Dsrna, use thereof and preparation method therefor

Abstract

Provided are a modifier for inhibiting the expression and / or activity of GPR75, such as a double-stranded RNA (dsRNA) active agent or an antisense polynucleotide preparation. Further provided are a method for inhibiting the expression and / or activity of GPR75 by using the modifier, and a method for preventing and treating GPR75-related diseases (such as obesity or lipometabolism-related diseases) 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. 202411737450.9, filed on November 29, 2024, the entire contents of which are incorporated herein by reference.

[0003] This disclosure 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 G protein-coupled receptor 75 (GPR75). This disclosure also relates to methods for inhibiting GPR75 expression and / or activity using such modulators, and methods for preventing and treating GPR75-related diseases in subjects (such as obesity or lipid metabolism-related diseases).

[0004] Background of the Invention

[0005] G protein-coupled receptor 75 (GPR75) is a member of the G protein-coupled receptor (GPCR) family, one of the largest families of membrane protein receptors in the human genome. GPCRs play a role in a variety of physiological and pathological functions, including neurotransmission, hormonal responses, and intercellular signaling. GPR75 is a cell surface receptor that activates guanine nucleotide-binding proteins (G proteins) upon ligand binding. The GPR75 protein contains 540 amino acid residues and has the typical structural features of a GPCR, namely seven transmembrane α-helical segments, an N-terminal N-glycosylation site, and numerous C-terminal serine and threonine phosphorylation sites. Amino acid sequence analysis revealed that GPR75 is most closely associated with the well-known Caenorhabditis elegans neuropeptide Y receptor (24% homology), rat glycopeptide receptor type 3 (25% homology), and porcine growth hormone secretagogue receptor type 1b (25% homology) (Tarttelin et al. (1999) Biochem Biophys Res Commun. 260:174–180). GPR75 is classified as a Gq-coupled class A orphan receptor, and its activation is associated with increased intracellular calcium and IP1 accumulation. GPR75 is expressed in many tissues; in the brain, it is expressed in the neocortex, entorhinal cortex, hippocampus, thalamus, and hypothalamus.

[0006] Currently, three ligands of GPR75 are known: 20-HETE, CCL5, and RANTES. 20-HETE triggers signaling pathways including PI3K / Akt and RAS / MAPK via GPR75, leading to enhanced invasiveness of prostate cancer cells. Furthermore, these signaling pathways activate NF-κB, which plays a crucial role in various pathways of cancer development, such as proliferation, migration, and apoptosis.

[0007] GPR75 is associated with neuroprotection, metabolic regulation, and the development of metabolic diseases such as obesity and diabetes. Current treatments for obesity and metabolic diseases do have some drawbacks. For example, while GLP-1 (glucagon-like peptide-1) drugs are excellent at controlling weight and improving metabolism, they can also cause side effects such as nausea, vomiting, and pancreatitis. Furthermore, some patients may experience muscle loss while using GLP-1 drugs, and weight rebound is common after discontinuation. Many existing drugs act on multiple targets through non-specific pathways, increasing the risk of adverse reactions. This non-specific action can lead to a range of side effects, thus limiting the use and efficacy of these drugs.

[0008] GPR75 plays an important role in a variety of diseases, including obesity, cancer, and metabolic syndrome. Inhibition of GPR75 in humans can increase insulin sensitivity and glucose tolerance, as well as reduce body fat storage.

[0009] In conclusion, GPR75 has significant potential and role in metabolic regulation and obesity treatment, and is considered a promising new mechanism for treating obesity and lipid metabolism-related diseases. Therefore, it is a promising therapeutic target that is expected to provide a more specific and safer treatment approach.

[0010] At present, there is still a need for inhibitors with better activity that can effectively regulate GPR75 expression and / or activity for the treatment of obesity or lipid metabolism-related diseases. Summary of the Invention

[0011] This disclosure provides a specific RNA interference (RNAi) agent capable of effectively reducing the expression and / or activity of GPR75. By designing dsRNAs targeting GPR75, this disclosure can specifically inhibit the expression of the GPR75 gene, contributing to reduced weight gain and improved obesity-related metabolic status, and / or representing a breakthrough in reducing side effects, improving treatment specificity and durability. In some embodiments, the GPR75-targeting dsRNAs of this disclosure can more effectively knock out GPR75, more efficiently promote lipolysis and energy expenditure, exhibiting high specificity and targeting, specifically inhibiting GPR75 gene expression, reducing side effects, improving treatment safety and / or improving treatment efficacy.

[0012] In some embodiments, this disclosure provides a double-stranded ribonucleic acid (dsRNA) activator for inhibiting the expression of the GPR75 target gene in tissues or 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. In some embodiments, these dsRNA activators further include, for example, one or more ligands conjugated to at least one strand of the dsRNA via a linker, the ligands being capable of targeted delivery of the dsRNA to a specific tissue or cell, such as lipids, like C16-C22 fatty acid chains or C16-C22 hydrocarbon chains, capable of delivering the dsRNA molecule to the target tissue or cell. In some embodiments, the ligands are suitable for delivering the dsRNA activator to tissues of the nervous system (e.g., tissues of the central nervous system, such as brain tissue, such as the cortex or cerebellum; or, for example, neurons), spinal tissues (e.g., cervical, lumbar, and thoracic vertebrae), muscles such as skeletal or cardiac muscle, ocular or adipose tissue; or to cells of said tissues.

[0013] This disclosure also provides a cell comprising the above-described dsRNA activator.

[0014] This disclosure provides a pharmaceutical composition comprising the above-described dsRNA activator and optionally a pharmaceutically acceptable carrier.

[0015] This disclosure provides a pharmaceutical combination comprising the above-described dsRNA activator and one or more other therapeutic agents, said other therapeutic agents being any therapeutic agent effective, for example, in preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), covering a variety of therapeutic agents for treating obesity or lipid metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions).

[0016] This disclosure provides the use of the dsRNA activators and / or pharmaceutical compositions and / or drug combinations described herein in the preparation of medicaments for treating GPR75-related diseases and / or conditions, such as diseases and / or conditions caused by abnormal expression of the GPR75 gene.

[0017] This disclosure provides the use of the dsRNA active agent described herein in the preparation of a medicament for inhibiting GPR75 gene expression in tissues or cells, preferably inhibiting GPR75 expression in target tissues or cells. This disclosure also provides a method for preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), the method comprising administering an effective amount of the dsRNA active agent and / or pharmaceutical composition and / or combination of drugs described herein to a subject in need.

[0018] On the other hand, this disclosure provides a method for inhibiting GPR75 gene expression, the method comprising contacting cells with an effective amount of the dsRNA activator and / or pharmaceutical composition and / or drug combination described herein, optionally maintaining the cells generated in this step for a period of time sufficient to degrade the mRNA transcript of the GPR75 gene, thereby inhibiting the expression of the GPR75 gene in the cells.

[0019] In some embodiments, the dsRNA activator disclosed herein can be delivered to any tissue or organ or cell, such as tissues of the nervous system (e.g., tissues of the central nervous system, such as brain tissue, such as the cortex or cerebellum, or such as neurons), spinal tissues (e.g., cervical, lumbar, and thoracic vertebrae), muscles such as skeletal or cardiac muscle, eye tissue, or adipose tissue.

[0020] In some embodiments, the dsRNA activator of this disclosure can be delivered to tissues of the nervous system (e.g., tissues of the central nervous system, such as brain tissue, such as the cortex or cerebellum, or such as neurons), spinal tissues (e.g., cervical, lumbar, and thoracic vertebrae), muscle tissues such as skeletal muscle or cardiac tissue, eye tissues or adipose tissue; or cells of said tissues.

[0021] In some embodiments, the dsRNA activator disclosed herein can be delivered to cells of brain or spinal tissue (e.g., cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae), nervous system cells, muscle cells such as skeletal muscle cells or cardiomyocytes, eye cells, or fat cells. Attached Figure Description

[0022] Figure 1 shows the dose curves of in vitro screening of the compounds of this disclosure at different concentrations.

[0023] Figure 2 shows the knockdown levels of hGPR75 mRNA in different regions of the mouse brain.

[0024] Invention Details

[0025] Before this disclosure is described in detail below, it should be understood that this disclosure 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 this disclosure, 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 disclosure pertains.

[0028] For the purpose of interpreting this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate.

[0029] It should be understood that the terminology used in this document is for the purpose of describing specific implementation schemes only and is not intended to be restrictive.

[0030] The term "about," when used in conjunction with a numerical value, means to cover a range of numerical values ​​having a lower limit that is 5% (e.g., 4%, 3%, 2%, or 1%) smaller than the specified numerical value and an upper limit that is 5% (e.g., 4%, 3%, 2%, or 1%) larger than the specified numerical value. When the term "about" is used in conjunction with a numerical range, such as in "about 0.1-2.5nM," it is intended to cover a numerical range whose lower limit is 5%, 4%, 3%, 2%, or 1% smaller than the lower limit of the numerical range associated with "about," and whose upper limit is 5%, 4%, 3%, 2%, or 1% larger than the upper limit of the numerical range associated with "about." When the term "about" is used in conjunction with multiple numerical values ​​or numerical ranges, it indicates that each of the stated numerical values ​​or numerical ranges is used in conjunction with the term "about." For example, the expression "about 0.1, 0.2, 0.3" is intended to cover the cases of "about 0.1," "about 0.2," and "about 0.3." As used herein, the term “and / or” means any one of the options or two or more or all of the options.

[0031] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0032] 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.

[0033] The term "regulator" as used in this article refers to a molecule that can modulate, for example, reduce or inhibit GPR75 expression and / or activity, such as RNAi activators or dsRNA activators.

[0034] As used herein, the term "G protein-coupled receptor 75" ("GPR75") refers to the gene and polypeptide well known in the art as "possible G protein-coupled receptor 75", "WI-31133", "GPRchr2", and "WI31133". GPR75 binds to 20-HETE and interferes with insulin signaling, which leads to obesity. The term "GPR75" includes human GPR75, whose amino acid and nucleotide sequences are available, for example, in GenBank accession number NM_006794.4 (SEQ ID NO:286); mouse GPR75, whose amino acid and nucleotide sequences are available, for example, in GenBank accession number NM_175490.4; and rat GPR75, whose amino acid and nucleotide sequences are available, for example, in GenBank accession number NM_001109096.1. The term "GPR75" also includes the macaque (Macaca mulatta) GPR75, whose amino acid and nucleotide sequences are available, for example, in GenBank accession number NM_001204509.2. Other examples of GPR75 mRNA sequences are readily available using websites such as GenBank, UniProt, OMIM, and the Macaque Genome Project. An exemplary GPR75 nucleotide sequence is also available in SEQ ID NO:286. SEQ ID NO:287 is the reverse complementary sequence of SEQ ID NO:286. More information about GPR75 is available, for example, in the NCBI Gene Database www.ncbi.nlm.nih.gov / gene / 10936. The entire contents of the aforementioned GenBank accession number and gene database number are incorporated herein by reference as of the date of this application.

[0035] As used herein, the terms “G protein-coupled receptor 75” and “GPR75” also refer to naturally occurring DNA sequence variants of the GPR75 gene. Many sequence variants of the GPR75 gene have been identified and are available in, for example, NCBI dbSNP and UniProt (see, for example, https: / / www.ncbi.nlm.nih.gov / snp / ?term=GPR75), the entire contents of which are incorporated herein by reference as of the date of this application.

[0036] GPR75 nucleotide sequence_NM_006794.4 (SEQ ID NO:286)

[0037] GPR75 nucleotide sequence_NM_006794.4 reverse complementary sequence (SEQ ID NO:287)

[0038] As used herein, a “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during GPR75 gene transcription, containing 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 GPR75 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 this disclosure, the target sequence length is preferably at least 18, 19, 20, or 21 nucleotides. In some embodiments of this disclosure, the target sequence length is about 19 to about 23 nucleotides. In some embodiments of this disclosure, the target sequence length is about 20 or about 21 nucleotides.

[0039] “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 characterized in this disclosure, 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 substituted moieties are suitable for the compositions and methods characteristic of this disclosure.

[0040] When this article refers to a "nucleotide sequence", it means a continuous nucleotide sequence, which can be a natural nucleotide or a modified nucleotide.

[0041] As used interchangeably herein, the terms “dsRNA,” “dsRNA activator,” “double-stranded RNA,” and “double-stranded RNA molecule” refer to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands having “sense” and “antisense” orientations relative to the target RNA (i.e., the GPR75 gene), respectively. In some embodiments of this disclosure, 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 this disclosure 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.

[0042] The term "siRNA" as used herein refers to a class of double-stranded RNA molecules that can mediate the silencing of a 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.

[0043] 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., GPR75 mRNA).

[0044] As used herein, the terms “sense strand” or “lazy strand” or “sense strand” refer to an oligonucleotide strand containing a region substantially complementary to the antisense strand as defined herein, which can complement the antisense strand to form a double-stranded region of dsRNA.

[0045] 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 final 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 dsRNA, or between the antisense strand and target sequence of RNAi or dsRNA, the terms "complementarity" or "complementarity" should be understood to cover not only 100% complementarity (i.e., complete complementarity) but also less than 100% complementarity (i.e., substantially complementarity), that is, the presence of base mismatches in the complementary double-stranded nucleic acid region that do not substantially affect the RNAi or dsRNA'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).

[0046] In this document, for the purposes of this disclosure, the expression “complementary” or “complementarity” associated with double-stranded RNAi activators (such as dsRNAs like siRNA described herein) is preferably not less than 70%, meaning that at least 70% of the base positions in the double-stranded region formed by complementary hybridization are complementary, i.e., the number of mismatched positions in the continuous nucleotide sequence forming the double-stranded region is less than 30%. For example, for a 21-base-pair double-stranded region, not less than 70% complementarity means that the double-stranded region forms no more than 6, 5, 4, 3, 2, 1, or 0 mismatched base pairs during hybridization. Preferably, the presence of insertions and deletions is not allowed when calculating the complementarity of the continuous nucleotide sequence in the double-stranded region. Accordingly, in this document, the expression associated with RNAi activators, “complementary (antisense) sequence” to the target sequence, or “complementary (sense) sequence” to a portion of the antisense sequence, can be “completely complementary” or “substantially complementary.” “Completely complementary” means that the two sequences have 100% complementarity. When the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may contain one or more, but typically no more than 30%, 20%, or 10% mismatched base pairs in the hybridized duplex, and still retain the ability to hybridize under conditions most relevant to their final application (e.g., repressing gene expression via the RISC pathway).

[0047] As used herein, “perfect complementarity” means complete complementarity between two strands. It should be understood that when referring to complete complementarity of complementary or double-stranded regions, it means complete complementarity between two identical nucleotide strands that match during alignment. Therefore, it should be understood that when the two oligonucleotides of an RNAi or dsRNA 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 sense oligonucleotide strand and a 21-nucleotide antisense oligonucleotide strand can still be considered “perfectly complementary” if the longer antisense oligonucleotide contains a 19-nucleotide sequence that is completely complementary to the shorter sense oligonucleotide.

[0048] As used herein, the term "complementary region" refers to a region on the antisense strand that is complementary (substantially complementary or fully complementary) to a sequence defined herein (e.g., a target sequence, such as the GPR75 mRNA target sequence). In cases where the complementary region is substantially complementary to the target sequence, mismatches can be located within the interior or terminal regions of the molecule. 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 active agent of this disclosure contains nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA active agent of this disclosure contains no more than 4 mismatches with the target mRNA; for example, the antisense strand contains 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 this disclosure is mismatched with the target mRNA, for example, the 5' end of the antisense strand of the double-stranded RNA activator of this disclosure 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 this disclosure 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.

[0049] 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.

[0050] "Flat-ended" or "blunt-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 dsRNAs disclosed herein cover dsRNAs with blunt ends at both the 5' and 3' ends.

[0051] 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.

