQ ID NO:296 / 302, or SEQ ID NO:297 / 303, 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.

[0297] In some specific embodiments, the dsRNA activator comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand respectively comprise or consist of the modified nucleotide sequences shown in SEQ ID NO:293 / 299, SEQ ID NO:294 / 300, SEQ ID NO:295 / 301, SEQ ID NO:296 / 302 or SEQ ID NO:297 / 303, wherein the 3' end of the sense strand nucleotide sequence shown in any one of SEQ ID NOs:299-303 is conjugated with an L96 ligand via a phosphate ester bond.

[0298] In some specific implementations, the dsRNA activator comprises a sense strand and an antisense strand, wherein

[0299] 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:299, and the antisense strand comprises or is composed of a modified nucleotide sequence as shown in SEQ ID NO:293.

[0300] The sense strand comprises or is composed of the modified nucleotide sequence shown in SEQ ID NO:300, 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:294.

[0301] The sense strand comprises or is composed of the modified nucleotide sequence shown in SEQ ID NO:301, 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:295.

[0302] The sense strand consists of a modified nucleotide sequence, as shown in SEQ ID NO:302, with an L96 structural ligand conjugated to its 3' end, and the antisense strand consists of a modified nucleotide sequence, as shown in SEQ ID NO:296; or

[0303] 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:303, and the antisense strand consists of a modified nucleotide sequence, as shown in SEQ ID NO:297.

[0304] In some specific embodiments, the dsRNA activator comprises an antisense strand and a sense strand, wherein the sense strand consists of a modified nucleotide sequence with an L96 ligand conjugated to its 3' end as shown in SEQ ID NO:300, and the antisense strand consists of a modified nucleotide sequence as shown in SEQ ID NO:294; or the sense strand consists of a modified nucleotide sequence with an L96 ligand conjugated to its 3' end as shown in SEQ ID NO:303, and the antisense strand consists of a modified nucleotide sequence as shown in SEQ ID NO:297.

[0305] In some embodiments, the present invention also relates to an RNA-induced silencing complex (RISC) comprising the antisense strand of any of the dsRNA activators described herein.

[0306] III. Preparation of dsRNA activators

[0307] dsRNA can be synthesized using standard methods known in the art. The double-stranded RNA activator 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 strand is 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.

[0308] 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:

[0309] 1. Template recognition:

[0310] 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.

[0311] 2. Transcription initiation:

[0312] 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.

[0313] 3. Transcription elongation:

[0314] 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.

[0315] 4. Cyclic Synthesis:

[0316] 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.

[0317] 5. Transcription termination:

[0318] 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.

[0319] 6. Post-processing:

[0320] 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.

[0321] 7. Purification and analysis:

[0322] 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.).

[0323] 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.

[0324] IV. Pharmaceutical Compositions

[0325] 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.

[0326] 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.

[0327] 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.

[0328] 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.

[0329] 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.

[0330] 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.

[0331] In some embodiments, the dsRNA activator according to the invention may be present in an unbuffered solution, for example, saline or water. In other embodiments, the dsRNA activator is present in a buffered solution, for example, wherein the buffered solution comprises acetate, citrate, prolyl, carbonate or phosphate or any combination thereof, such as phosphate-buffered saline (PBS).

[0332] The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. In some embodiments, said other active ingredients are, for example, various therapeutic agents for treating chronic inflammatory diseases. The active ingredients are suitably combined in amounts effective for the intended use.

[0333] In some embodiments, the pharmaceutical composition or formulation of the present invention may be contained in a vial or in a syringe.

[0334] V. Drug combinations and pillboxes

[0335] 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.

[0336] 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.

[0337] In one embodiment, the complete medicine box of the present invention comprises, within the same package:

[0338] - A first container containing a pharmaceutical composition comprising the dsRNA active agent of the present invention;

[0339] - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0340] In some implementations, other therapeutic agents, such as any therapeutic agent effective in preventing or treating CIDEB-related diseases and / or conditions, cover a wide range of therapeutic agents used to treat chronic inflammatory diseases.

[0341] VI. Uses and Methods

[0342] One aspect of the present invention provides a method for inhibiting the expression and / or activity of the CIDEB gene in cells, comprising reacting the cells with the dsRNA activator, pharmaceutical composition, or pharmaceutical combination product of the present invention, thereby inhibiting the expression of the CIDEB gene in the cells. In some embodiments, the cells are in a subject, optionally a human. In some embodiments, the cells are hepatocytes. In some embodiments, the subject suffers from a CIDEB-related disease and / or condition.

[0343] In some embodiments, contacting the cells with the dsRNA activator inhibits CIDEB expression by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, or about 93%. In some embodiments, inhibiting CIDEB expression reduces CIDEB protein levels in the subject's serum by at least 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, or about 93%. In some embodiments, administration of the dsRNA activator to the subject results in a decrease in CIDEB concentration or content (e.g., in the liver or hepatocytes), or a decrease in CIDEB protein accumulation or content in the subject's body (e.g., within the liver, or within hepatocytes). In some embodiments, administration of the dsRNA activator to the subject results in a decrease in CIDEB concentration or content in the subject's body fluids. In some embodiments, administration of the dsRNA activator to the subject results in a decrease in CIDEB expression or protein content in the subject's liver or hepatocytes.

[0344] 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 a CIDEB-related disease and / or condition.

[0345] 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 CIDEB-related diseases and / or conditions.