[0052] As used herein, the “internal position” of the sense or antisense strand refers to a position other than the positions of the 5' and 3' terminal nucleotides of the sense or antisense strand. Therefore, the nucleotide at the “internal position” of the sense or antisense strand is neither a 5' nor a 3' terminal nucleotide. In some embodiments, the nucleotide at the “internal position” of the sense or antisense strand is not a nucleotide at the overhang of the sense or antisense strand, but rather a nucleotide within the double-stranded region (nucleotides at both ends of the non-double-stranded region).

[0053] Generally, most nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also contain one or more modified ribonucleotides, such as deoxyribonucleotides or chemically modified nucleotides. 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 applicable to the active agents of this disclosure include all types of modifications disclosed herein or known in the art.

[0054] For naturally occurring oligonucleotides, internucleotide bonds include phosphate groups that form phosphodiester bonds between adjacent nucleosides. In this document, 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 this disclosure may contain one or more internucleotide bonds modified from natural phosphodiester bonds. Modified internucleotide bonds contemplated according to this disclosure 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 this disclosure is a thiophosphate bond.

[0055] As used herein, a "ligand moiety" refers to a chemical portion that conjugates to the double strand of dsRNA and is capable of altering the distribution, targeting, or half-life of the dsRNA. When "dsRNA" or "dsRNA activator" is mentioned herein, it also encompasses dsRNA containing a ligand moiety unless the context clearly contradicts this description. Similarly, when "siRNA" or "siRNA activator" is mentioned herein, it also encompasses siRNA containing a ligand moiety unless the context clearly contradicts this description. In some embodiments of this disclosure, the ligand moiety is a lipid or lipid-based molecule, such as a lipophilic moiety.

[0056] 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.

[0057] As used herein, the term “inhibition” is used interchangeably with “reduction,” “silence,” “downregulation,” and other similar terms, and includes any level of inhibition.

[0058] The expression "inhibit GPR75" refers to inhibiting the activity or expression of any GPR75 gene. The expression "inhibit GPR75 expression" refers to inhibiting the expression of any GPR75 gene, as well as variants or mutants of the GPR75 gene. Therefore, the GPR75 gene can be a wild-type GPR75 gene, a mutant GPR75 gene, or a transgenic GPR75 gene in the case of genetically manipulated cells, cell groups, or organisms.

[0059] "Inhibition of GPR75 gene expression" includes inhibition of any level of the GPR75 gene, such as at least partial repression of GPR75 gene expression. GPR75 gene expression can be assessed based on the level or change in the level of any variable associated with GPR75 gene expression, such as GPR75 mRNA level or GPR75 protein level. This level can be assessed in individual cells or in a group of cells (including, for example, samples derived from an individual). Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with GPR75 expression compared to a control level. The control level can be any type of control level utilized in the art, such as baseline levels before administration or levels determined from similar untreated or controlled (e.g., buffer-only control or inert agent control) individuals, cells, or samples.

[0060] As used herein, the term "GPR75-related disease or condition" refers to a disease or condition caused by or associated with abnormal expression and / or activity of GPR75. The term "GPR75-related disease or condition" includes diseases or conditions caused by abnormal expression of the GPR75 gene, or diseases, disorders, or ailments from which one may benefit by decreased GPR75 gene expression, replication, or protein activity. In some embodiments, a GPR75-related disease or condition is obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, encompassing obesity) or a disease related to fat metabolism. In some embodiments, a disorder from which one may benefit by decreased GPR75 gene expression, replication, or protein activity is obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, encompassing obesity). Therefore, the dsRNA activator of this disclosure can be used for weight management or to reduce weight or body fat.

[0061] As used herein, the term "obesity" refers to an abnormal or excessive accumulation of fat in an individual that may impair health, encompassing overweight, obesity, severe obesity, or extreme obesity. Various criteria exist for determining obesity, such as BMI, body fat percentage, waist circumference, and waist-to-hip ratio. The "obesity" of this disclosure encompasses any condition identified as exceeding a normal weight using any of these criteria.

[0062] The term "effective amount" refers to such an amount or dose of the dsRNA active agent or composition or combination of the present disclosure that, 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".

[0063] "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%.

[0064] "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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] The term "drug combination" refers to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that active ingredients (e.g., (i) the dsRNA active agent of this disclosure, 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 this disclosure 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.

[0070] 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 disease or condition described herein.

[0071] The term "other therapeutic agents" encompasses any therapeutic agent, other than the dsRNA active agents disclosed herein or pharmaceutical compositions containing them, that is effective in preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), and includes various therapeutic agents for weight management, for reducing weight or body fat, or for preventing or treating obesity or fat metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions).

[0072] 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, circumstances, illnesses, or diseases.

[0073] When used in this article, "prevention" includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition.

[0074] 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."

[0075] "Subject / Patient / Individual Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds that are naturally occurring and do not mix with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0076] II. dsRNA activator

[0077] This disclosure provides RNAi activators for inhibiting GPR75, 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 GPR75 gene in cells (such as tissues or cells in a subject, e.g., a mammal, such as a person with a GPR75-related disease or condition, such as overweight or obesity).

[0078] 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 (in its entirety incorporated herein by reference). 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 an antisense strand. It is understood that these molecular forms all fall within the scope of the RNAi activators disclosed herein. Furthermore, siRNA or dsRNA may contain modified nucleotides, and may also contain ligands that deliver siRNA or dsRNA to targeted tissues or cells in vivo. These dsRNAs or siRNAs with modified nucleotides, as well as dsRNAs and siRNAs with modified nucleotides and ligands, are also dsRNA activators of this disclosure.

[0079] In some embodiments, the dsRNA activator of this disclosure, such as siRNA, inhibits the expression of the GPR75 gene (e.g., the human GPR75 gene) at least 15%, about 20%, about 25%, 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%, or about 92%, as determined by, for example, PCR or by a protein-based method (e.g., by immunofluorescence analysis using, for example, Western blotting or flow cytometry). In some embodiments, the inhibition rate of expression is determined in suitable biological cell lines using, for example, concentrations of dsRNA such as siRNA of approximately 10 nM, 1 nM, or 0.1 nM, via the qPCR method provided herein. In some embodiments, the inhibition rate of expression is determined in suitable biological cell lines using, for example, serially diluted concentrations of dsRNA such as siRNA via the qPCR method provided herein.

[0080] 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 effectively inhibit the expression of the GPR75 gene in vivo, for example in the central nervous system such as brain tissue, such as in the cerebral cortex, hypothalamus, hippocampus or other brain tissues.

[0081] In some embodiments, the dsRNA activator of this disclosure, such as siRNA (including siRNA with modified nucleotides and siRNA with modified nucleotides and ligands), inhibits the expression of the GPR75 gene (e.g., human GPR75 gene) in vivo (e.g., in brain tissue) by at least about 5%, 10%, 15%, 20%, or 25%, for example, by detecting mouse brain tissue (e.g., cerebral cortex, hypothalamus, hippocampus, and / or other brain regions) extracted after a single intraventricular injection in mice, as described in Example 6.

[0082] In some embodiments, the dsRNA activator comprises an antisense strand containing a complementary region that is complementary (substantially complementary or fully complementary) to at least a portion (e.g., a target sequence) of the mRNA formed during GPR75 gene expression. In some embodiments, the complementary region is about 15 to 30 nucleotides in length, 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 complementary region is between 18 and 23 nucleotides in length. In some embodiments, the complementary region is 18, 19, 20, or 21 nucleotides in length. In some embodiments, the complementary region is at least 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the antisense strand is complementary to the mRNA target sequence starting from the second nucleotide from the 5' end. In some embodiments, the complementary region of the antisense strand comprises the second nucleotide from the 5' end to the 3rd, 2nd, or 1st nucleotide from the 3' end. In some embodiments, the complementary region of the antisense strand comprises all antisense strand nucleotides starting from the second nucleotide from the 5' end. In some embodiments, the complementary region of the antisense strand comprises at least nucleotides 2-16, 2-17, 2-18, 2-19, 2-20, or 2-21 from the 5' end of the antisense strand. In some embodiments, the complementary region of the antisense strand comprises at least 2-19 consecutive nucleotides from the 5' end of the antisense strand. In some embodiments, the complementary region of the antisense strand comprises, or consists of, consecutive nucleotides 2-19, 2-20, or 2-21 from the 5' end of the antisense strand.

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

[0084] In some embodiments, the antisense strand of the dsRNA includes 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 corresponding portion of the target sequence. The target sequence can be derived from the sequence of the mRNA formed during GPR75 gene expression. In some embodiments, the antisense complement is substantially complementary to the target sequence, for example, it is mismatched with the target sequence at 1, 2, 3, 4, or 5 nucleotides (preferably 1 or 2 nucleotides at the 5' and / or 3' ends, e.g., the first nucleotide at the 5' end of the antisense strand). In some embodiments, the antisense complement is completely complementary to the target sequence.

[0085] In some embodiments, the antisense strand of the dsRNA is completely complementary to the corresponding portion of the target sequence, starting from the second nucleotide from the 5' end. In some embodiments, the antisense strand of the dsRNA is completely complementary to the corresponding portion of the target sequence from the second nucleotide from the 5' end to the first, second, or third nucleotide from the 3' end. In some embodiments, the entire length of the antisense strand of the dsRNA, starting from the second nucleotide from the 5' end, is completely complementary to the corresponding portion of the target sequence. In some embodiments, nucleotides 2-16, 2-17, 2-18, 2-19, 2-20, or 2-21 of the antisense strand of the dsRNA, starting from the 5' end, are completely complementary to the corresponding portion of the target sequence. In some embodiments, consecutive nucleotides from positions 2-19 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 corresponding portion of 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.

[0086] When referring to "the corresponding portion of the target sequence" in this document, "the corresponding portion of the target sequence" means 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, counting from the 5' end, are completely complementary to the consecutive nucleotides from position 2 to 21, counting from the 5' end, the "corresponding portion of the target sequence" refers to the consecutive nucleotides from position 1 to 20, counting from the 5' end, of the target sequence.

[0087] In some implementations, the dsRNA described herein targets the sequence of the GPR75 gene at or near the location shown in Table 1 (e.g., NM_006794.4) of the GPR75 genome.

[0088] 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 GPR75 genome (e.g., NM_006794.4) mRNA or the nucleic acid sequence shown in SEQ ID NO:286 or its complementary sequence (e.g., the nucleic acid sequence shown in SEQ ID NO:287) genomic sequence.For example, the nucleic acid sequence shown in SEQ ID NO:286 includes sequences 251-271, 338-358, 346-366, 373-393, 440-460, 450-470, 451-471, 452-472, 453-473, 473-493, 474-494, 475-495, 476-496, 478-498, 479-499, 480-500, 481-501, 482-502, 483-503, 485-505, 486-506, 488-508, 489-509, 508-528, 542-562, 781-801, 790- 810, 821-841, 847-867, 861-881, 866-886, 904-924, 1056-1076, 1058-1078, 1059-1079, 1060-1080, 1065-1085, 1079-1099, 1100-1120, 1165-1185, 1184-1204, 1185-1205, 1277-1297, 1295-1315, 1296-1316, 1299-1319, 1300-1320, 1301-1321, 1302-1322, 1348-1368, 134 9-1369, 1350-1370, 1351-1371, 1352-1372, 1366-1386, 1370-1390, 1371-1391, 1372-1392, 1374-1394, 1429-1449, 1448-1468, 1486-1506, 1526-1546, 1540-1560, 1567-1587, 1623-1643, 1635-1655, 1705-1725, 1733-1753, 1747-1767, 1748-1768, 1749-1769, 1751-1771, The sequences corresponding to positions 1752-1772, 1786-1806, 1818-1838, 1819-1839, 1969-1989, 1971-1991, 1972-1992, 1973-1993, 1974-1994, 1975-1995, 1977-1997, 1978-1998, 1979-1999, 1980-2000, 1981-2001, 1983-2003, 1984-2004, 1985-2005, 1986-2006, 1988-2008, 2024-2044, or 2025-2045.

[0089] In a preferred embodiment, 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, for example, the sequence corresponding to positions 1349-1369 of the nucleic acid sequence shown in SEQ ID NO:286, in the GPR75 genome (e.g., NM_006794.4) mRNA or the nucleic acid sequence shown in SEQ ID NO:286 or its complementary sequence (e.g., the nucleic acid sequence shown in SEQ ID NO:287).

[0090] In a preferred embodiment, 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, for example, the sequence corresponding to positions 1984-2004 of the nucleic acid sequence shown in SEQ ID NO:286, in the GPR75 genome (e.g., NM_006794.4) mRNA or the nucleic acid sequence shown in SEQ ID NO:286 or its complementary sequence (e.g., the nucleic acid sequence shown in SEQ ID NO:287).

[0091] In some implementations, the dsRNA described herein targets a nucleotide sequence (target sequence) of the mRNA of the GPR75 gene selected from the following:

[0092] (i) A series of consecutive nucleotides of GPR75 mRNA at or near the location of the GPR75 genome (e.g., NM_006794.4) mRNA as shown in Table 1, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 consecutive nucleotides at or near the location;

[0093] (ii) The 251st, 338th, 346th, 373rd, 440th, 450th, 451st, 452nd, 453rd, 473rd, 474th, 475th, 476th, 478th, 479th, 480th, 481st, 482nd, 483rd, 485th, 486th, 488th, 489th, 508th, 542nd, 781st, 790th, and 82nd positions of the GPR75 genome (e.g., NM_006794.4) mRNA or the nucleic acid sequence shown in SEQ ID NO:286 or its complementary sequence (e.g., the nucleic acid sequence shown in SEQ ID NO:287). 1, 847, 861, 866, 904, 1056, 1058, 1059, 1060, 1065, 1079, 1100, 1165, 1184, 1185, 1277, 1295, 1296, 1299, 1300, 1301, 1302, 1348, 1349, 1350, 1351, 135 2. At least 15-35 consecutive nucleotides starting at position 1366, 1370, 1371, 1372, 1374, 1429, 1448, 1486, 1526, 1540, 1567, 1623, 1635, 1705, 1733, 1747, 1748, 1749, 1751, 1752, 1786, 1818, 1819, 1969, 1971, 1972, 1973, 1974, 1975, 1977, 1978, 1979, 1980, 1981, 1983, 1984, 1985, 1986, 1988, 2024, or 2025, for example, 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides;

[0094] (iii) GPR75 genomic mRNA (e.g., NM_006794.4) or the nucleic acid sequence shown in SEQ ID NO:286 or its complementary sequence (e.g., SEQ ID NO:286).The nucleic acid sequence shown in NO:287 contains sequences 251-271, 338-358, 346-366, 373-393, 440-460, 450-470, 451-471, 452-472, 453-473, 473-493, 474-494, 475-495, 476-496, 478-498, 479-499, 480-500, 481-501, 482-502, 483-503, 485-505, 486-506, 488-508, 489-509, 508-528, 542-562, 781-801, 790-810, and 821-8 41, 847-867, 861-881, 866-886, 904-924, 1056-1076, 1058-1078, 1059-1079, 1060-1080, 1065-1085, 1079-1099, 1100-1120, 1165-1185, 1184-1204, 1185-1205, 1277-1297, 1295-1315, 1296-1316, 1299-1319, 1300-1320, 1301-1321, 1302-1322, 1348-1368, 1349-1369, 1350-1370, 1351- 1371, 1352-1372, 1366-1386, 1370-1390, 1371-1391, 1372-1392, 1374-1394, 1429-1449, 1448-1468, 1486-1506, 1526-1546, 1540-1560, 1567-1587, 1623-1643, 1635-1655, 1705-1725, 1733-1753, 1747-1767, 1748-1768, 1749-1769, 1751-1771, 1752-1772, 1786-1806, 1818-1838, 1819- The 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, or 35th consecutive nucleotides of the sequence corresponding to positions 1839, 1969-1989, 1971-1991, 1972-1992, 1973-1993, 1974-1994, 1975-1995, 1977-1997, 1978-1998, 1979-1999, 1980-2000, 1981-2001, 1983-2003, 1984-2004, 1985-2005, 1986-2006, 1988-2008, 2024-2044, or 2025-2045;

[0095] (iv) GPR75 genome (e.g., NM_006794).4) mRNA or the nucleic acid sequence shown in SEQ ID NO:286 or its complementary sequence (e.g., SEQ ID NO:286). Nucleic acid sequences shown in NO:287, including sequences 251-271, 338-358, 346-366, 373-393, 440-460, 450-470, 451-471, 452-472, 453-473, 473-493, 474-494, 475-495, 476-496, 478-498, 479-499, 480-500, 481-501, 482-502, 483-503, 485-505, 486-506, 488-508, 489-509, 508-528, 542-562, 781-801, 790-810, and 821-84. 1. 847-867, 861-881, 866-886, 904-924, 1056-1076, 1058-1078, 1059-1079, 1060-1080, 1065-1085, 1079-1099, 1100-1120, 1165-1185, 1184-1204, 1185-1205, 1277-1297, 1295-1315, 1296-1316, 1299-1319, 1300-1320, 1301-1321, 1302-1322, 1348-1368, 1349-1369, 1350-1370, 1351-1 371, 1352-1372, 1366-1386, 1370-1390, 1371-1391, 1372-1392, 1374-1394, 1429-1449, 1448-1468, 1486-1506, 1526-1546, 1540-1560, 1567-1587, 1623-1643, 1635-1655, 1705-1725, 1733-1753, 1747-1767, 1748-1768, 1749-1769, 1751-1771, 1752-1772, 1786-1806, 1818-1838, 1819- The sequence corresponding to positions 1839, 1969-1989, 1971-1991, 1972-1992, 1973-1993, 1974-1994, 1975-1995, 1977-1997, 1978-1998, 1979-1999, 1980-2000, 1981-2001, 1983-2003, 1984-2004, 1985-2005, 1986-2006, 1988-2008, 2024-2044, or 2025-2045, consisting of any 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides, preferably the nucleotide sequence corresponding to the positions mentioned above; or...