[0346] 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 to uses for preparing a medicament for said treatment.

[0347] In some embodiments, the disease or condition is associated with increased expression or activity of CIDEB. In some embodiments, the disease or condition is an indication for benefiting from a reduction in the expression and / or activity of cell death-induced DFFA-like effector B (CIDEB).

[0348] In some embodiments, "increased CIDEB expression or activity" refers to increased CIDEB expression (e.g., nucleic acid or protein levels) or activity in the liver or hepatocytes of the subject. In some embodiments, the liver or hepatocytes of patients with CIDEB-related diseases and / or conditions have moderate to high levels of CIDEB expression. In some embodiments, the amount of CIDEB in the liver or hepatocytes of patients with CIDEB-related diseases and / or conditions is higher than the amount of CIDEB in the liver or body fluids of healthy controls. In some embodiments, increased CIDEB expression or activity leads to lipid droplet accumulation in the liver of patients with CIDEB-related diseases and / or conditions.

[0349] In some embodiments, the CIDEB-related disease and / or condition is a chronic inflammatory disease, such as a chronic inflammatory disease of the liver and other tissues. In one instance, the chronic inflammatory disease is chronic inflammatory liver disease. In another instance, chronic inflammatory liver disease is selected from hepatic steatosis, hepatitis, liver fibrosis, fatty liver disease (steatohepatitis), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and cirrhosis. In one embodiment, the chronic inflammatory disease is a chronic inflammatory liver disease, such as inflammation of the liver, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury, hepatocellular necrosis, hepatocellular carcinoma, insulin insensitivity, and / or diabetes.

[0350] In some embodiments, subjects with CIDEB-related diseases and / or conditions, or whose cells exhibit abnormal CIDEB expression or activity. In some embodiments, the subjects (particularly adult subjects) have CIDEB overexpression. In some embodiments, the subjects have (e.g., elevated levels, such as nucleic acid or protein levels or activity) of CIDEB (e.g., compared to healthy subjects). In some embodiments, the subjects' biological samples (e.g., blood, serum, tissues such as liver tissue, or cells such as hepatocytes) have (e.g., elevated levels, such as nucleic acid or protein levels or activity) of CIDEB (e.g., compared to biological samples from healthy subjects (e.g., corresponding tissues or cells in healthy subjects)). In some embodiments, the subjects have cells that overexpress CIDEB, such as hepatocytes. In some embodiments, the individual's cells (e.g., hepatocytes) overexpress CIDEB, for example, moderately or highly. In some embodiments, abnormal CIDEB expression refers to higher CIDEB expression in cells (e.g., hepatocytes) compared to CIDEB expression in control cells (e.g., healthy cells in the corresponding tissue of a healthy individual, such as healthy hepatocytes). In some implementation schemes, aberrant CIDEB expression refers to higher CIDEB expression in liver tissue or hepatocytes compared to control tissues or cells (e.g., corresponding tissues or corresponding healthy hepatocytes of a healthy individual).

[0351] In this article, “CIDEB overexpression or upregulation” refers to an increase in the protein level (e.g., protein concentration or accumulation) of CIDEB compared to the corresponding tissues or cells of healthy individuals.

[0352] 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.

[0353] 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.

[0354] 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.

[0355] In some embodiments, the method or use further includes determining the level of CIDEB (e.g., protein or nucleic acid level, such as mRNA level) in the subject's body, liver, or hepatocytes prior to administration of the dsRNA active agent. In some embodiments, the level of CIDEB is compared with the level of CIDEB (e.g., protein or nucleic acid level, such as mRNA level) in a healthy subject's body, liver, or hepatocytes. If the level of CIDEB in the subject's body, liver, or hepatocytes is higher than the level of CIDEB in a healthy control, the subject is then administered the dsRNA active agent or a composition, drug, formulation, or combination product containing it.

[0356] 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).

[0357] In one implementation, the subject has the disease described herein or is at risk of having the disease described herein.

[0358] 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 which the administration of the dsRNA active agent or composition or drug or formulation of the present invention may occur before, simultaneously with, and / or after the administration of other therapeutic agents and / or active agents.

[0359] 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 can be any therapeutic agent that is effective, for example, in preventing or treating CIDEB-related diseases and / or conditions, covering a wide range of therapeutic agents for treating chronic inflammatory diseases. Example

[0360] Example 1: siRNA Synthesis

[0361] RNA solid-phase synthesis is a commonly used technique for synthesizing RNA molecules, allowing for the stepwise construction of RNA strands on a solid support. This method is characterized by high throughput, high efficiency, and automation, and is widely used in biotechnology and research fields. The following describes the synthesis of the CIDEB RNAi agent duplexes shown in Table 2 according to the following method: all RNA and 2'-modified phosphoramidites were purchased from Shanghai Zhaowei Technology Development Co., Ltd. Specifically, the following 2'-O-methylphosphoramidites were used: (5'-O-dimethoxytriphenylmethyl-N6-(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites, 5'-O-dimethoxytriphenylmethyl-N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites, (5'-O-dimethoxytriphenylmethyl-N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites, (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.

[0362] 1. Preparation of siRNA without ligand linkage

[0363] 1.1 Synthesis of the Justice Chain (SS Chain)

[0364] The oligonucleotide was synthesized using a solid-phase phosphoramide method, employing a blank CPG solid support as the starting cycle. Nucleoside monomers or nucleotide analog monomers were sequentially linked from the 3'-5' direction according to the nucleotide arrangement of the positive strand. Each linkage of a nucleoside monomer or nucleotide analog monomer involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for 5 μmol oligonucleotides were as follows:

[0365] The nucleoside monomer or nucleotide analog monomer was provided in a 0.05 mol / L acetonitrile solution. The reaction conditions were identical for each step: 25°C. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was activated twice using a 0.25 mol / L ETT-acetonitrile solution. Capping was performed twice using a 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile mixture (10:14:76, v / v / v). Oxidation was performed twice using a 0.05 mol / L iodine / tetrahydrofuran / pyridine / water mixture (70 / 20 / 10, v / v / v). Thiolation was performed twice using a 0.2 mol / L PADS mixture of acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0366] 1.2 Synthesis of the antisense chain (AS chain)

[0367] The solid-phase phosphoramide synthesis method utilizes a blank CPG solid-phase support as the starting cycle, and nucleoside monomers or nucleotide analog monomers are sequentially linked from the 3'-5' direction according to the nucleotide arrangement sequence of the antisense strand. Each linkage of a nucleoside monomer or nucleotide analog monomer involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for 5 μmol oligonucleotides of the antisense strand are the same as those for the sense strand.

[0368] 1.3 Purification and Annealing of Oligonucleotides

[0369] 1.3.1 Ammonolysis

[0370] The synthesized solid support (sense or antisense chain) was added to a 5 mL centrifuge tube, and 3% diethylamine / ammonia (v / v) was added. The mixture was reacted in a constant temperature water bath at 35℃ (or 55℃) for 16 hours (or 8 hours). After filtration, the solid support was washed three times with ethanol / water, 1 mL each time. The filtrate was concentrated by centrifugation and the crude product was purified.

[0371] 1.3.2 Purification

[0372] 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 either filtered DI water or 100mM ammonium bicarbonate and 20% acetonitrile at pH 6.7.

[0373] 1.3.3 Annealing

[0374] According to Table 2, the sense strand (SS strand) and antisense strand (AS strand) were mixed at a molar ratio (SS strand / AS strand = 1 / 1.05), heated in a water bath to 70-95°C, held for 3-5 min, and then naturally cooled to room temperature. The system was then lyophilized to obtain the product. Some RNAi agents were 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.

[0375] Table 1: Antisense and sense strands of unmodified CIDEB double-stranded mRNA

[0376] Table 2*: Antisense and sense strands of CIDEB-modified double-stranded mRNA

[0377] 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.

[0378] Preparation of GalNAc-siRNA conjugates:

[0379] Preparation of GalNAc-siRNA conjugates (Table 3): The synthesis method of GalNAc-siRNA conjugates can be referred to WO2023003922A1.

[0380] 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, XD000308.1 corresponds to XD000308.

[0381] Table 3*: Antisense and sense strands of CIDEB-modified double-stranded mRNA

[0382] 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.

[0383] Example 2: Inhibition of human CIDEB in Hep3B cells by siRNA

[0384] Hep3B2.1-7 cells (CyberKang (Shanghai) Biotechnology Co., Ltd., Cat#iCell-h091) were cultured in EMEM medium (ATCC, Cat#30-2003) containing 10% fetal bovine serum at 37°C and 5% CO2.

[0385] 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 Hep3B cells were added, with 15,000 cells per well, resulting in a final siRNA concentration of 10 nM. Twenty-four hours after transfection, cellular RNA was extracted 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 CIDEB mRNA levels. CIDEB mRNA levels were corrected for GAPDH internal control mRNA levels. CIDEB mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of residual CIDEB mRNA expression relative to the negative siRNA control group. The calculation formula is as follows:

[0386] ΔCt = Ct(target gene) – Ct(internal reference gene)

[0387] ΔCt = ΔCt (drug administration group) - ΔCt (negative siRNA control group)

[0388] CIDEB mRNA relative expression level = 2 -ΔΔCt ×100%

[0389] Table 3: Single-dose screening of dsRNA drugs targeting CIDEB in Hep3B cells

[0390] Table 3 shows the single-point inhibition of CIDEB mRNA after transfection with the siRNA molecules in Table 2 in Hep3B.

[0391] Example 3: Inhibition of human CIDEB in Hep3B cells by siRNA

[0392] Hep3B2.1-7 cells (CyberKang (Shanghai) Biotechnology Co., Ltd., Cat#iCell-h091) were cultured in EMEM medium (ATCC, Cat#30-2003) 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 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 Hep3B cells were added, with 15,000 cells per well, resulting in a final siRNA concentration of 10 nM. Each siRNA and negative control was analyzed in two wells. Twenty-four hours after transfection, cellular RNA was extracted 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 CIDEB mRNA levels. CIDEB mRNA levels were corrected for GAPDH internal control mRNA levels. CIDEB mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of residual CIDEB mRNA expression relative to the negative siRNA (negative control, PBS) control group. The calculation formula is as follows:

[0394] ΔCt = Ct(target gene) – Ct(internal reference gene)

[0395] ΔCt = ΔCt (drug administration group) - ΔCt (negative siRNA control group)

[0396] CIDEB mRNA relative expression level = 2 -ΔΔCt ×100%

[0397] Figure 1 shows the single-point inhibition of CIDEB mRNA after transfection with siRNA molecules in Hep3B, indicating that all detected siRNA molecules can effectively inhibit the expression level of mRNA in Hep3B.