[0096] (v) A nucleotide sequence that contains or consists of the nucleotide sequences shown in any of SEQ ID NO:191-285.

[0097] In some implementations, the dsRNA described herein targets a nucleotide sequence (target sequence) of the mRNA of the GPR75 gene selected from the following:

[0098] (i) At least 15-35 consecutive nucleotides, such as 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides, starting from position 1349 of the GPR75 genome (e.g., NM_006794.4) mRNA or the nucleic acid sequence shown in SEQ ID NO:286 or its complementary sequence (e.g., the nucleic acid sequence shown in SEQ ID NO:287);

[0099] (ii) 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 consecutive nucleotides containing the sequence corresponding to positions 1349-1369 in the GPR75 genome (e.g., NM_006794.4) mRNA or the nucleic acid sequence shown in SEQ ID NO:286 or its complementary sequence (e.g., the nucleic acid sequence shown in SEQ ID NO:287);

[0100] (iii) Any 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of the sequence corresponding to positions 1349-1369 in the GPR75 genome (e.g., NM_006794.4) mRNA or the nucleic acid sequence shown in SEQ ID NO:286 or its complementary sequence (e.g., the nucleic acid sequence shown in SEQ ID NO:287), preferably the nucleotide sequence corresponding to the positions mentioned above; or

[0101] (iv) A nucleotide sequence that contains or consists of the nucleotide sequence shown in SEQ ID NO:241.

[0102] In some implementations, the dsRNA described herein targets a nucleotide sequence (target sequence) of the mRNA of the GPR75 gene selected from the following:

[0103] (i) At least 15-35 consecutive nucleotides, such as 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides, starting from position 1984 of the GPR75 genome (e.g. NM_006794.4) mRNA or the nucleic acid sequence shown in SEQ ID NO:286 or its complementary sequence (e.g. the nucleic acid sequence shown in SEQ ID NO:287);

[0104] (ii) 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 consecutive nucleotides containing the sequence corresponding to positions 1984-2004 in the GPR75 genome (e.g., NM_006794.4) mRNA or the nucleic acid sequence shown in SEQ ID NO:286 or its complementary sequence (e.g., the nucleic acid sequence shown in SEQ ID NO:287);

[0105] (iii) Any 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of the sequence corresponding to positions 1984-2004 in the GPR75 genome (e.g., NM_006794.4) mRNA or the nucleic acid sequence shown in SEQ ID NO:286 or its complementary sequence (e.g., the nucleic acid sequence shown in SEQ ID NO:287), preferably the nucleotide sequence corresponding to the positions mentioned above; or

[0106] (iv) A nucleotide sequence that contains or consists of the nucleotide sequence shown in SEQ ID NO:280.

[0107] In some embodiments, the target sequence comprises, or consists of, a continuous sequence of GPR75 mRNA at or near the location shown in Table 1 of the GPR75 genome (e.g., NM_006794.4). In some embodiments, the mRNA sequence targeted by the dsRNA described herein (i.e., the GPR75 gene target sequence) comprises, or consists of, the target sequence corresponding to the location shown in Table 1 of the GPR75 mRNA. In some embodiments, the mRNA sequence targeted by the dsRNA described herein (i.e., the GPR75 gene target sequence) comprises, or consists of, the nucleotide sequence shown in any of SEQ ID NO:191-285.

[0108] 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 position in the GPR75 genome (e.g., NM_006794.4) shown in Table 1 or the target sequence disclosed in Table 2. In some embodiments, the antisense strand of the dsRNA has the same number of nucleotides as or differs from the GPR75 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 GPR75 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 GPR75 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 one, two, or three nucleotides. In some embodiments, the region of the dsRNA antisense strand of the GPR75 gene target sequence (e.g., the target sequence corresponding to the position shown in Table 1 or the target sequence shown in Table 2) excluding the first and / or second nucleotides at the 5' end is completely complementary to the region of the target sequence excluding the first and / or second nucleotides at the 3' end. In some embodiments, the region of the dsRNA antisense strand of the GPR75 gene target sequence (e.g., the target sequence corresponding to the position shown in Table 1 or the target sequence shown in Table 2) excluding the first nucleotide at the 5' end is completely complementary to the region of the target sequence excluding the first nucleotide at the 3' end. In some embodiments, the first nucleotide at the 5' end of the dsRNA antisense strand is U or A, for example, U, for example, to facilitate recognition by the Ago2 protein to form the RICS complex.

[0109] In some implementations, the dsRNA comprises two complementary RNA strands that hybridize under conditions that will cause the dsRNA to hybridize to form a double-stranded structure (also known as a double-stranded region), namely the antisense strand and the sense strand.

[0110] In some embodiments, the length of the sense strand and the antisense strand is independently 15-30 nucleotides, such as 17-27 nucleotides, 19-25 nucleotides, 18-25 nucleotides, 18-24 nucleotides, 18-23 nucleotides, 19-23 nucleotides, 19-22 nucleotides, or 19-21 nucleotides. In some embodiments, the length of the antisense strand or the sense strand is independently no more than 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides. In some embodiments, the length of the antisense strand or the sense strand is independently not less than 15, 16, 17, 18, 19, 20, or 21 nucleotides. In some embodiments, the length of the sense strand is 18-20 nucleotides (e.g., 18, 19, or 20 nucleotides), and the length of the antisense 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.

[0111] 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 implementations, the length of the double-stranded region is 18, 19, 20, or 21 nucleotide pairs, for example, 19 nucleotide pairs.

[0112] 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), for example, located at the 5' and / or 3' ends of the sense and / or antisense strands or within the sense and / or antisense strands. In some embodiments, the length of the completely complementary double-stranded region is at least 13, 14, 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 implementations, the length of the fully complementary double-stranded region is 16, 17, 18, 19, 20, or 21 nucleotides, for example, 18 or 19 nucleotides.

[0113] In some embodiments, the number of completely complementary nucleotide pairs in the double-stranded region is at least 15, 16, 17, 18, or 19 nucleotide pairs. In some embodiments, the number of completely complementary nucleotide pairs in the 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 nucleotide pairs. In some embodiments, the number of completely complementary nucleotide pairs in the double-stranded region is 16, 17, 18, 19, 20, or 21 nucleotide pairs, for example, 18 or 19 nucleotide pairs.

[0114] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, for example, 1 to 4, 2 to 4, 1 to 3, 2 to 3, 1, 2, 3, or 4 nucleotides. In some embodiments, dsRNA having at least one nucleotide overhang has better repressive properties relative to its blunt-ended counterpart. The nucleotide overhang may include or consist of nucleotide / nucleoside analogs comprising deoxynucleotides / nucleosides. The overhang may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide may be present at the 5' end, 3' end, or both ends of the antisense strand or sense strand of the dsRNA.

[0115] 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.

[0116] 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.

[0117] In some embodiments, the sense strand includes a 5' overhang with at least 1, 2, or 3 nucleotides, and / or the antisense strand includes a 3' overhang with at least 1, 2, or 3 nucleotides.

[0118] In some embodiments, the antisense strand of the dsRNA has a 3' overhang, for example, a 3' overhang of 2 nucleotides, and a blunt end at the 5' end.

[0119] In some embodiments, this disclosure relates to a double-stranded RNA (dsRNA) activator for inhibiting the expression of GPR75, 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 a corresponding portion of the target sequence of the GPR75 gene at least from position 2 to 19 (e.g., positions 2 to 20 or 2 to 21 or the full length) starting from the 5' end, for example, wherein the antisense strand is completely complementary to the target sequence of the GPR75 gene 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.

[0120] In some embodiments, the dsRNA activator of this disclosure 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 18 or 19 nucleotides, for example, at 19 consecutive nucleotides (e.g., at the first to 19th consecutive nucleotides of the antisense strand).

[0121] In some embodiments, the dsRNA of this disclosure comprises a sense strand and an antisense strand forming a double-stranded region, wherein

[0122] (i) The positive strand contains or is 19 nucleotides.

[0123] (ii) The antisense strand comprises or is 21 nucleotides and is completely complementary to the target sequence of the GPR75 gene in the region excluding the first nucleotide counting from the 5' end, wherein the first nucleotide counting from the 5' end of the antisense strand is A or U, for example U; and / or

[0124] (iii) The antisense strand contains a 3' overhang of 2 nucleotides compared to the sense strand, and wherein the sense strand and the antisense strand are completely complementary at consecutive nucleotides from position 1 to position 19 of the antisense strand.

[0125] In one aspect of this disclosure, the disclosure relates to a double-stranded ribonucleic acid (dsRNA) activator for inhibiting the expression of GPR75, 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 the nucleotide sequence of any antisense strand 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.

[0126] In some embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand comprises 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 wherein the antisense strand comprises 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 sense strand in Table 1, 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, wherein the sense strand comprises 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 comprises 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, wherein the first nucleotide at the 5' end of the antisense strand is A or U, for example U, and wherein the sense strand and antisense strand in Table 1 are sense strands and antisense strands under the same siRNA name.

[0128] In some embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand comprises 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 comprises 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, and wherein the sense strand and antisense strand in Table 1 are sense strands and antisense strands under the same siRNA name.

[0129] 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: 96-190. In some specific embodiments, the antisense strand differs from the nucleotide sequence shown in any one of SEQ ID NO: 96-190 by no more than 1, 2, or 3 nucleotides. In some specific embodiments, the antisense strand comprises or consists of the nucleotide sequence shown in any one of SEQ ID NO: 96-190. In some specific embodiments, the antisense strand comprises or consists of the nucleotide sequence shown in SEQ ID NO: 146. In some specific embodiments, the antisense strand comprises or consists of the nucleotide sequence shown in SEQ ID NO: 185.

[0130] In some specific embodiments, the positive strand comprises a nucleotide sequence of at least 15, 16, 17, 18, or 19 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO:1-95. In some specific embodiments, the positive strand differs from the nucleotide sequence shown in any one of SEQ ID NO:1-95 by no more than 1, 2, or 3 nucleotides. In some specific embodiments, the positive strand comprises or consists of the nucleotide sequence shown in any one of SEQ ID NO:1-95. In some specific embodiments, the positive strand comprises or consists of the nucleotide sequence shown in SEQ ID NO:51. In some specific embodiments, the positive strand comprises or consists of the nucleotide sequence shown in SEQ ID NO:90.

[0131] In some embodiments, the dsRNA activator comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence selected from any of the nucleotide sequences of the sense strand in Table 1, and the antisense strand comprising a nucleotide sequence selected from any of the nucleotide sequences of the antisense strand in Table 1. In some embodiments, the combination of the nucleotide sequences of the antisense and sense strands in the dsRNA activator is as shown in Table 1 for any combination of nucleotide sequences of the antisense and sense strands.

[0132] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the antisense strand and the sense strand respectively comprise SEQ ID NO:96 / SEQ ID NO:1, SEQ ID NO:97 / SEQ ID NO:2, SEQ ID NO:98 / SEQ ID NO:3, SEQ ID NO:99 / SEQ ID NO:4, SEQ ID NO:100 / SEQ ID NO:5, SEQ ID NO:101 / SEQ ID NO:6, SEQ ID NO:102 / SEQ ID NO:7, SEQ ID NO:103 / SEQ ID NO:8, SEQ ID NO:104 / SEQ ID NO:9, SEQ ID NO:105 / SEQ ID NO:10, SEQ ID NO:106 / SEQ ID NO:11, SEQ ID NO:107 / SEQ ID NO:12, SEQ ID NO:108 / SEQ ID NO:13, SEQ ID NO:109 / SEQ ID NO:14, SEQ ID NO:110 / SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:10 ... NO:111 / SEQ ID NO:16, SEQ ID NO:112 / SEQ ID NO:17, SEQ ID NO:113 / SEQ ID NO:18, SEQ ID NO:114 / SEQ ID NO:19, SEQ ID NO:115 / SEQ ID NO:20, SEQ ID NO:116 / SEQ ID NO:21, SEQ ID NO:117 / SEQ ID NO:22, SEQ ID NO:118 / SEQ ID NO:23, SEQ ID NO:119 / SEQ ID NO:24, SEQ ID NO:120 / SEQ ID NO:25, SEQ ID NO:121 / SEQ ID NO:26, SEQ ID NO:122 / SEQ ID NO:27, SEQ ID NO:123 / SEQ ID NO:28, SEQ ID NO:124 / SEQ ID NO:29, SEQ ID NO:125 / SEQ ID NO:30, SEQ ID NO:126 / SEQ ID NO:31, SEQ ID NO:127 / SEQ ID NO:32, SEQ ID NO:128 / SEQ ID NO:33, SEQ ID NO:129 / SEQ ID NO:34, SEQ ID NO:130 / SEQ ID NO:35, SEQ ID NO:131 / SEQ IDNO:36、SEQ ID NO:132 / SEQ ID NO:37、SEQ ID NO:133 / SEQ ID NO:38、SEQ ID NO:134 / SEQ ID NO:39、SEQ ID NO:135 / SEQ ID NO:40、SEQ ID NO:136 / SEQ ID NO:41、SEQ ID NO:137 / SEQ ID NO:42、SEQ ID NO:138 / SEQ ID NO:43、SEQ ID NO:139 / SEQ ID NO:44、SEQ ID NO:140 / SEQ ID NO:45、SEQ ID NO:141 / SEQ ID NO:46、SEQ ID NO:142 / SEQ ID NO:47、SEQ ID NO:143 / SEQ ID NO:48、SEQ ID NO:144 / SEQ ID NO:49、SEQ ID NO:145 / SEQ ID NO:50、SEQ ID NO:146 / SEQ ID NO:51、SEQ ID NO:147 / SEQ ID NO:52、SEQ ID NO:148 / SEQ ID NO:53、SEQ ID NO:149 / SEQ ID NO:54、SEQ ID NO:150 / SEQ ID NO:55、SEQ ID NO:151 / SEQ ID NO:56、SEQ ID NO:152 / SEQ ID NO:57、SEQ ID NO:153 / SEQ ID NO:58、SEQ ID NO:154 / SEQ ID NO:59、SEQ ID NO:155 / SEQ ID NO:60、SEQ ID NO:156 / SEQ ID NO:61、SEQ ID NO:157 / SEQ ID NO:62、SEQ ID NO:158 / SEQ ID NO:63、SEQ ID NO:159 / SEQ ID NO:64、SEQ ID NO:160 / SEQ ID NO:65、SEQ ID NO:161 / SEQ ID NO:66、SEQ ID NO:162 / SEQ ID NO:67、SEQ ID NO:163 / SEQ ID NO:68、SEQ ID NO:164 / SEQ ID NO:69、SEQ ID NO:165 / SEQ ID NO:70、SEQ ID NO:166 / SEQ ID NO:71、SEQ ID NO:167 / SEQ ID NO:72、SEQ ID NO:168 / SEQ IDNO:73, SEQ ID NO:169 / SEQ ID NO:74, SEQ ID NO:170 / SEQ ID NO:75, SEQ ID NO:171 / SEQ ID NO:76, SEQ ID NO:172 / SEQ ID NO:77, SEQ ID NO:173 / SEQ ID NO:78, SEQ ID NO:174 / SEQ ID NO:79, SEQ ID NO:175 / SEQ ID NO:80、SEQ ID NO:176 / SEQ ID NO:81、SEQ ID NO:177 / SEQ ID NO:82、SEQ ID NO:178 / SEQ ID NO:83、SEQ ID NO:179 / SEQ ID NO:84、SEQ ID NO:180 / SEQ ID NO:85、SEQ ID NO:181 / SEQ ID NO:86、SEQ ID NO:182 / SEQ ID NO:87, SEQ ID NO:183 / SEQ ID NO:88, SEQ ID NO:184 / SEQ ID NO:89, SEQ At least 15, 16, 17, 18, or 19 consecutive nucleotides in the nucleotide sequences shown in SEQ ID NO:185 / SEQ ID NO:90, SEQ ID NO:186 / SEQ ID NO:91, SEQ ID NO:187 / SEQ ID NO:92, SEQ ID NO:188 / SEQ ID NO:93, SEQ ID NO:189 / SEQ ID NO:94, or SEQ ID NO:190 / SEQ ID NO:95, 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 or U, for example U. In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the antisense strand and the sense strand each comprise at least 15, 16, 17, 18, or 19 consecutive nucleotides from the nucleotide sequence shown in SEQ ID NO:51 / SEQ ID NO:146, optionally the antisense strand further having a 3' overhang of 1 or 2 nucleotides, optionally the first nucleotide at the 5' end of the antisense strand being A or U, for example U. In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand each comprise SEQ ID NO:90 / SEQ ID NO:146.The nucleotide sequence shown in NO:185 contains at least 15, 16, 17, 18, or 19 consecutive nucleotides, and optionally the antisense strand also has a 3' overhang of 1 or 2 nucleotides, and optionally the first nucleotide at the 5' end of the antisense strand is A or U, for example, U.