[0398] Example 4: Inhibition of human CIDEB in Hep3B cells by siRNA

[0399] Hep3B2.1-7 cells (CyberKang (Shanghai) Biotechnology Co., Ltd., Cat#iCell-h091) were cultured in EMEM medium (ATCC, Cat#30-2003) containing 10% fetal bovine serum at 37°C and 5% CO2.

[0400] siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat#13778150). The specific method was 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 Hep3B cells were added, with 15,000 cells per well. siRNA was serially diluted, starting at 10 nM and 10-fold, for a total of 7 dilution points. Each siRNA, each dilution, and the negative control were analyzed in 2 wells. Twenty-four hours after transfection, cellular RNA was extracted 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 CIDEB mRNA levels. CIDEB mRNA levels were corrected for GAPDH internal control mRNA levels. CIDEB mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of residual CIDEB mRNA expression relative to the negative siRNA (negative control, PBS) control group. The calculation formula is as follows:

[0401] ΔCt = Ct(target gene) – Ct(internal reference gene)

[0402] ΔCt = ΔCt (drug administration group) - ΔCt (negative siRNA control group)

[0403] CIDEB mRNA relative expression level = 2 -ΔΔCt ×100%

[0404] Figure 2 shows the inhibition of CIDEB mRNA after transfection with siRNA molecules in Hep3B.

[0405] Example 5: Inhibition of human CIDEB in primary human hepatocytes by siRNA

[0406] In human primary hepatocytes (PHH, Huizhiheyuan), siRNA activity was screened using a concentration gradient.

[0407] Following the product instructions, siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat#13778150) at a final concentration of 10 nM. An equal volume of diluted Lipofectamine RNAiMAX and siRNA was mixed in Opti-MEM medium (GIBCO, Cat#31985070) and incubated at room temperature for 20 min. 20 μl of the diluted siRNA was added to each well of a 96-well plate, followed by 80 μl of revived cryopreserved human primary hepatocytes, with 40,000 cells per well. A serial dilution of siRNA was used, starting at 600 nM and followed by 5-fold dilutions for a total of 9 dilution points. Twenty-four hours after transfection, RNA was extracted from the 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 CIDEB mRNA levels. CIDEB mRNA levels were corrected for based on GAPDH internal control mRNA levels. CIDEB mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of residual CIDEB mRNA expression in cells treated with the negative siRNA control. The calculation formula is as follows:

[0408] ΔCt = Ct(target gene) – Ct(internal reference gene)

[0409] ΔCt = ΔCt (drug administration group) - ΔCt (negative siRNA control group)

[0410] CIDEB mRNA relative expression level = 2 -ΔΔCt ×100%

[0411] Figure 3 shows the inhibition of CIDEB mRNA after transfection with siRNA molecules in PHH.

[0412] Example 6: Inhibition of monkey CIDEB in primary monkey hepatocytes by siRNA

[0413] Activity screening of siRNA was performed in primary monkey hepatocytes using a concentration gradient.

[0414] Following the product instructions, siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat#13778150) at a final concentration of 10 nM. An equal volume of diluted Lipofectamine RNAiMAX and siRNA was mixed in Opti-MEM medium (GIBCO, Cat#31985070) and incubated at room temperature for 20 min. 20 μL of the mixture was added to each well of a 96-well plate, followed by 80 μL of revived cryopreserved primary monkey hepatocytes, with a cell density of 40,000 cells per well. A serial dilution of siRNA was used, starting at 500 nM and followed by 5-fold dilutions for a total of 9 dilution points. Twenty-four hours after transfection, RNA was extracted from the 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 CIDEB mRNA levels. CIDEB mRNA levels were corrected for based on GAPDH internal control mRNA levels. CIDEB mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of residual CIDEB mRNA expression in cells treated with the negative siRNA control. The calculation formula is as follows:

[0415] ΔCt = Ct(target gene) – Ct(internal reference gene)

[0416] ΔCt = ΔCt (drug administration group) - ΔCt (negative siRNA control group)

[0417] CIDEB mRNA relative expression level = 2 -ΔΔCt ×100%

[0418] Figure 4 shows the inhibition of CIDEB mRNA after transfection with siRNA molecules in PCH.

[0419] Example 7: Inhibition of mouse CIDEB by siRNA in mice

[0420] This experiment preliminarily screened drugs by knocking down the CIDEB gene in the liver of transgenic hCIDEB mice.

[0421] hCIDEB mice:

[0422] male hCIDEB mice, aged 35-43 days, were purchased from Shanghai Southern Model Biotechnology Co., Ltd., SPF grade, and inspected by Suzhou Xishan Biotechnology Co., Ltd., with certificate numbers 20190002050030 and 20190002050048. The mice were acclimatized for 3 days after arrival before the study began.

[0423] Administration:

[0424] This experiment included 7 groups: PBS group, XD000308.1, XD000314.1, XD000321.1, XD000332.1, XD000350.1, and XD000368.1 (BMK), with 6 mice in each group. The right ear of each mouse was tagged. The specific dosage and frequency of the single subcutaneous administration are shown in Table 1. The PBS group served as the control group and was administered the drug on day 0 of the experiment. On days 7 and 21, the mice were euthanized with carbon dioxide, and their liver tissue was collected and stored in PBS on wet ice.

[0425] Table 5: Dosing conditions for each group

[0426] Figure 5 shows the inhibition of CIDEB mRNA in transgenic hCIDEB mice. The results indicate that after 7 days of 5mpk administration, the remaining mRNA levels of XD000321.1 and XD000332.1 in mice reached approximately 25%, while the remaining CIDEB mRNA levels of XD000308.1, XD000314.1, and XD000350.1 were below 10%. After 21 days of 5mpk administration, the remaining mRNA level of XD000350.1 in mice was below 5%, demonstrating a very good knockdown effect. The remaining CIDEB mRNA levels of XD000308.1 and XD000314.1 reached approximately 30%, an increase compared to the previous 7 days. The remaining CIDEB mRNA levels of XD000321.1 and XD000332.1 were also lower. The remaining percentages reached approximately 60% and 100%, respectively. In summary, both XD000314.1 and XD000350.1 exhibited very stable knockdown effects in mice over a long period of time, with knockdown efficiencies comparable to those of BMK XD000368.1.

[0427] Example 8: Inhibition of mouse CIDEB by siRNA in mice

[0428] This experiment preliminarily screened drugs by knocking down the CIDEB gene in the liver of transgenic hCIDEB mice.

[0429] hCIDEB mice:

[0430] Five-week-old male hCIDEB mice were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., SPF grade, and inspected by Suzhou Xishan Biotechnology Co., Ltd., certificate number A202408270233. The mice were acclimatized for three days after arrival before the study began.

[0431] Administration:

[0432] This experiment included four groups: PBS group, XD000314.1, XD000350.1, and XD000368.1, with six mice in each group. The right ear of each mouse was tagged. The specific dosage and frequency of the single subcutaneous administration are shown in Table 6. The PBS group served as the control group and was administered the drug on day 0 of the experiment. On days 7 and 21, the mice were euthanized with carbon dioxide, and their liver tissue was collected and stored in PBS on wet ice.

[0433] Table 6: Dosing conditions for each group

[0434] Figure 6 shows the inhibition of CIDEB mRNA in transgenic hCIDEB mice. The results show that after 7 days of 1mpk administration, the remaining CIDEB mRNA in XD000350.1 reached about 20%, and that in XD000314.1 reached about 50%, similar to BMK. After 21 days of 1mpk administration, the remaining mRNA in XD000350.1 reached about 40%, and that in XD000314.1 reached about 60%, showing an increase compared to the previous 7 days. However, the remaining CIDEB mRNA in BMK XD000368.1 reached about 100%. In summary, the efficacy of 1mpk administration of XD000314.1 and XD000350.1 in mice is insufficient, only showing a certain knockdown effect.

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 in a target sequence, and the complementary region and the at least 15 consecutive nucleotides in the target sequence contain a mismatch of no more than 3, 2, or 1 nucleotide, wherein the target sequence is selected from... (i) The target sequence corresponding to positions 1420-1440 or 2052-2072 of CIDEB mRNA; (ii) The target sequence shown in SEQ ID NO:124 or SEQ ID NO:158; (iii) The target sequence shown in any one of SEQ ID NO: 117-174; or (iv) The target sequences corresponding to the positions in CIDEB mRNA shown in Table 1; Optionally, the dsRNA activator is used to inhibit the expression of the gene encoding CIDEB.

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 to a target sequence encoding a CIDEB mRNA, and comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differing from any antisense nucleotide sequence in the antisense strands listed in Table 1 by no more than 3, 2, or 1 nucleotide. Optionally, the dsRNA activator is used to inhibit the expression of a gene encoding CIDEB, and optionally, the target sequence of the mRNA is as defined in claim 1.

3. The dsRNA activator of claim 2, wherein the antisense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides differing from the nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:42 by no more than 3, 2 or 1 nucleotides.

4. The dsRNA activator according to any one of claims 1-3, wherein the length of the complementary region to the target sequence comprising the antisense strand is between 15 and 30 nucleotides, 18 and 30 nucleotides, or 18 and 23 nucleotides, for example, the length of the complementary region is 18, 19, 20, or 21 nucleotides; Optionally, the complementary region comprises at least the following nucleotides, starting from the 5' end of the antisense strand: nucleotides 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21, for example, nucleotides 2-19.

5. The dsRNA activator according to any one of claims 1 to 4, wherein the antisense strand has the same number of nucleotides as the target sequence and is mismatched with the target sequence at least 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; or the antisense strand is completely complementary to the target sequence.

6. The dsRNA activator according to any one of claims 1 to 5, wherein the first nucleotide at the 5' end of the antisense strand is A or U, for example, U.

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, 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.

8. The dsRNA activator according to claim 7, wherein the positive strand comprises at least 15, 16, 17, 18 or 19 consecutive nucleotides that differ from the nucleotide sequence shown in SEQ ID NO:66 or SEQ ID NO:100 by no more than 3 nucleotides.

9. The dsRNA activator according to any one of claims 1 to 8, wherein the double-stranded region formed by the sense strand and the antisense strand 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 nucleotide pairs, 17 and 24 nucleotide pairs, 17 and 23 nucleotide pairs, 18 and 23 nucleotide pairs, or 19 and 22 nucleotide pairs, for example, the length of the fully complementary double-stranded region is 16, 17, 18, or 19 nucleotide pairs, for example, 19 nucleotide pairs.