[0133] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the antisense strand and the sense strand respectively comprise SEQ ID NO:96 / SEQ ID NO:1, SEQ ID NO:97 / SEQ ID NO:2, SEQ ID NO:98 / SEQ ID NO:3, SEQ ID NO:99 / SEQ ID NO:4, SEQ ID NO:100 / SEQ ID NO:5, SEQ ID NO:101 / SEQ ID NO:6, SEQ ID NO:102 / SEQ ID NO:7, SEQ ID NO:103 / SEQ ID NO:8, SEQ ID NO:104 / SEQ ID NO:9, SEQ ID NO:105 / SEQ ID NO:10, SEQ ID NO:106 / SEQ ID NO:11, SEQ ID NO:107 / SEQ ID NO:12, SEQ ID NO:108 / SEQ ID NO:13, SEQ ID NO:109 / SEQ ID NO:14, SEQ ID NO:110 / SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:10 ... NO:111 / SEQ ID NO:16, SEQ ID NO:112 / SEQ ID NO:17, SEQ ID NO:113 / SEQ ID NO:18, SEQ ID NO:114 / SEQ ID NO:19, SEQ ID NO:115 / SEQ ID NO:20, SEQ ID NO:116 / SEQ ID NO:21, SEQ ID NO:117 / SEQ ID NO:22, SEQ ID NO:118 / SEQ ID NO:23, SEQ ID NO:119 / SEQ ID NO:24, SEQ ID NO:120 / SEQ ID NO:25, SEQ ID NO:121 / SEQ ID NO:26, SEQ ID NO:122 / SEQ ID NO:27, SEQ ID NO:123 / SEQ ID NO:28, SEQ ID NO:124 / SEQ ID NO:29, SEQ ID NO:125 / SEQ ID NO:30, SEQ ID NO:126 / SEQ ID NO:31, SEQ ID NO:127 / SEQ ID NO:32, SEQ ID NO:128 / SEQ ID NO:33, SEQ ID NO:129 / SEQ ID NO:34, SEQ ID NO:130 / SEQ ID NO:35, SEQ ID NO:131 / SEQ IDNO:36、SEQ ID NO:132 / SEQ ID NO:37、SEQ ID NO:133 / SEQ ID NO:38、SEQ ID NO:134 / SEQ ID NO:39、SEQ ID NO:135 / SEQ ID NO:40、SEQ ID NO:136 / SEQ ID NO:41、SEQ ID NO:137 / SEQ ID NO:42、SEQ ID NO:138 / SEQ ID NO:43、SEQ ID NO:139 / SEQ ID NO:44、SEQ ID NO:140 / SEQ ID NO:45、SEQ ID NO:141 / SEQ ID NO:46、SEQ ID NO:142 / SEQ ID NO:47、SEQ ID NO:143 / SEQ ID NO:48、SEQ ID NO:144 / SEQ ID NO:49、SEQ ID NO:145 / SEQ ID NO:50、SEQ ID NO:146 / SEQ ID NO:51、SEQ ID NO:147 / SEQ ID NO:52、SEQ ID NO:148 / SEQ ID NO:53、SEQ ID NO:149 / SEQ ID NO:54、SEQ ID NO:150 / SEQ ID NO:55、SEQ ID NO:151 / SEQ ID NO:56、SEQ ID NO:152 / SEQ ID NO:57、SEQ ID NO:153 / SEQ ID NO:58、SEQ ID NO:154 / SEQ ID NO:59、SEQ ID NO:155 / SEQ ID NO:60、SEQ ID NO:156 / SEQ ID NO:61、SEQ ID NO:157 / SEQ ID NO:62、SEQ ID NO:158 / SEQ ID NO:63、SEQ ID NO:159 / SEQ ID NO:64、SEQ ID NO:160 / SEQ ID NO:65、SEQ ID NO:161 / SEQ ID NO:66、SEQ ID NO:162 / SEQ ID NO:67、SEQ ID NO:163 / SEQ ID NO:68、SEQ ID NO:164 / SEQ ID NO:69、SEQ ID NO:165 / SEQ ID NO:70、SEQ ID NO:166 / SEQ ID NO:71、SEQ ID NO:167 / SEQ ID NO:72、SEQ ID NO:168 / SEQ IDNO:73, SEQ ID NO:169 / SEQ ID NO:74, SEQ ID NO:170 / SEQ ID NO:75, SEQ ID NO:171 / SEQ ID NO:76, SEQ ID NO:172 / SEQ ID NO:77, SEQ ID NO:173 / SEQ ID NO:78, SEQ ID NO:174 / SEQ ID NO:79, SEQ ID NO:175 / SEQ ID NO:80、SEQ ID NO:176 / SEQ ID NO:81、SEQ ID NO:177 / SEQ ID NO:82、SEQ ID NO:178 / SEQ ID NO:83、SEQ ID NO:179 / SEQ ID NO:84、SEQ ID NO:180 / SEQ ID NO:85、SEQ ID NO:181 / SEQ ID NO:86、SEQ ID NO:182 / SEQ ID NO:87, SEQ ID NO:183 / SEQ ID NO:88, SEQ ID NO:184 / SEQ ID NO:89, SEQ The nucleotide sequences shown in SEQ ID NO:185 / SEQ ID NO:90, SEQ ID NO:186 / SEQ ID NO:91, SEQ ID NO:187 / SEQ ID NO:92, SEQ ID NO:188 / SEQ ID NO:93, SEQ ID NO:189 / SEQ ID NO:94, or SEQ ID NO:190 / SEQ ID NO:95, or are composed of the nucleotide sequences shown therein. In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand each comprise the nucleotide sequence shown in SEQ ID NO:51 / SEQ ID NO:146, or are composed of the nucleotide sequences shown therein. In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand each comprise the nucleotide sequence shown in SEQ ID NO:90 / SEQ ID NO:185, or are composed of the nucleotide sequences shown therein.

[0134] For the purpose of inhibiting target mRNA expression, as those skilled in the art will 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 this disclosure 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 respect to the antisense strand, for example, at least 70%, at least 80%, at least 90%, or 100% complementary; having one or more additional nucleotides forming a protrusion or loop relative to the consecutive nucleotides of the antisense strand in the duplex region; and having one or more nucleotide gaps or vacancies relative to the consecutive nucleotides of the antisense strand in the duplex region. Similarly, for the purpose of inhibiting target mRNA expression, as those skilled in the art will understand, the antisense strand, serving as a guide RNAi for the specific binding of the target mRNA, may also contain sequences that are not 100% complementary to a consecutive nucleotide region of the target mRNA; 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 purposes of this disclosure, in some aspects, when considering the sequence motif of the antisense strand to the consecutive nucleotide complementarity of the target gene sequence, the presence of insertions and deletions is preferably not permitted. With regard to the sense and antisense strands of this disclosure, 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 at at least 18 or 19 consecutive nucleotides, for example, perfectly complementary. In a preferred embodiment, the antisense strand and the sense strand are complementary at 19 nucleotides (e.g., 19 consecutive complementary nucleotides, such as the first to 19 consecutive nucleotides from the 5' end of the antisense strand), for example, completely complementary.

[0135] 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.

[0136] In some embodiments, at least one nucleotide of the dsRNA activator of this disclosure is a modified nucleotide. In some embodiments, in the dsRNA activator of this disclosure, 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.

[0137] In some embodiments, the dsRNA activator of this disclosure comprises at least one modified nucleotide. Those skilled in the art will recognize that the dsRNA molecule according to this disclosure may be unmodified (i.e., comprising naturally occurring RNA nucleosides) but may also be (and preferably) modified, provided that it retains the desired functional activity (i.e., the ability to form the desired double-stranded structure and to allow or mediate specific degradation of the target RNA via a RISC pathway). Such RNA modification may 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 / or 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 to increase the physical stability of the double-stranded structure formed between antisense and sense nucleic acids).

[0138] 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.

[0139] In some embodiments, in the dsRNA activator of this disclosure, 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, all or substantially all nucleotides of the dsRNA activator are modified nucleotides. As described herein, a dsRNA activator in which substantially all nucleotides are modified nucleotides refers to a dsRNA activator having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides that are 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 two or fewer (i.e., 0, 1, or 2) nucleotides are ribonucleotides. As used herein, an antisense strand in which substantially all nucleotides are modified nucleotides refers to an antisense strand in which two or fewer (i.e., 0, 1, or 2) nucleotides are ribonucleotides.

[0141] 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.

[0142] In some embodiments, nucleotide modifications suitable for the dsRNA activator of this disclosure encompass modifications to nucleoside bases, ribose moieties, and / or the phosphate backbone. Exemplary modifications can be found in PCT Publication WO 200370918, some of which are known in the art and are incorporated herein by reference in their entirety.

[0143] 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. All of these modified nucleoside bases are within the scope of consideration in this disclosure.

[0144] 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.

[0145] In some embodiments, the dsRNA activator of this disclosure 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 this disclosure comprises at least one 2'-modified nucleotide. In some embodiments, the 2'-modification is selected from 2'-deoxy, 2'-fluorinated, 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 this disclosure comprises at least one 2'-modified nucleoside selected from the group consisting of 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.

[0146] In some embodiments, the dsRNA activator according to this disclosure may optionally also include 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'-vinylphosphonate (5'-VP) such as 5'-(E)-vinylphosphonate (5'-(E)-VP), 5'-methylphosphonate (5'-MP), (S)-5'-C-methyl analogues, and 5'-thiophosphate (5'-PS).

[0147] 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.

[0148] In some embodiments, at least one of the modified nucleotides in the dsRNA activator is selected from the group consisting of: nonlocked nucleotides (UNA), locked nucleotides (LNA), HNA, threonucleotides (TNA), CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, and ethylene glycol; and combinations thereof. In some embodiments, at least one of the modified nucleotides in the dsRNA activator is selected from the group consisting of: deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-hexadecyl modified nucleotides, nucleotides comprising 2'-phosphate groups, and nucleotides comprising thiophosphate groups; and combinations thereof.

[0149] In some embodiments, the antisense strand of the dsRNA activator of this disclosure contains 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 from the 5' end of the antisense strand of the dsRNA activator of this disclosure are 2'-methoxy modified nucleotides. In some embodiments, the sense strand of the dsRNA activator of this disclosure contains 2'-methoxy (2'-O-methyl) modified nucleotides, for example, all nucleotides are 2'-methoxy modified nucleotides or 1-19 nucleotides are 2'-methoxy modified nucleotides, for example, 15 or 16 nucleotides are 2'-methoxy modified nucleotides. In some embodiments, the nucleotides at positions 1-6 and 10-19 of the sense strand of the dsRNA activator of this disclosure are 2'-methoxy modified nucleotides. In some embodiments, the nucleotides at positions 1-5 and 10-19 of the sense strand from the 5' end of the dsRNA activator of this disclosure are 2'-methoxy modified nucleotides.

[0150] In some embodiments, the antisense strand of the dsRNA activator disclosed herein has nucleotides 1, 3-5, 7-13, 15, and 17-21 from the 5' end that are 2'-methoxy modified nucleotides, and the sense strand has nucleotides 1-6 and 10-19 from the 5' end that are 2'-methoxy modified nucleotides.

[0151] In some embodiments, the antisense strand of the dsRNA activator disclosed herein has nucleotides 1, 3-5, 7-13, 15, and 17-21 from the 5' end that are 2'-methoxy modified nucleotides, and the sense strand has nucleotides 1-5 and 10-19 from the 5' end that are 2'-methoxy modified nucleotides.

[0152] In some embodiments, the antisense strand of the dsRNA activator of this disclosure 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 this disclosure are 2'-fluorinated nucleotides. In some embodiments, the sense strand of the dsRNA activator of this disclosure 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 this disclosure are 2'-fluorinated nucleotides.

[0153] In some embodiments, in the dsRNA activator of this disclosure, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand, counting from the 5' end, are 2'-fluorinated nucleotides, and the nucleotides at positions 7-9 of the sense strand, counting from the 5' end, are 2'-fluorinated nucleotides.

[0154] In some embodiments, in the dsRNA activator of this disclosure, the antisense strand has nucleotides at positions 2, 6, 14, and 16 from the 5' end that are 2'-fluorinated nucleotides and the remaining nucleotides are 2'-methoxyinated nucleotides; and the sense strand has nucleotides at positions 7-9 from the 5' end that are 2'-fluorinated nucleotides and the remaining nucleotides are 2'-methoxyinated nucleotides, or nucleotides at positions 1-5 and 10-19 from the 5' end that are 2'-methoxyinated nucleotides.

[0155] 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.

[0156] 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.

[0157] 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 the first three nucleotides counting 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 the first three nucleotides counting from the 5' end and between the first three nucleotides counting from the 3' end of the antisense strand. In some embodiments, the dsRNA activator contains two phosphate-thioester nucleoside bonds in the sense strand and four phosphate-thioester nucleoside bonds in the antisense strand. In some embodiments, the dsRNA activator contains two phosphate-thioester nucleoside bonds in the sense strand, located between the first to third nucleotides from the 5' end of the sense strand; and contains four phosphate-thioester nucleoside bonds in the antisense strand, located between the first to third nucleotides from the 5' end of the antisense strand and between the first to third nucleotides from the 3' end of the antisense strand.