10. The dsRNA activator according to any one of claims 1 to 9, wherein the length of the sense strand and the antisense strand is each independently 15-30 nucleotides, for example 17-27 nucleotides, for example 19-25 nucleotides, for example 19-23 nucleotides, or for example 19-21 nucleotides, for example, the length of the sense strand is 19 nucleotides, and the length of the antisense strand is 21 nucleotides.

11. The dsRNA activator according to any one of claims 1 to 10, 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.

12. The dsRNA activator according to any one of claims 1 to 11, 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 target sequence encoding the CIDEB mRNA 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 over 19 nucleotides, for example, completely complementary over 19 consecutive nucleotides; for example, the sense strand and the antisense strand are completely complementary over consecutive nucleotides from the 5' end to the 19th position.

13. The dsRNA activator according to any one of claims 1 to 12, wherein the dsRNA activator comprises a sense strand and an antisense strand, the sense strand and the antisense strand respectively comprising nucleotide sequences of the sense strand and the antisense strand selected from any combination of sense strands and antisense strands in Table 1, or respectively composed of said nucleotide sequences.

14. The dsRNA activator according to any one of claims 1 to 13, wherein the dsRNA activator comprises an antisense strand and a sense strand.The antisense chain and the justice chain respectively include SEQ ID NO:1 / 59, SEQ ID NO:2 / 60, SEQ ID NO:3 / 61, SEQ ID NO:4 / 62, SEQ ID NO:5 / 63, SEQ ID NO:6 / 64, SEQ ID NO:7 / 65, SEQ ID NO:8 / 66, SEQ ID NO:9 / 67, SEQ ID NO:10 / 68, SEQ ID NO:11 / 69, SEQ ID NO:12 / 70, SEQ ID NO:13 / 71, SEQ ID NO:14 / 72, SEQ ID NO:15 / 73, SEQ ID NO:16 / 74, SEQ ID NO:17 / 75, SEQ ID NO:18 / 76, SEQ ID NO:19 / 77, SEQ ID NO:20 / 78, SEQ ID NO:21 / 79, SEQ ID NO:22 / 80, SEQ ID NO:23 / 81, SEQ ID NO:24 / 82, SEQ ID NO:25 / 83, SEQ ID NO:26 / 84, SEQ ID NO:27 / 85, SEQ ID NO:28 / 86, SEQ ID NO:29 / 87, SEQ ID NO:30 / 88, SEQ ID NO:31 / 89, SEQ ID NO:32 / 90, SEQ ID NO:33 / 91, SEQ ID NO:34 / 92, SEQ ID NO:35 / 93, SEQ ID NO:36 / 94, SEQ ID NO:37 / 95, SEQ ID NO:38 / 96, SEQ ID NO:39 / 97, SEQ ID NO:40 / 98, SEQ ID NO:41 / 99, SEQ ID NO:42 / 100, SEQ ID NO:43 / 101, SEQ ID NO:44 / 102, SEQ ID NO:45 / 103, SEQ ID NO:46 / 104, SEQ ID NO:47 / 105, SEQ ID NO:48 / 106, SEQ The nucleotide sequences shown in SEQ ID NO:49 / 107, SEQ ID NO:50 / 108, SEQ ID NO:51 / 109, SEQ ID NO:52 / 110, SEQ ID NO:53 / 111, SEQ ID NO:54 / 112, SEQ ID NO:55 / 113, SEQ ID NO:56 / 114, SEQ ID NO:57 / 115, or SEQ ID NO:58 / 116, or each of the nucleotide sequences shown, are composed of the nucleotide sequences shown. Optionally, the antisense strand and the sense strand respectively comprise the nucleotide sequence shown in SEQ ID NO:8 and the nucleotide sequence shown in SEQ ID NO:66; or the antisense strand and the sense strand respectively comprise the nucleotide sequence shown in SEQ ID NO:42 and the nucleotide sequence shown in SEQ ID NO:

100.

15. The dsRNA activator according to any one of claims 1 to 14, wherein at least one nucleotide in the dsRNA activator is a modified nucleotide, for example, wherein 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.

16. The dsRNA activator according to claim 15, wherein all nucleotides of the sense strand are modified nucleotides; all nucleotides of the antisense strand are modified nucleotides; or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

17. The dsRNA activator according to claim 15 or 16, wherein 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, 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 inter-bond is located between the 1st and 3rd nucleotides at the 5' end of the sense strand, and the thiophosphate nucleotide inter-bond is located between the 1st and 3rd nucleotides at the 5' end and between the 1st and 3rd nucleotides at the 3' end of the antisense strand. Optionally, the nucleotides at positions 2, 6, 14, and 16, counting from the 5' end of the antisense strand, are 2'-fluorinated nucleotides, and the nucleotides at positions 7-9, counting from the 5' end of the sense strand, are 2'-fluorinated nucleotides.

18. The dsRNA activator according to claim 17, wherein the modified nucleotides in the antisense and sense strands have the following modification patterns: antisense chain: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm, and Chain of Justice: NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm; in Nf = any nucleotide modified with 2'-fluorine; Nm = any 2'-methoxynucleotide; 's' indicates that the two nucleotides are linked by a phosphate thioester bond.