[0158] In some embodiments, the dsRNA activator's sense strand contains four phosphate-thioester nucleoside bonds. In some embodiments, these two phosphate-thioester nucleoside bonds are located between the first three nucleotides from the 5' end and between the first three nucleotides from the 3' 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 the first three nucleotides from the 5' end and between the first three nucleotides from the 3' end of the antisense strand. In some embodiments, the dsRNA activator contains four phosphate-thioester nucleoside bonds in both the sense and antisense strands. In some embodiments, the dsRNA activator contains four phosphate-thioester nucleoside bonds in the sense strand, with two of these bonds located between nucleotides at positions 1-3 from the 5' end and 1-3 from the 3' end of the sense strand; and contains four phosphate-thioester nucleoside bonds in the antisense strand, with these bonds located between nucleotides at positions 1-3 from the 5' end and 1-3 from the 3' end of the antisense strand.

[0159] In some embodiments, the 5' end nucleotide of the antisense strand has a 5'-vinyl phosphonate (5'-VP) modification, such as a 5'-(E)-VP modification.

[0160] Other modifications applicable to the dsRNA activator of this disclosure may also be found in, for example, the modifications listed in WO2023044094A1, WO2023245060A2, or WO2018 / 027106, which are incorporated herein by reference in their entirety.

[0161] In some implementations, the modified nucleotides in the sense and antisense strands of the dsRNA activator have the following modification patterns:

[0162] antisense chain:

[0163] NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0164] and / or

[0165] Chain of Justice:

[0166] NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm,

[0167] The symbols mentioned herein are as defined herein.

[0168] Nf = any 2'-fluorine modified nucleotide

[0169] Nfs = any 2'-fluorine modified nucleoside-3' thiophosphate;

[0170] Nm = any 2'-methoxynucleotide;

[0171] Nms = any 2'-methoxynucleoside-3'-thiophosphate;

[0172] 's' indicates that the two nucleotides are linked by a phosphate thioester bond.

[0173] In some implementations, the modified nucleotides in the sense and antisense strands of the dsRNA activator have the following modification patterns:

[0174] antisense chain:

[0175] VPNmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0176] and / or

[0177] Chain of Justice:

[0178] NmsNmsNmNmNmNNfNfNfNmNmNmNmNmNmNmNmNmsNmsNm,

[0179] in,

[0180] Nf = any 2'-fluorine modified nucleotide

[0181] Nfs = any 2'-fluorine modified nucleoside-3' thiophosphate;

[0182] Nm = any 2'-methoxynucleotide;

[0183] Nms = any 2'-methoxynucleoside-3'-thiophosphate;

[0184] N = any form of nucleotide

[0185] s indicates that the two nucleotides are linked by a phosphate thioester bond; VP indicates that the 5' end nucleotide of the antisense strand has a 5'-vinyl phosphonate (5'-VP) modification, such as 5'-(E)-VP modification;

[0186] For example, the 5' nucleotide VPNms of the antisense strand can be VPUms, with the following structure:

[0187] Preferably, the 6th nucleotide from the 5' end of the positive strand is conjugated with an aliphatic chain, such as a hydrocarbon group or hydrocarbon chain, which can serve as a ligand, for example, C. 16 -C 22 Hydrocarbon chains, such as those containing C 16 -C 22 Nucleoside monomers with saturated hydrocarbon chains, such as Uhd, Chd, Ahd, or Ghd.

[0188] In some embodiments, the antisense strand of the dsRNA activator comprises or is composed of any of the modified nucleotide sequences in the antisense strands of Table 2, and / or the sense strand comprises or is composed of any of the modified nucleotide sequences in the sense strands of Table 2.

[0189] In some implementations, the combination of modified antisense and sense strands in the dsRNA activator is shown in Table 2 as any combination of antisense and sense strands.

[0190] In some embodiments, the dsRNA activator is any of the dsRNA activators shown in Table 2.

[0191] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand respectively comprise SEQ ID NO:288 / SEQ ID NO:383, SEQ ID NO:289 / SEQ ID NO:384, SEQ ID NO:290 / SEQ ID NO:385, SEQ ID NO:291 / SEQ ID NO:386, SEQ ID NO:292 / SEQ ID NO:387, SEQ ID NO:293 / SEQ ID NO:388, SEQ ID NO:294 / SEQ ID NO:389, SEQ ID NO:295 / SEQ ID NO:390, SEQ ID NO:296 / SEQ ID NO:391, SEQ ID NO:297 / SEQ ID NO:392, SEQ ID NO:298 / SEQ ID NO:393, SEQ ID NO:299 / SEQ ID NO:394, SEQ ID NO:300 / SEQ ID NO:395, SEQ ID NO:301 ... NO:396, SEQ ID NO:302 / SEQ ID NO:397, SEQ ID NO:303 / SEQ ID NO:398, SEQ ID NO:304 / SEQ ID NO:399, SEQ ID NO:305 / SEQ ID NO:400, SEQ ID NO:306 / SEQ ID NO:401, SEQ ID NO:307 / SEQ ID NO:402, SEQ ID NO:308 / SEQ ID NO:403, SEQ ID NO:309 / SEQ ID NO:404, SEQ ID NO:310 / SEQ ID NO:405, SEQ ID NO:311 / SEQ ID NO:406, SEQ ID NO:312 / SEQ ID NO:407, SEQ ID NO:313 / SEQ ID NO:408, SEQ ID NO:314 / SEQ ID NO:409、SEQ ID NO:315 / SEQ ID NO:410、SEQ ID NO:316 / SEQ ID NO:411, SEQ ID NO:317 / SEQ ID NO:412, SEQ ID NO:318 / SEQ ID NO:413, SEQ ID NO:319 / SEQ ID NO:414, SEQ ID NO:320 / SEQ ID NO:415, SEQ ID NO:321 / SEQ IDNO:416、SEQ ID NO:322 / SEQ ID NO:417、SEQ ID NO:323 / SEQ ID NO:418、SEQ ID NO:324 / SEQ ID NO:419、SEQ ID NO:325 / SEQ ID NO:420、SEQ ID NO:326 / SEQ ID NO:421、SEQ ID NO:327 / SEQ ID NO:422、SEQ ID NO:328 / SEQ ID NO:423、SEQ ID NO:329 / SEQ ID NO:424、SEQ ID NO:330 / SEQ ID NO:425、SEQ ID NO:331 / SEQ ID NO:426、SEQ ID NO:332 / SEQ ID NO:427、SEQ ID NO:333 / SEQ ID NO:428、SEQ ID NO:334 / SEQ ID NO:429、SEQ ID NO:335 / SEQ ID NO:430、SEQ ID NO:336 / SEQ ID NO:431、SEQ ID NO:337 / SEQ ID NO:432、SEQ ID NO:338 / SEQ ID NO:433、SEQ ID NO:339 / SEQ ID NO:434、SEQ ID NO:340 / SEQ ID NO:435、SEQ ID NO:341 / SEQ ID NO:436、SEQ ID NO:342 / SEQ ID NO:437、SEQ ID NO:343 / SEQ ID NO:438、SEQ ID NO:344 / SEQ ID NO:439、SEQ ID NO:345 / SEQ ID NO:440、SEQ ID NO:346 / SEQ ID NO:441、SEQ ID NO:347 / SEQ ID NO:442、SEQ ID NO:348 / SEQ ID NO:443、SEQ ID NO:349 / SEQ ID NO:444、SEQ ID NO:350 / SEQ ID NO:445、SEQ ID NO:351 / SEQ ID NO:446、SEQ ID NO:352 / SEQ ID NO:447、SEQ ID NO:353 / SEQ ID NO:448、SEQ ID NO:354 / SEQ ID NO:449、SEQ ID NO:355 / SEQ ID NO:450、SEQ ID NO:356 / SEQ ID NO:451、SEQ IDNO:357 / SEQ ID NO:452, SEQ ID NO:358 / SEQ ID NO:453, SEQ ID NO:359 / SEQ ID NO:454, SEQ ID NO:360 / SEQ ID NO:455, SEQ ID NO:361 / SEQ ID NO:456, SEQ ID NO:362 / SEQ ID NO:457, SEQ ID NO:363 / SEQ ID NO:458, SEQ ID NO:364 / SEQ ID NO:459, SEQ ID NO:365 / SEQ ID NO:460, SEQ ID NO:366 / SEQ ID NO:461, SEQ ID NO:367 / SEQ ID NO:462, SEQ ID NO:368 / SEQ ID NO:463, SEQ ID NO:369 / SEQ ID NO:464, SEQ ID NO:370 / SEQ ID NO:465、SEQ ID NO:371 / SEQ ID NO:466、SEQ ID NO:372 / SEQ ID NO:467, SEQ ID NO:373 / SEQ ID NO:468, SEQ ID NO:374 / SEQ ID NO:469, SEQ ID NO:375 / SEQ ID NO:470, SEQ ID NO:376 / SEQ ID NO:471, SEQ ID NO:377 / SEQ ID NO:472, SEQ ID NO:378 / SEQ ID NO:473, SEQ ID NO:379 / SEQ ID NO:474, SEQ ID NO:380 / SEQ ID NO:475, SEQ ID NO:381 / SEQ ID NO:476 or SEQ ID NO:382 / SEQ ID The nucleotide sequence shown in NO:477 contains at least 15, 16, 17, 18, or 19 consecutive modified nucleotides, 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, e.g., Um, or Am, e.g., Um.

[0192] Optionally, it also has one or more of the following modifications: the first nucleotide at the 5' end of the antisense strand is modified with 5'-vinylphosphonate (5'-VP), for example, 5'-(E)-VP; for example, VPUm or VPAm; the 6th nucleotide from the 5' end of the sense strand is conjugated with an aliphatic chain, such as a hydrocarbon group or hydrocarbon chain, which can serve as a ligand, for example, C 16-C 22 Hydrocarbon chains, such as those containing C 16 -C 22 Nucleoside monomers with saturated hydrocarbon chains, such as Uhd, Chd, Ahd, or Ghd; and / or phosphate thioester bonds between the first to third nucleotides starting from the 3' end of the sense chain.

[0193] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand each comprise at least 15, 16, 17, 18, or 19 consecutive modified nucleotides of the nucleotide sequence shown in SEQ ID NO:338 / SEQ ID NO:433. Optionally, the antisense strand further comprises a 3' overhang of one or two nucleotides. Optionally, the first nucleotide at the 5' end of the antisense strand is a modified A or U, such as a modified U, for example, Um or Am, for example, Um. Optionally, it also comprises one or more of the following modifications: the first nucleotide at the 5' end of the antisense strand is modified with 5'-vinylphosphonate (5'-VP), for example, 5'-(E)-VP; for example, VPUm or VPAm; the sixth nucleotide from the 5' end of the sense strand is conjugated with an aliphatic chain, such as a hydrocarbon group or hydrocarbon chain, which can serve as a ligand, for example, C. 16 -C 22 Hydrocarbon chains, such as those containing C 16 -C 22 Nucleoside monomers with saturated hydrocarbon chains, such as Uhd, Chd, Ahd, or Ghd; and / or phosphate thioester bonds between the first to third nucleotides starting from the 3' end of the sense chain. In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand each comprise at least 15, 16, 17, 18, or 19 consecutive modified nucleotides of the nucleotide sequence shown in SEQ ID NO:377 / SEQ ID NO:472, optionally the antisense strand further 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, optionally further having one or more of the following modifications: the first nucleotide at the 5' end of the antisense strand being modified with 5'-vinylphosphonate (5'-VP), such as 5'-(E)-VP modification; for example, VPUm or VPAm; the 6th nucleotide from the 5' end of the sense strand being conjugated with an aliphatic chain, such as a hydrocarbon group or hydrocarbon chain, which can serve as a ligand, such as C. 16 -C 22 Hydrocarbon chains, such as those containing C 16 -C 22Nucleoside monomers with saturated hydrocarbon chains, such as Uhd, Chd, Ahd, or Ghd; and / or phosphate thioester bonds between the first to third nucleotides starting from the 3' end of the sense chain.

[0194] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand respectively comprise SEQ ID NO:288 / SEQ ID NO:383, SEQ ID NO:289 / SEQ ID NO:384, SEQ ID NO:290 / SEQ ID NO:385, SEQ ID NO:291 / SEQ ID NO:386, SEQ ID NO:292 / SEQ ID NO:387, SEQ ID NO:293 / SEQ ID NO:388, SEQ ID NO:294 / SEQ ID NO:389, SEQ ID NO:295 / SEQ ID NO:390, SEQ ID NO:296 / SEQ ID NO:391, SEQ ID NO:297 / SEQ ID NO:392, SEQ ID NO:298 / SEQ ID NO:393, SEQ ID NO:299 / SEQ ID NO:394, SEQ ID NO:300 / SEQ ID NO:395, SEQ ID NO:301 / SEQ ID NO:392, SEQ ID NO:298 / SEQ ID NO:393, SEQ ID NO:299 / SEQ ID NO:394, SEQ ID NO:300 / SEQ ID NO:395, SEQ ID NO:301 / SEQ ID NO:392, SEQ ID NO:298 / SEQ ID NO:393, SEQ ID NO:299 / SEQ ID NO:394, SEQ ID NO:300 / SEQ ID NO:395, SEQ ID NO:301 / SEQ ID NO:393 ... NO:396, SEQ ID NO:302 / SEQ ID NO:397, SEQ ID NO:303 / SEQ ID NO:398, SEQ ID NO:304 / SEQ ID NO:399, SEQ ID NO:305 / SEQ ID NO:400, SEQ ID NO:306 / SEQ ID NO:401, SEQ ID NO:307 / SEQ ID NO:402, SEQ ID NO:308 / SEQ ID NO:403, SEQ ID NO:309 / SEQ ID NO:404, SEQ ID NO:310 / SEQ ID NO:405, SEQ ID NO:311 / SEQ ID NO:406, SEQ ID NO:312 / SEQ ID NO:407, SEQ ID NO:313 / SEQ ID NO:408, SEQ ID NO:314 / SEQ ID NO:409、SEQ ID NO:315 / SEQ ID NO:410、SEQ ID NO:316 / SEQ ID NO:411, SEQ ID NO:317 / SEQ ID NO:412, SEQ ID NO:318 / SEQ ID NO:413, SEQ ID NO:319 / SEQ ID NO:414, SEQ ID NO:320 / SEQ ID NO:415, SEQ ID NO:321 / SEQ IDNO:416、SEQ ID NO:322 / SEQ ID NO:417、SEQ ID NO:323 / SEQ ID NO:418、SEQ ID NO:324 / SEQ ID NO:419、SEQ ID NO:325 / SEQ ID NO:420、SEQ ID NO:326 / SEQ ID NO:421、SEQ ID NO:327 / SEQ ID NO:422、SEQ ID NO:328 / SEQ ID NO:423、SEQ ID NO:329 / SEQ ID NO:424、SEQ ID NO:330 / SEQ ID NO:425、SEQ ID NO:331 / SEQ ID NO:426、SEQ ID NO:332 / SEQ ID NO:427、SEQ ID NO:333 / SEQ ID NO:428、SEQ ID NO:334 / SEQ ID NO:429、SEQ ID NO:335 / SEQ ID NO:430、SEQ ID NO:336 / SEQ ID NO:431、SEQ ID NO:337 / SEQ ID NO:432、SEQ ID NO:338 / SEQ ID NO:433、SEQ ID NO:339 / SEQ ID NO:434、SEQ ID NO:340 / SEQ ID NO:435、SEQ ID NO:341 / SEQ ID NO:436、SEQ ID NO:342 / SEQ ID NO:437、SEQ ID NO:343 / SEQ ID NO:438、SEQ ID NO:344 / SEQ ID NO:439、SEQ ID NO:345 / SEQ ID NO:440、SEQ ID NO:346 / SEQ ID NO:441、SEQ ID NO:347 / SEQ ID NO:442、SEQ ID NO:348 / SEQ ID NO:443、SEQ ID NO:349 / SEQ ID NO:444、SEQ ID NO:350 / SEQ ID NO:445、SEQ ID NO:351 / SEQ ID NO:446、SEQ ID NO:352 / SEQ ID NO:447、SEQ ID NO:353 / SEQ ID NO:448、SEQ ID NO:354 / SEQ ID NO:449、SEQ ID NO:355 / SEQ ID NO:450、SEQ ID NO:356 / SEQ ID NO:451、SEQ IDNO:357 / SEQ ID NO:452, SEQ ID NO:358 / SEQ ID NO:453, SEQ ID NO:359 / SEQ ID NO:454, SEQ ID NO:360 / SEQ ID NO:455, SEQ ID NO:361 / SEQ ID NO:456, SEQ ID NO:362 / SEQ ID NO:457, SEQ ID NO:363 / SEQ ID NO:458, SEQ ID NO:364 / SEQ ID NO:459, SEQ ID NO:365 / SEQ ID NO:460, SEQ ID NO:366 / SEQ ID NO:461, SEQ ID NO:367 / SEQ ID NO:462, SEQ ID NO:368 / SEQ ID NO:463, SEQ ID NO:369 / SEQ ID NO:464, SEQ ID NO:370 / SEQ ID NO:465、SEQ ID NO:371 / SEQ ID NO:466、SEQ ID The modified nucleotide sequences shown in SEQ ID NO:372 / SEQ ID NO:467, SEQ ID NO:373 / SEQ ID NO:468, SEQ ID NO:374 / SEQ ID NO:469, SEQ ID NO:375 / SEQ ID NO:470, SEQ ID NO:376 / SEQ ID NO:471, SEQ ID NO:377 / SEQ ID NO:472, SEQ ID NO:378 / SEQ ID NO:473, SEQ ID NO:379 / SEQ ID NO:474, SEQ ID NO:380 / SEQ ID NO:475, SEQ ID NO:381 / SEQ ID NO:476 or SEQ ID NO:382 / SEQ ID NO:477, or each of the modified nucleotide sequences shown.

[0195] Optionally, it also has one or more of the following modifications: the first nucleotide at the 5' end of the antisense strand is modified with 5'-vinylphosphonate (5'-VP), for example, 5'-(E)-VP; for example, VPUm or VPAm; the 6th nucleotide from the 5' end of the sense strand is conjugated with an aliphatic chain, such as a hydrocarbon group or hydrocarbon chain, which can serve as a ligand, for example, C 16 -C 22 Hydrocarbon chains, such as those containing C 16 -C 22Nucleoside monomers with saturated hydrocarbon chains, such as Uhd, Chd, Ahd, or Ghd; and / or phosphate thioester bonds between the first to third nucleotides starting from the 3' end of the sense chain.

[0196] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand each comprise the nucleotide sequences shown in SEQ ID NO:338 / SEQ ID NO:433 or are composed of the modified nucleotide sequences shown; optionally, it also has one or more of the following modifications: the first nucleotide at the 5' end of the antisense strand is modified with 5'-vinylphosphonate (5'-VP), for example, 5'-(E)-VP; for example, VPUm or VPAm; the 6th nucleotide from the 5' end of the sense strand is conjugated with an aliphatic chain, such as a hydrocarbon group or hydrocarbon chain, which can serve as a ligand, for example, C 16 -C 22 Hydrocarbon chains, such as those containing C 16 -C 22 The saturated hydrocarbon chain nucleoside monomer, such as Uhd, Chd, Ahd, or Ghd; and / or a phosphate thioester bond between the first to third nucleotides from the 3' end of the sense strand. In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand each comprise the nucleotide sequences shown in SEQ ID NO:377 / SEQ ID NO:472 or each consists of the modified nucleotide sequences shown; optionally, it also has one or more of the following modifications: the first nucleotide at the 5' end of the antisense strand is modified with 5'-vinylphosphonate (5'-VP), such as 5'-(E)-VP; for example, VPUm or VPAm; the sixth nucleotide from the 5' end of the sense strand is conjugated with an aliphatic chain, such as a hydrocarbon group or hydrocarbon chain, which can serve as a ligand, such as C 16 -C 22 Hydrocarbon chains, such as those containing C 16 -C 22 Nucleoside monomers with saturated hydrocarbon chains, such as Uhd, Chd, Ahd, or Ghd; and / or phosphate thioester bonds between the first to third nucleotides starting from the 3' end of the sense chain.

[0197] As used herein (including 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, these monomers, when present in oligonucleotides, are interconnected by 5'-3'-phosphodiester bonds, as will be readily understood by those skilled in the art:

[0198] T = 5'-methyluridine-3'-phosphate

[0199] Nf = any 2'-fluorine modified nucleotide

[0200] Nfs = any 2'-fluorine-modified nucleoside-3'-thiophosphate ester

[0201] Af = 2'-Fluoroadenosine-3'-phosphate

[0202] Afs = 2'-Fluoroadenosine-3'-Thiophosphate

[0203] Cf = 2'-Fluorocytidine-3'-phosphate

[0204] Cfs = 2'-Fluorocytidine-3'-Thiophosphate

[0205] Gf = 2'-Fluoroguanosine-3'-phosphate

[0206] Gfs = 2'-Fluoroguanosine-3'-Thiophosphate

[0207] Tf = 2'-Fluoro-5'-methyluridine-3'-phosphate

[0208] Tfs = 2'-Fluoro-5'-methyluridine-3'-thiophosphate

[0209] Uf = 2'-fluorouridine-3'-phosphate

[0210] Ufs = 2'-fluorouridine-3'-thiophosphate

[0211] Nm = any 2'-methoxynucleotide

[0212] Nms = any 2'-methoxynucleoside-3'-thiophosphate

[0213] Am = 2'-methoxyadenosine-3'-phosphate

[0214] Ams = 2'-methoxyadenosine-3'-thiophosphate

[0215] Tm = 2'-methoxythymidine-3'-phosphate

[0216] Tms = 2'-methoxythymidine-3'-thiophosphate

[0217] Um = 2'-methoxyuridine-3'-phosphate

[0218] Ums = 2'-methoxyuridine-3'-thiophosphate

[0219] Gm = 2'-methoxyguanosine-3'-phosphate

[0220] Gms = 2'-methoxyguanosine-3'-thiophosphate

[0221] Cm = 2'-methoxycytidine-3'-phosphate

[0222] Cms = 2'-methoxycytidine-3'-thiophosphate

[0223] Nhd = any nucleotide modified with 2′-O-hexadecyl;

[0224] VPN = any nucleotide modified with 5'-vinylphosphonate (5'-VP, 5'-vinyl phosphate), preferably with 5'-(E)-VP modification, such as VPUms, which is a 2'-methoxyuridine-3'-thiophosphate with 5'-(E)-VP modification, and its structural formula is as follows:

[0225] Uhd, Chd, Ahd, and Ghd represent 2′-O-hexadecyl-modified monomers A, C, G, and U, respectively, with the following structures:

[0226] In some embodiments, the dsRNA activator of this disclosure further includes a ligand. When this disclosure refers to "dsRNA" or "dsRNA activator," it also encompasses dsRNA conjugated to a ligand.

[0227] As described herein, a "ligand" refers to a chemical moiety conjugated to 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.

[0228] 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, descriptions are provided in Vajinder Kumar, Targeted delivery of oligonucleotides using multivalent protein–carbohydrate interactions, Cite this: Chem. Soc. Rev., 2023, 52, 1273; Rosemary Kanasty, Delivery materials for siRNAtherapeutics, NATURE MATERIALS, VOL 12, NOVEMBER 2013; Wanyi Tai, Current Aspects of siRNABioconjugate for In Vitro and In Vivo Delivery, Molecules 2019, 24, 2211; doi:10.3390 / molecules24122211; and WO 93 / 07883 and WO 2013 / 033230, which are incorporated herein by reference.

[0229] In some embodiments, the ligand used for the RNAi active agent of this disclosure 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.

[0230] In some embodiments, the ligand is a sugar, 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 ligand is a monosaccharide.

[0231] In some embodiments, the ligand 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, the GalNAc ligand is used as a ligand to target a dsRNA activator to specific cells. In some embodiments, the GalNAc ligand targets dsRNA to hepatocytes, for example by acting as a ligand for the desialylate glycoprotein receptor 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.

[0232] Commonly used ligands that conjugate with dsRNA can also be found in, for example, those disclosed in WO2023044094A1, WO2023245060A2, or WO2018 / 027106, or WO2012083185A2, WO2015021092, WO2018044350A1, etc., the full contents of which are hereby incorporated herein by reference.

[0233] In some embodiments, the dsRNA activator of this disclosure includes a GalNAc or GalNAc derivative linked to the dsRNA activator. In some embodiments, the dsRNA activator of this disclosure includes 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.

[0234] 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 nucleotide of the dsRNA is conjugated to the 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. Preferably, the ligand is conjugated to the 3' end of the sense strand of the dsRNA activator.

[0235] In some embodiments, the ligand is L96, see, for example, WO2009073809 and WO2009082607, which are incorporated herein by reference in their entirety.

[0236] In some implementations, the ligand is

[0237] In some embodiments, the dsRNA activator comprises the ligand-conjugated nucleotide, as illustrated in the following schematic diagram.

[0238] And X is O or S, and the double-stranded structure is a nucleotide chain of dsRNA, for example, containing a double-stranded region formed by a sense strand and an antisense strand.

[0239] In some embodiments, the ligand is a ligand capable of targeted delivery of dsRNA to target tissues or cells. In some embodiments, the dsRNA activator containing said ligand is suitable for delivery to target tissues or cells.

[0240] In some embodiments, the dsRNA disclosed herein can target any tissue or organ or cell, such as the nervous system (e.g., the central nervous system, such as the brain, such as the cortex or cerebellum, such as the cerebral cortex, hypothalamus, hippocampus, and other brain regions; such as neurons), spinal tissue (e.g., cervical, lumbar, and thoracic vertebrae), muscle tissue such as skeletal muscle or cardiac muscle, eye tissue, or adipose tissue. In some embodiments, the tissue is selected from tissues of the nervous system (e.g., tissues of the central nervous system such as brain tissue, such as the cortex or cerebellum, such as the cerebral cortex, hypothalamus, hippocampus, and other brain regions; or such as neurons), spinal tissue (e.g., cervical, lumbar, and thoracic vertebrae), muscle tissue such as skeletal muscle or cardiac muscle, eye tissue, or adipose tissue. In some embodiments, the cells are cells derived from the target tissue. In some embodiments, the target cells are selected from cells of the brain or spinal tissue (e.g., cortex, cerebellum, cerebral cortex, hypothalamus, hippocampus, cervical, lumbar, and thoracic vertebrae), nervous system cells (e.g., neurons), muscle cells such as skeletal muscle cells or cardiac muscle cells, eye cells, or adipose cells.

[0241] In some embodiments, the ligand is a lipid or lipid-based molecule. In some embodiments, the ligand is a lipophilic moiety. In some embodiments, the lipid is a fatty chain, such as a C16-C22 lipid, or a C16-C22 hydrocarbon chain. In some embodiments, the lipid is capable of targeted delivery of dsRNA to the target tissues or target cells described herein. In some embodiments, the ligand is, for example, a lipid capable of delivering dsRNA molecules to the target tissues or target cells described herein.

[0242] In some implementations, such lipids or lipid-based molecules bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands allow the conjugate to distribute to target tissues, such as non-renal target tissues 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).

[0243] In some embodiments, the ligand is a lipid nanoparticle, such as a 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.

[0244] In some implementations, antibodies can also be used as ligands for the delivery of dsRNA, such as antibody-oligonucleotide conjugates (AOCs).

[0245] In some implementations, polymer matrix copolymerization can also be used to deliver dsRNA, such as the LODER (Local Drug EluteR) delivery platform.

[0246] In some embodiments, the ligand is attached to the 5' and / or 3' terminal nucleotides of the sense strand and optionally the 5' and / or 3' terminal nucleotides of the antisense strand of the disclosed dsRNA, optionally via a phosphate thioester group or a phosphate group, for example via a nucleoside internucleotide bond. In some embodiments, the dsRNA activator is conjugated to the ligand via a phosphate ester group or a phosphate thioester group, for example, the phosphate thioester internucleotide bond is located at the 3' end of the sense strand or antisense strand; or the phosphate thioester internucleotide bond is located at the 5' end of the sense strand or antisense strand; or the phosphate thioester internucleotide bond is located at both the 5' and 3' ends of the sense strand, and / or the phosphate thioester internucleotide bond is located at both the 5' and 3' ends of the antisense strand.

[0247] In some cases, the ligand 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.

[0248] In some implementations, when a ligand is attached to the end of a dsRNA (such as siRNA) nucleotide chain, the ligand is typically attached to a phosphate group of the nucleotide; when a ligand is attached to an internal sequence of a dsRNA (such as siRNA) nucleotide, the ligand is typically attached to a sugar ring or base of the ribose.

[0249] The ligand can be directly linked to the nucleotide double strand of the dsRNA of this disclosure or linked via a linker portion (e.g., a adapter). In some embodiments of this disclosure, the dsRNA of this disclosure may optionally include an adapter located between the nucleotide strand of the dsRNA and the ligand. In some embodiments, the adapter is a biocleavable adapter. In some embodiments, the adapter need not be biocleavable. In some embodiments, the adapter may include a branching region. Hereinafter, the term "branching region" means a compound portion capable of covalently coupling two or more entities together. In some embodiments, adapters having branching regions can be used to conjugate multiple entities, such as N-acetylgalactosamine moieties, to the oligonucleotide of the dsRNA of this disclosure. Adapters having branching regions that can be used for this purpose are known in the art and include, but are not limited to, amino acids (including natural and non-natural amino acids), peptides and their derivatives, glycounits and their derivatives, aromatic-substituted compounds and their derivatives, substituted hydrocarbon groups and their derivatives, triazole-containing derivatives, etc. See, for example, CN104651408A, CN113286888A, WO2015 / 173208 and WO2023 / 076451.

[0250] Any ligand capable of delivering dsRNA molecules to target cells or tissues is suitable for the dsRNA activators disclosed herein, such as lipophilic / lipophilic moieties, like lipids such as fatty chains, lipophilic moieties disclosed in WO2019 / 217459A1, C22 hydrocarbon chains or ligands containing said C22 hydrocarbon chains as described in WO2023064530A1, Lipid PK / PD modulators as described in WO2024 / 148329A1, ligands disclosed in WO2024235124A1, high-affinity targeting ligands, liposomes, nucleic acid aptamers, or cholesterol. The entire contents of the aforementioned patents or patent applications are incorporated herein by reference.

[0251] In some embodiments, the dsRNA activator of this disclosure may comprise a lipophilic / lipophilic moiety, such as a lipid or fatty chain, conjugated to one or both chains of the dsRNA activator for delivery of dsRNA to the target tissues or cells described herein. In some embodiments, the lipophilic moiety is a C16-C22 fatty chain, such as a C16-C22 hydrocarbon chain, such as a saturated or unsaturated fatty chain. In some embodiments, the one or more fatty chains, such as C16 hydrocarbon chains (e.g., saturated or unsaturated), are conjugated to one or more internal positions of at least one chain of the dsRNA activator. In some specific embodiments, the lipophilic moiety, such as a fatty chain, may be conjugated to the 6th nucleotide of the dsRNA activator. In some embodiments, the 6th position of the positive strand of the dsRNA activator of this disclosure, counting from the 5' end, is conjugated with a lipophilic moiety, such as a fatty chain, such as a C16 hydrocarbon chain (e.g., saturated or unsaturated), such as a nucleoside monomer containing a C16 saturated alkyl group. In some embodiments, the sixth position of the dsRNA activator of this disclosure, counting from the 5' end of the positive strand, is a nucleotide in which the ribose is etherified by a 2′-hydroxyl group by a hexadecyl (C16) long-chain alkyl group, such as 2′-O-hexadecyluridine (Uhd), 2′-O-hexadecylcytidine (Chd), 2′-O-hexadecyladenosine (Ahd), or 2′-O-hexadecylguanosine (Ghd).