19. The dsRNA activator according to any one of claims 15 to 18, 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 sense strand and the antisense strand each comprise modified nucleotide sequences of the sense strand and the antisense strand selected from any combination of sense strands and antisense strands in Table 2, or each consist of the modified nucleotide sequences. Optionally, the dsRNA activator comprises a combination of modified antisense and sense strands as shown in Table 2; Optionally, the dsRNA activator comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand respectively comprise SEQ ID NO:177 / 235, SEQ ID NO:178 / 236, SEQ ID NO:179 / 237, SEQ ID NO:180 / 238, SEQ ID NO:181 / 239, SEQ ID NO:182 / 240, SEQ ID NO:183 / 241, SEQ ID NO:184 / 242, SEQ ID NO:185 / 243, SEQ ID NO:186 / 244, SEQ ID NO:187 / 245, SEQ ID NO:188 / 246, SEQ ID NO:189 / 247, SEQ ID NO:190 / 248, SEQ ID NO:191 / 249, SEQ ID NO:192 / 250, SEQ ID NO:193 / 251, SEQ ID NO:194 / 252, SEQ ID NO:19 ...8 / NO:195 / 253, SEQ ID NO:196 / 254, SEQ ID NO:197 / 255, SEQ ID NO:198 / 256, SEQ ID NO:199 / 257, SEQ ID NO:200 / 258, SEQ ID NO:201 / 259, SEQ ID NO:202 / 260, SEQ ID NO:203 / 261, SEQ ID NO:204 / 262, SEQ ID NO:205 / 263, SEQ ID NO:206 / 264, SEQ ID NO:207 / 265, SEQ ID NO:208 / 266, SEQ ID NO:209 / 267, SEQ ID NO:210 / 268, SEQ ID NO:211 / 269, SEQ ID NO:212 / 270, SEQ ID NO:213 / 271、SEQ ID NO:214 / 272、SEQ ID NO:215 / 273, SEQ ID NO:216 / 274, SEQ ID NO:217 / 275, SEQ ID NO:218 / 276, SEQ ID NO:219 / 277, SEQ ID NO:220 / 278, SEQ ID NO:221 / 279, SEQ ID NO:222 / 280, SEQ ID NO:223 / 281, SEQ ID NO:224 / 282, SEQ ID NO:225 / 283, SEQ ID NO:226 / 284, SEQ ID NO:227 / 285, SEQ ID NO:228 / 286, SEQ ID NO:229 / 287, SEQ IDThe modified nucleotide sequences shown in NO:230 / 288, SEQ ID NO:231 / 289, SEQ ID NO:232 / 290, SEQ ID NO:233 / 291 or SEQ ID NO:234 / 292, or each of the modified nucleotide sequences shown; Optionally, the antisense strand and the sense strand respectively comprise the nucleotide sequence shown in SEQ ID NO:184 and the nucleotide sequence shown in SEQ ID NO:242; or the antisense strand and the sense strand respectively comprise the nucleotide sequence shown in SEQ ID NO:218 and the nucleotide sequence shown in SEQ ID NO:

276.

20. The dsRNA activator according to any one of claims 1 to 19, further comprising a ligand, for example, the nucleotide sequence of the dsRNA activator being conjugated to the ligand via a phosphate ester group or a thiophosphate ester group, for example, the thiophosphate ester nucleotide bond being located at the 3' end of the sense strand or antisense strand; or the thiophosphate ester nucleotide bond being located at the 5' end of the sense strand or antisense strand; or the thiophosphate ester nucleotide bond being located at both the 5' end and the 3' end of the sense strand, and / or the thiophosphate ester nucleotide bond being located at both the 5' end and the 3' end of the antisense strand; Optionally, the ligand is an asialic acid glycoprotein receptor (ASGPR) ligand capable of delivering dsRNA molecules to liver tissue or hepatocytes, 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.

21. The dsRNA activator according to any one of claims 1 to 20, 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 antisense strand and the sense strand each comprise either one of the combinations of sense strands and antisense strands selected from Table 3, or a modified nucleotide sequence of the sense strand with an L96 ligand structure conjugated at the 3' end via a phosphate ester bond, or each comprises the nucleotide sequence. 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 an antisense strand and a sense strand, wherein the antisense strand and the sense strand respectively comprise or consist of the modified nucleotide sequences shown in SEQ ID NO:293 / 299, SEQ ID NO:294 / 300, SEQ ID NO:295 / 301, SEQ ID NO:296 / 302 or SEQ ID NO:297 / 303; Optionally, the dsRNA activator comprises a sense strand and an antisense strand, wherein The positive strand comprises or consists of a modified nucleotide sequence as shown in SEQ ID NO:299, with the 3' end conjugated to an L96 structural ligand, and The antisense strand comprises or consists of the modified nucleotide sequence shown in SEQ ID NO:293; The sense strand comprises or is composed of the modified nucleotide sequence shown in SEQ ID NO:300, 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:

294. The sense strand comprises or is composed of the modified nucleotide sequence shown in SEQ ID NO:301, 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:

295. The sense strand consists of a modified nucleotide sequence, as shown in SEQ ID NO:302, with an L96 structural ligand conjugated to its 3' end, and the antisense strand consists of a modified nucleotide sequence, as shown in SEQ ID NO:296; or The sense strand consists of a modified nucleotide sequence with an L96 ligand attached to the 3' end, as shown in SEQ ID NO:303, and the antisense strand consists of a modified nucleotide sequence, as shown in SEQ ID NO:

297. Optionally, the sense strand consists of a modified nucleotide sequence with an L96 ligand conjugated to the 3' end as shown in SEQ ID NO:300, and the antisense strand consists of a modified nucleotide sequence as shown in SEQ ID NO:294; or the sense strand consists of a modified nucleotide sequence with an L96 ligand conjugated to the 3' end as shown in SEQ ID NO:303, and the antisense strand consists of a modified nucleotide sequence as shown in SEQ ID NO:

297.