[0252] In some embodiments, the modified nucleotide in the sense and antisense strands of the dsRNA activator, wherein the ribose 2′-hydroxyl group at position 6 is etherified by a hexadecyl (C16) long-chain alkyl group, has the following modification pattern:

[0253] antisense chain:

[0254] VPNmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0255] and / or

[0256] Chain of Justice:

[0257] NmsNmsNmNmNmNhdNfNfNfNmNmNmNmNmNmNmNmNmsNmsNm,

[0258] in,

[0259] Nf = any 2'-fluorine modified nucleotide

[0260] Nfs = any 2'-fluorine modified nucleoside-3' thiophosphate;

[0261] Nm = any 2'-methoxynucleotide;

[0262] Nms = any 2'-methoxynucleoside-3'-thiophosphate;

[0263] Nhd = any nucleotide modified with 2′-O-hexadecyl;

[0264] s indicates that the two nucleotides are linked by a phosphate thioester bond; VP indicates that the 5' end nucleotide of the antisense strand has a 5'-vinyl phosphonate (5'-VP) modification, such as 5'-(E)-VP modification;

[0265] For example, the 5' nucleotide VPNms of the antisense strand can be VPUms, with the following structure:

[0266] For example, Nhd is selected from Uhd, Chd, Ahd, and Ghd, which represent 2′-O-hexadecyl-modified A, C, G, and U monomers, respectively, and their structures are as follows:

[0267] In some embodiments, the lipophilic moiety is aliphatic, cyclic (e.g., alicyclic), or polycyclic, such as polycyclic compounds, such as steroids (e.g., sterols), or straight-chain or branched aliphatic hydrocarbons. Exemplary lipophilic moieties are lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geraniol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytriphenylmethyl, or phenoxazine. Suitable lipophilic moieties also include those containing a saturated or unsaturated C4-C30 hydrocarbon chain (e.g., C4-C30 alkyl or alkenyl) and optional functional groups selected from the group consisting of: hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. These functional groups can be used to attach the lipophilic moieties to dsRNA activators. In some embodiments, the lipophilic moieties contain a saturated or unsaturated C6-C18 hydrocarbon chain (e.g., straight-chain C6-C18 alkyl or alkenyl). In one embodiment, the lipophilic moieties contain a saturated or unsaturated C16 hydrocarbon chain (e.g., straight-chain C16 alkyl or alkenyl). In some embodiments, the lipophilic moiety is a C6-C30 acid (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid, vitamin A, vitamin E, cholesterol, etc.) or a C6-C30 alcohol (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecaneol, dodecaneol, tridecaneol, tetradecaneol, pentadecaneol, hexadecaneol, heptadecanol, octadecaneol, oleyl alcohol, linolenic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid, retinol, vitamin E, cholesterol, etc.). The lipophilic moiety can be conjugated to the dsRNA activator via direct attachment to the ribose of the dsRNA activator. Alternatively, the lipophilic moiety may be conjugated to the dsRNA activator via a linker or vector. Exemplary lipophilic moieties can be found in the lipophilic moiety disclosed in WO2019 / 217459A1 or the C22 hydrocarbon chain or ligand containing the C22 hydrocarbon chain described in WO2023064530A1, both of which are incorporated herein by reference in their entirety.

[0268] In some embodiments, the lipophilic moiety is a C22 hydrocarbon chain. In some embodiments, the dsRNA activator of this disclosure may comprise a C22 hydrocarbon chain, as illustrated in, for example, WO2023064530A1, which is incorporated herein by reference in its entirety. In some embodiments, one or more C22 hydrocarbon chains (e.g., saturated or unsaturated) are conjugated to one or more internal positions of at least one chain of the dsRNA activator. In some embodiments, the C22 hydrocarbon chain is saturated or unsaturated. In some embodiments, the C22 hydrocarbon chain is linear or branched. In some embodiments, one or more C22 hydrocarbon chains are aliphatic, alicyclic, or polycyclic compounds. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, such as oxygen or nitrogen atoms. One or more C22 hydrocarbon chains can be linked to a dsRNA activator by any method known in the art, including through functional groups already present in the lipophilic moiety or introduced into the dsRNA activator, such as hydroxyl groups (e.g., -CO-CH2-OH). Functional groups already present in the C22 hydrocarbon chain or introduced into the dsRNA activator include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. The conjugation of the dsRNA activator and the C22 hydrocarbon chain can occur, for example, by forming an ether or carboxylic acid or carbamoyl ester bond between a hydroxyl group and an alkyl R-, alkylyl RCO-, or a substituted carbamoyl RNHCO-. The alkyl R can be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chain or branched; and saturated or unsaturated). The alkyl R can be butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, decadecyl, hexadecyl, heptadecanyl, or octadecyl, etc. In some embodiments, the C22 hydrocarbon chain is conjugated to a dsRNA activator via a linker, said linker including ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphate diester, sulfonamide bond, click reaction product (e.g., triazole from an azide-alkyne cycloaddition reaction), or carbamate.

[0269] In one embodiment, the one or more C22 hydrocarbon chains are C22 acids, for example, the C22 acids are selected from the following: docosanoic acid, 6-octyltetradecanoic acid, 10-hexylhexadecanoic acid, all-cis 7,10,13,16,19-docosapentaenoic acid, all-cis 4,7,10,13,16,19-docosahexaenoic acid, all-cis 13,16-docosadienoic acid, all-cis 7,10,13,16-docosatetraenoic acid, all-cis 4,7,10,13,16-docosapentaenoic acid, and cis-13-docosaenoic acid. In one embodiment, the one or more C22 hydrocarbon chains are C22 alcohols, for example, selected from the following: 1-docosahexaenoic acid, 6-octyltetradecane-1-ol, 10-hexylhexadecane-1-ol, cis-13-docosaen-1-ol, docosane-9-ol, docosane-2-ol, docosane-10-ol, docosane-11-ol, and cis-4,7,10,13,16,19-docosahexaenoic acid. In one embodiment, the one or more C22 hydrocarbon chains are not cis-4,7,10,13,16,19-docosahexaenoic acid. In one embodiment, the one or more C22 hydrocarbon chains are not cis-4,7,10,13,16,19-docosahexaenoic acid. In one embodiment, the one or more C22 hydrocarbon chains are neither cis-4,7,10,13,16,19-docosahexaenoic acid nor cis-4,7,10,13,16,19-docosahexaol. In one embodiment, the one or more C22 hydrocarbon chains are C22 amides, for example, selected from the following: (E)-docosa-4-enamide, (E)-docosa-5-enamide, (Z)-docosa-9-enamide, (E)-docosa-11-enamide, 12-docosa-enamide, (Z)-docosa-13-enamide, (Z)-N-hydroxy-13-docosa-enamide, (E)-docosa-14-enamide, 6-cis-docosa-enamide, 14-docosa-enamide, docosa-11-enamide, (4E,13E)-docosa-4,13-dieneamide, and (5E,13E)-docosa-5,13-dieneamide.

[0270] In some embodiments, more than one C22 hydrocarbon chain can be incorporated into the dsRNA activator, particularly when the C22 hydrocarbon chain has low lipophilicity or hydrophobicity. In one embodiment, two or more C22 hydrocarbon chains are incorporated into the same chain of the dsRNA activator. In one embodiment, each chain of the dsRNA activator contains one or more C22 hydrocarbon chains. In one embodiment, two or more C22 hydrocarbon chains are incorporated into the same position (i.e., the same nucleotide base, the same sugar moiety, or the same nucleotide internucleotide bond) of the dsRNA activator. This can be achieved, for example, by conjugating two or more saturated or unsaturated C22 hydrocarbon chains via a carrier, and / or by conjugating two or more C22 hydrocarbon chains via a branching linker, and / or by conjugating two or more C22 hydrocarbon chains via one or more linkers, wherein one or more linkers continuously connect the C22 hydrocarbon chains. One or more C22 hydrocarbon chains can be conjugated to the dsRNA activator by directly attaching to the ribose of the dsRNA activator. Alternatively, one or more C22 hydrocarbon chains may be conjugated to the dsRNA activator via a linker or carrier. In some embodiments, one or more C22 hydrocarbon chains may be conjugated to the dsRNA activator via one or more linkers. In one embodiment, the one or more C22 hydrocarbon chains are conjugated to the dsRNA activator via linkers comprising ethers, thioethers, ureas, carbonates, amines, amides, maleimide-thioethers, disulfides, phosphodiesters, sulfonamide bonds, click reaction products (e.g., triazoles from azide-alkyne cycloaddition reactions), or carbamates.

[0271] In some embodiments, lipids suitable for delivering the dsRNA of this disclosure to the target tissues or target cells described herein include, for example, Lipid PK / PD modulators, such as those disclosed in WO2024 / 148329A1, which is incorporated herein by reference in its entirety.

[0272] In some embodiments, the terminal nucleotide of one strand of the dsRNA activator disclosed herein is conjugated to a lipid such as a Lipid PK / PD modulator. In some embodiments, the lipid is conjugated to a 5' terminal nucleotide of one strand or a 3' terminal nucleotide of one strand, or to both a 5' terminal nucleotide and a 3' terminal nucleotide of one strand. In some embodiments, the lipid is internally conjugated to one or more nucleotides of one or both strands (e.g., at the 2' position).

[0273] In some embodiments, the lipid-containing dsRNA activator of this disclosure is a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0274] Where R Z1 and R Z3Each is an independent bond or end-capping residue; R Z2 The dsRNA contained in this disclosure; Y is a bond or linker that connects at least one L 1 Connect to L 2 (If it exists), or connected to R Z1 ;Y 1 It is a key or connector that will have at least one L 3 Connect to L 4 (If it exists), or connected to R Z3 L 2 and L 3 Each is an independent connector; q is 1, 2, or 3, subject to price allowance; t is 1, 2, or 3, subject to price allowance; and L 1 and L 4 Each of the lipids independently comprises about 10 to about 50 carbon atoms. In some embodiments, the definitions of the various groups in formula (I) are as defined in WO2024 / 148329A1.

[0275] In some embodiments, the lipids suitable for conjugation with the dsRNA of the present disclosure are selected from the Lipid PK / PD modulators shown in Table 2 of WO2024 / 148329A1, such as LP-4-a, LP-18-a, LP-128-a, LP-151-a, LP-183-a, LP-200-a, LP-208-a, LP-211-a, LP-232-a, LP-242-a, LP-243-a, LP-244-a, LP-245-a, LP-249-a, LP-274-a, LP-295-a, LP-310-a, LP-359-a, LP-361-a, LP-371-a, LP-374-a, LP-375-a, LP-377-a, LP-378-a, LP-379-a, LP-380-a, LP-403-a, LP-404-a, LP-412-a, LP-413-a, LP-416-a.LP-416-a, LP-424-a, LP-425-a, LP-426-a, LP-427-a.LP-428-a, LP-432-a, LP-433-a, LP-444-a, LP-445-a, LP-446-a, LP-447-a, LP-453-a, LP-455-a, LP-457-a, LP-458-a, LP-459-a, LP-460-a, LP-461-a, LP-468-a, LP-469-a, LP-470-a, LP-473-a, LP-474-a, CNR1 SM2-a or CNR1 SM2-a; or the formulas LP-4-b, LP-18-b, LP-128-b, LP-151-b, LP-183-b, LP-200-b, LP-208-b, LP-211-b, LP-232-b, LP-242-b, LP-243-b, LP-244-b, LP-245-b, LP-249-b, LP-274-b, LP-295-b, LP-310-b, LP-359-b, LP-361-b, LP-371-b, LP-374-b, LP-375-b, LP-377-b, LP-378-b, LP-379-b, LP-380-b, LP-403-b, LP-404-b, LP-412-b, LP-413-b, LP-416-a.LP-416-b, LP-424-b, LP-425-b, LP-426-b, LP-427-b shown in Table 3.The portions of LP-428-b, LP-432-b, LP-433-b, LP-444-b, LP-445-b, LP-446-b, LP-447-b, LP-453-b, LP-455-b, LP-457-b, LP-458-b, LP-459-b, LP-460-b, LP-461-b, LP-468-b, LP-469-b, LP-470-b, LP-473-b, LP-474-b, and CNR1 SM2-b or CNR1 SM2-1-b, excluding the connected R.

[0276] In some embodiments, the dsRNA activator comprising the Lipid PK / PD modulator is selected, for example, from the compounds shown in Table 3 of WO2024 / 148329A1 or their salts, wherein R represents the dsRNA of this disclosure.

[0277] In some embodiments, the dsRNA activator of this disclosure may further comprise a targeting ligand capable of targeted delivery to the target tissues or target cells described herein. In some embodiments, the targeting ligand encompasses targeting dsRNA to any tissue or organ or cell, such as the nervous system (e.g., the central nervous system, such as the brain, such as the cortex or cerebellum; such as neurons), spinal tissue (e.g., cervical, lumbar, and thoracic vertebrae), muscle tissue such as skeletal or cardiac muscle, ocular tissue, or adipose tissue. In some embodiments, the tissue is selected from tissues of the nervous system (e.g., tissues of the central nervous system, such as brain tissue, such as the cortex or cerebellum, such as the cerebral cortex, hypothalamus, hippocampus, and other brain regions; or such as neurons), spinal tissue (e.g., cervical, lumbar, and thoracic vertebrae), muscle tissue such as skeletal or cardiac muscle, ocular tissue, or adipose tissue. In some embodiments, the cells are cells derived from the target tissue. In some embodiments, the target cells are selected from cells of brain or spinal tissue (e.g., cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae), nervous system cells (e.g., neurons), muscle cells such as skeletal muscle cells or cardiomyocytes, eye cells, or fat cells.

[0278] In some embodiments, the dsRNA activator of this disclosure may be conjugated to a ligand capable of targeted delivery to the nervous system, such as the central nervous system (CNS). In one embodiment, the targeting ligand is selected from the group consisting of: Angiopep-2, lipoprotein receptor-associated protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter, LDL receptor ligand, trans-retinol, RGD peptide, LDL receptor ligand, CD63 ligand, CD36, and carbohydrate-based ligands. In some embodiments, the ligand targeted to the central nervous system (CNS) is selected from the group consisting of: angiopeptide-2 (Angiopep-2), lipoprotein receptor-associated protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter, and LDL receptor ligand. In some embodiments, the ligand targeted to the eye is selected from the group consisting of trans-retinol, RGD peptides, LDL receptor ligands, and carbohydrate-based ligands. In one embodiment, the targeting ligand is an RGD peptide, such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH or Cyclo(-Arg-Gly-Asp-D-Phe-Cys).

[0279] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand each comprise at least 15, 16, 17, 18, or 19 consecutive modified nucleotides of the nucleotide sequences SEQ ID NO:478 / SEQ ID NO:480 shown in Table 3.

[0280] In some specific embodiments, the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise or consist of the nucleotide sequences shown in Table 3, SEQ ID NO:478 / SEQ ID NO:480, respectively.

[0281] In some embodiments, the combination of the antisense and sense strands in the dsRNA activator of this disclosure is shown as any combination of antisense and sense strands in Table 3.

[0282] In some embodiments, this disclosure also relates to an RNA-induced silencing complex (RISC) comprising the antisense strand of any of the dsRNA activators described herein.

[0283] III. Preparation of dsRNA activators

[0284] The dsRNA disclosed herein can be synthesized using standard methods known in the art. The double-stranded RNA activator of this disclosure 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.

[0285] In some embodiments, the dsRNA of this disclosure is prepared via RNA solid-phase synthesis. 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. In some embodiments, RNA solid-phase synthesis comprises the following basic methods and steps:

[0286] 1. Template recognition:

[0287] 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.

[0288] 2. Transcription initiation:

[0289] 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.

[0290] 3. Transcription elongation:

[0291] 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.

[0292] 4. Cyclic Synthesis:

[0293] 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.

[0294] 5. Transcription termination:

[0295] 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.

[0296] 6. Post-processing:

[0297] 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.

[0298] 7. Purification and analysis:

[0299] 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.).