22. A cell containing a dsRNA activator according to any one of claims 1 to 21.

23. A pharmaceutical composition comprising a dsRNA activator according to any one of claims 1 to 21 and a pharmaceutically acceptable carrier.

24. The pharmaceutical composition of claim 23, wherein the dsRNA activator is in an unbuffered solution, for example, saline or water; or The dsRNA activator is in a buffer solution, such as an acetate, citrate, prolyl, carbonate, or phosphate, or any combination thereof, such as phosphate buffer solution (PBS).

25. A pharmaceutical combination comprising a dsRNA activator according to any one of claims 1 to 21 and one or more other therapeutic agents, said other therapeutic agents being any therapeutic agent effective, for example, in preventing or treating CIDEB-related diseases and / or conditions, covering a variety of therapeutic agents for treating chronic inflammatory diseases (e.g., chronic inflammatory liver disease, such as those selected from hepatic steatosis, hepatitis, liver fibrosis, fatty liver disease (steatohepatitis), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and cirrhosis).

26. A method for inhibiting the expression of the CIDEB gene in cells, the method comprising contacting the cells with a dsRNA activator according to any one of claims 1 to 21, or a pharmaceutical composition according to claim 23 or 24, or a pharmaceutical composition according to claim 25, thereby inhibiting the expression of the CIDEB gene in the cells.

27. The method of claim 26, wherein the cells are in a subject, optionally a human; or the cells are hepatocytes.

28. The method of claim 27, wherein the subject suffers from a CIDEB-related disease and / or condition, such as a chronic inflammatory disease, such as chronic inflammatory liver disease, selected from liver fat accumulation, hepatitis, liver fibrosis, fatty liver disease (steatohepatitis), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and cirrhosis.

29. The method according to any one of claims 26 to 28, wherein contacting the cells with the dsRNA activator inhibits CIDEB expression by at least 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, or about 93%, or inhibits CIDEB expression to reduce CIDEB protein levels in the serum of the subject by at least 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, or about 93%.

30. A method for preventing or treating in a subject an indication or CIDEB-related disease and / or condition that would benefit from reduced CIDEB expression and / or activity, comprising administering to the subject a dsRNA activator of any one of claims 1-21 or a pharmaceutical composition of claim 23 or 24 or a pharmaceutical composition of claim 25.

31. The method of claim 30, wherein the CIDEB-related condition is a chronic inflammatory disease, such as chronic inflammatory liver disease, selected from liver fat accumulation, hepatitis, liver fibrosis, fatty liver disease (steatohepatitis), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and cirrhosis.

32. The method of claim 30 or 31, wherein administration of the dsRNA activator to the subject results in a decrease in CIDEB concentration or content (e.g., in the liver or hepatocytes), or a decrease in CIDEB protein accumulation or content in the subject (e.g., in the liver or hepatocytes).

33. The method of any one of claims 26 to 32, further comprising applying to the cells or administering to the subject one or more other therapeutic agents, said other therapeutic agents being any therapeutic agent effective, for example, in preventing or treating CIDEB-related diseases and / or conditions, covering therapeutic agents for treating chronic inflammatory diseases, such as chronic inflammatory liver disease, such as those selected from various therapeutic agents for hepatic steatosis, hepatitis, liver fibrosis, fatty liver disease (steatohepatitis), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and cirrhosis.

34. The method according to any one of claims 26 to 32, further comprising determining the level of CIDEB in a sample from the cells or the subject.

35. Use of the dsRNA activator of any one of claims 1-21, or the pharmaceutical composition of claim 23 or 24, or the pharmaceutical composition of claim 25 in the preparation of a medicament for the prevention or treatment of an indication or CIDEB-related disease and / or condition in which the patient would benefit from reduced CIDEB expression and / or activity.

36. The use of claim 35, wherein the CIDEB-related condition is a chronic inflammatory disease, such as chronic inflammatory liver disease, selected from liver fat accumulation, hepatitis, liver fibrosis, fatty liver disease (steatohepatitis), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and cirrhosis.

37. The use of claim 35 or 36, wherein administration of the dsRNA activator or the drug to the subject results in a decrease in CIDEB concentration or content (e.g., in the liver or hepatocytes), or a decrease in CIDEB protein accumulation or content in the subject (e.g., in the liver or hepatocytes).

38. The use of any one of claims 35 to 37, wherein the medicament is used in combination with one or more other therapeutic agents, said other therapeutic agents being any therapeutic agent effective, for example, in preventing or treating CIDEB-related diseases and / or conditions, covering therapeutic agents for treating chronic inflammatory diseases, such as chronic inflammatory liver disease, such as those selected from various therapeutic agents for hepatic steatosis, hepatitis, liver fibrosis, fatty liver disease (steatohepatitis), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and cirrhosis.

39. Use according to any one of claims 35 to 38, wherein the level of CIDEB in a sample from the subject is determined prior to administration of the drug.

40. A kit comprising the dsRNA activator of any one of claims 1-21, or the pharmaceutical composition of claim 23 or 24, or the pharmaceutical composition of claim 25.

41. A vial or syringe comprising the dsRNA active agent of any one of claims 1-21, or the pharmaceutical composition of claim 23 or 24, or the pharmaceutical composition of claim 25.

42. 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 21.

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