[0300] 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.

[0301] IV. Pharmaceutical Compositions

[0302] In some embodiments, this disclosure provides compositions comprising the dsRNA active agent of this disclosure 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 this disclosure, and a combination of one or more other therapeutic agents.

[0303] In some embodiments, the other therapeutic agents, such as any therapeutic agent effective in preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), cover a variety of therapeutic agents for weight management, for reducing weight or body fat, or for preventing or treating obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, covering obesity) or lipid metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions).

[0304] This disclosure also includes compositions (including pharmaceutical compositions) comprising the dsRNA active agent of this disclosure 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.

[0305] 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.

[0306] 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.

[0307] The compositions disclosed herein 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.

[0308] A medicament comprising the dsRNA active agent of the present disclosure can be prepared by mixing the dsRNA active agent of the present disclosure having the desired purity with one or more optional pharmaceutical excipients.

[0309] In some embodiments, the dsRNA activator according to this disclosure may be present in a non-buffered solution, preferably saline or water. In other embodiments, the dsRNA activator is 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).

[0310] The pharmaceutical compositions or formulations disclosed herein may also comprise 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, such as other therapeutic agents, such as any therapeutic agent effective in preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), cover a variety of therapeutic agents for weight management, for reducing weight or body fat, or for preventing or treating obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, encompassing obesity) or lipid metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions). The active ingredients are suitably combined in amounts effective for the intended use.

[0311] In some embodiments, the pharmaceutical compositions or formulations disclosed herein may be contained in vials or in syringes.

[0312] V. Drug combinations and pillboxes

[0313] In some embodiments, this disclosure also provides pharmaceutical combinations or pharmaceutical combination products comprising the dsRNA active agent of this disclosure, and one or more other therapeutic agents.

[0314] Another object of this disclosure is to provide a kit containing the drug combination of this disclosure, preferably in the form of drug dosage units. This allows the dosage units to be provided according to a dosing regimen or drug administration interval.

[0315] In one embodiment, the kit of this disclosure comprises, within the same package:

[0316] - A first container containing a pharmaceutical composition comprising the dsRNA active agent disclosed herein;

[0317] - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0318] In some embodiments, the other therapeutic agents, such as any therapeutic agent effective in preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), cover a variety of therapeutic agents for weight management, for reducing weight or body fat, or for preventing or treating obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, covering obesity) or lipid metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions).

[0319] VI. Uses and Methods

[0320] One aspect of this disclosure provides a method for inhibiting the expression and / or activity of the GPR75 gene in cells, comprising reacting the cells with a dsRNA activator, pharmaceutical composition, or pharmaceutical combination product of this disclosure, thereby inhibiting the expression of the GPR75 gene in the cells. In some embodiments, the cells are in a subject. In some embodiments, the cells are cells in the central nervous system, such as brain tissue. In some embodiments, the cells are cells of nervous system tissues (e.g., central nervous system tissues such as brain tissue, such as the cortex or cerebellum, such as the cerebral cortex, hypothalamus, hippocampus, and other brain regions; or such as neurons), spinal tissues (e.g., cervical, lumbar, and thoracic vertebrae), muscle tissues such as skeletal muscle or cardiac muscle, ocular tissues, or adipose tissue. In some embodiments, the subject suffers from GPR75-related diseases and / or conditions.

[0321] In some embodiments, contacting the cells with the dsRNA activator inhibits GPR75 expression (e.g., mRNA level) by at least about 15%, about 20%, about 25%, 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%, or about 92%.

[0322] In some embodiments, administration of the dsRNA activator to a subject results in a decrease in GPR75 concentration or content (e.g., in target tissues or target cells as described herein), or a decrease in GPR75 protein accumulation or content in the subject's body (e.g., in target tissues or target cells as described herein). In some embodiments, administration of the dsRNA activator to a subject results in a decrease in GPR75 concentration or content in the subject's body fluids.

[0323] In some implementations, administration of the dsRNA activator to a subject results in a decrease in GPR75 expression or protein content in the subject's target tissues (e.g., central nervous system tissues, such as brain tissues like the cerebral cortex, hypothalamus, hippocampus, or other brain tissues) or target cells described herein.

[0324] In some embodiments, the target tissue is selected from tissues of the nervous system (e.g., tissues of the central nervous system such as brain tissue, such as the cortex or cerebellum, such as the cerebral cortex, hypothalamus, hippocampus, and other brain regions; or such as neurons), spinal tissues (e.g., cervical, lumbar, and thoracic vertebrae), muscle tissues such as skeletal muscle or cardiac muscle, eye tissues, or adipose tissue. In some embodiments, the target cells are cells derived from the target tissue. In some embodiments, the target cells are selected from cells of the brain or spinal tissues (e.g., cortex, cerebellum, cerebral cortex, hypothalamus, hippocampus, cervical, lumbar, and thoracic vertebrae), nervous system cells (e.g., neurons), muscle cells such as skeletal muscle cells or cardiac muscle cells, eye cells, or adipose cells.

[0325] This disclosure provides, in one aspect, methods for preventing or treating diseases and / or conditions in subjects, including administering an effective amount of the disclosed dsRNA active agent, pharmaceutical composition, pharmaceutical combination, or kit to the subject. In some embodiments, the disease and / or condition is a GPR75-related disease and / or condition. In some embodiments, the GPR75-related disease and / or condition is obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, encompassing obesity) or a lipid metabolism-related disease. In some embodiments, treating GPR75-related diseases and / or conditions encompasses preventing or treating obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, encompassing obesity), such as performing weight management such as reducing weight, reducing body fat, or controlling weight gain, and / or improving obesity-related metabolic conditions. In some embodiments, this disclosure provides methods for performing weight management, such as reducing weight, reducing body fat, or controlling weight gain, and improving obesity-related metabolic conditions in subjects, including administering an effective amount of the disclosed dsRNA active agent, pharmaceutical composition, pharmaceutical combination, or kit to the subject.

[0326] In some embodiments, this disclosure relates to the dsRNA active agent, pharmaceutical composition, pharmaceutical combination or kit of the disclosure for use in therapies, such as for the prevention or treatment of GPR75-related diseases and / or conditions, such as obesity (e.g., overweight, obesity, severe obesity or extreme obesity, encompassing obesity) or lipid metabolism-related diseases.

[0327] In some embodiments, this disclosure relates to methods of preventing or treating diseases and / or conditions, such as GPR75-related diseases and / or conditions mentioned herein (e.g., obesity (e.g., overweight, obese, severely obese, or extremely obese, encompassing obesity) or lipid metabolism-related diseases), or uses for said prevention or treatment, or uses for preparing medicaments for said prevention or treatment.

[0328] In some embodiments, the disease and / or condition is associated with increased expression or activity of GPR75. In some embodiments, the disease and / or condition is caused by gene expression of GPR75 (e.g., aberrant expression). In some embodiments, the disease and / or condition is a condition or indication that benefits from reduced GPR75 expression and / or activity. In some embodiments, the disease and / or condition is obesity, such as overweight, obesity, severe obesity, or extreme obesity, encompassing obesity disorders.

[0329] In some embodiments, this disclosure relates to the dsRNA active agent, pharmaceutical composition, pharmaceutical combination or kit of the disclosure for use in therapies, such as for weight management, such as reducing weight, reducing body fat or controlling weight gain, and / or improving obesity-related metabolic conditions.

[0330] In some embodiments, this disclosure relates to methods for weight management, such as reducing weight, reducing body fat, or controlling weight gain, and / or improving obesity-related metabolic conditions, using the dsRNA active agents, pharmaceutical compositions, drug combinations, or kits of this disclosure.

[0331] In some embodiments, this disclosure relates to the use of the dsRNA active agent, pharmaceutical composition, pharmaceutical combination or kit of this disclosure for weight management, such as reducing weight, reducing body fat or controlling weight gain, and / or improving obesity-related metabolic conditions.

[0332] In some embodiments, this disclosure relates to the use of the dsRNA active agent, pharmaceutical composition, pharmaceutical combination or kit of this disclosure for the preparation of a medicament for weight management, such as reducing weight, reducing body fat or controlling weight gain, and / or improving obesity-related metabolic conditions.

[0333] In some embodiments, "increased expression or activity of GPR75" refers to increased expression (e.g., nucleic acid or protein level) or activity of GPR75 in the subject's tissues or cells, such as the target tissues described herein (e.g., central nervous system tissues, such as brain tissues, such as the cerebral cortex, hypothalamus, hippocampus, or other brain tissues) or target cells described herein. In some embodiments, patients with GPR75-related diseases and / or conditions have intermediate or high levels of GPR75 expression in the target tissues described herein (e.g., central nervous system tissues, such as brain tissues, such as the cerebral cortex, hypothalamus, hippocampus, or other brain tissues) or target cells described herein. In some embodiments, patients with GPR75-related diseases and / or conditions have higher levels of GPR75 in the target tissues described herein (e.g., central nervous system tissues, such as brain tissues, such as the cerebral cortex, hypothalamus, hippocampus, or other brain tissues) or target cells described herein than in the corresponding tissues or cells of healthy controls.

[0334] The dsRNA active agent or composition or drug or formulation comprising thereof disclosed herein 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 and / or conditions mentioned herein. Therefore, this disclosure also relates to combination therapy of the dsRNA active agent or composition or drug or formulation comprising thereof with one or more other therapies.

[0335] In other respects, this disclosure provides for the use of the dsRNA active agent of this disclosure 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 and / or conditions mentioned herein.

[0336] In some embodiments, the method or use further includes determining the level of GPR75 (e.g., protein or nucleic acid level, such as mRNA level) in a subject or in the target tissue or target cells described herein prior to administration of the dsRNA active agent. In some embodiments, the level of GPR75 is compared with the level of GPR75 (e.g., protein or nucleic acid level, such as mRNA level) in a healthy subject or in a healthy corresponding tissue or corresponding cell, and if the level of GPR75 in the subject or in the target tissue or target cells is higher than the level of GPR75 in a healthy control, the subject is then administered the dsRNA active agent or a composition, drug, formulation, or combination product containing it.

[0337] In some embodiments, the target tissue refers to any tissue or organ or cell within the subject, such as the nervous system (e.g., the central nervous system, such as the brain, such as the cortex or cerebellum, like the cerebral cortex, hypothalamus, hippocampus, or other brain regions; e.g., neurons), spinal tissue (e.g., cervical, lumbar, and thoracic vertebrae), muscle tissue such as skeletal muscle or cardiac muscle, ocular tissue, or adipose tissue. In some embodiments, the "target tissue" is selected from tissues of the nervous system (e.g., tissues of the central nervous system, such as brain tissue, such as the cortex or cerebellum, like the cerebral cortex, hypothalamus, hippocampus, or other brain regions; or e.g., neurons), spinal tissue (e.g., cervical, lumbar, and thoracic vertebrae), muscle tissue such as skeletal muscle or cardiac muscle, ocular tissue, or adipose tissue. In some embodiments, the target cells are cells derived from the target tissue. In some embodiments, the “target cells” are selected from cells of brain or spinal cord tissues (e.g., cortex, cerebellum, cerebral cortex, hypothalamus, hippocampus or other brain tissues, cervical, lumbar and thoracic vertebrae), nervous system cells (e.g., neurons), muscle cells such as skeletal muscle cells or cardiomyocytes, eye cells or fat cells.

[0338] 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).

[0339] In one implementation, the subject has the disease and / or condition described herein or is at risk of having the disease and / or condition described herein.

[0340] The combination therapies disclosed herein cover 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 disclosure may occur before, simultaneously with, and / or after the administration of other therapeutic agents and / or active agents.

[0341] In some embodiments, any therapeutic agent that can be combined with or combined with other therapeutic agents such as those disclosed in this disclosure (dsRNA active agent, drug, formulation, or composition) for the prevention or treatment of GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene) covers a variety of therapeutic agents for weight management, for reducing weight or body fat, or for the prevention or treatment of obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, covering obesity) or lipid metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions).

[0342] VII. Specific Implementation Plan

[0343] 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, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of a target sequence, and the complementary region contains a mismatch of no more than 3, 2, or 1 nucleotide with the at least 15 consecutive nucleotides of the target sequence, wherein the target sequence is selected from...

[0344] (i) The target sequence corresponding to positions 1349-1369 or 1984-2004 of GPR75 mRNA;

[0345] (ii) The target sequence shown in SEQ ID NO:280 or SEQ ID NO:241;

[0346] (iii) The target sequence shown in any one of SEQ ID NO: 191-285; or

[0347] (iv) The target sequences corresponding to the positions in GPR75 mRNA, such as NM_006794.4, shown in Table 1;

[0348] Optionally, the dsRNA activator is used to inhibit the expression of a gene encoding GPR75.

[0349] 2. The dsRNA activator according to embodiment 1, 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 the target sequence encoding the mRNA of GPR75, and comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides differing by no more than 3, 2 or 1 nucleotides from any antisense nucleotide sequence in the antisense strands in Table 1 or any antisense nucleotide sequence shown in SEQ ID NO:96-190.

[0350] 3. 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 GPR75 mRNA, and comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differing by no more than 3, 2, or 1 nucleotide from any antisense nucleotide sequence in the antisense strands listed in Table 1 or any antisense nucleotide sequence shown in SEQ ID NO:96-190;

[0351] Optionally, the dsRNA activator is used to inhibit the expression of a gene encoding GPR75.

[0352] 4. The dsRNA activator according to any one of embodiments 1 to 3, wherein the nucleotide sequence of the antisense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ from the nucleotide sequence shown in SEQ ID NO:185 or SEQ ID NO:146 by no more than 3, 2 or 1 nucleotides.

[0353] 5. The dsRNA activator according to any one of embodiments 1 to 4, wherein the antisense strand contains a complementary region of 15 to 30 nucleotides or 18 to 23 nucleotides in length to the target sequence, for example, the complementary region is 15, 16, 17, 18, 19, 20 or 21 nucleotides in length.

[0354] 6. The dsRNA activator according to any one of embodiments 1 to 5, wherein the complementary region comprises at least nucleotides 2-16, 2-17, 2-18, 2-19, 2-20, or 2-21, for example, nucleotides 2-19, 2-20, or 2-21, starting from the 5' end of the antisense strand.

[0355] 7. The dsRNA activator according to any one of embodiments 1 to 6, wherein the antisense strand 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, or the antisense strand is completely complementary to the target sequence.

[0356] 8. The dsRNA activator according to any one of embodiments 1 to 7, wherein the first nucleotide at the 5' end of the antisense strand is A or U, for example, U.

[0357] 9. The dsRNA activator according to any one of embodiments 1 to 8, 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, 2 or 1 nucleotides from any nucleotide sequence of the sense strand in Table 1 or any sense nucleotide sequence shown in SEQ ID NO:1-95.

[0358] 10. The dsRNA activator according to any one of embodiments 1 to 8, 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:51 or SEQ ID NO:90 by no more than 3 nucleotides.

[0359] 11. The dsRNA activator according to any one of embodiments 1 to 10, 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 mismatches.

[0360] 12. The dsRNA activator according to any one of embodiments 1 to 11, wherein the length of the fully complementary double-stranded region is between 15 and 25 nucleotide pairs, 16 and 24 nucleotide pairs, 17 and 23 nucleotide pairs, or 18 and 22 nucleotide pairs, for example, the length of the fully complementary double-stranded region is 15, 16, 17, 18, or 19 nucleotide pairs, for example, 19 nucleotide pairs.

[0361] 13. The dsRNA activator according to any one of embodiments 1 to 12, 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.

[0362] 14. The dsRNA activator according to any one of embodiments 1 to 13, 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.

[0363] 15. The dsRNA activator according to any one of embodiments 1 to 14, comprising a sense strand and an antisense strand forming a double-stranded region, wherein

[0364] (i) The positive strand contains or is 19 nucleotides.

[0365] (ii) The antisense strand comprises or is 21 nucleotides and is completely complementary to the target sequence encoding GPR75 mRNA in the region excluding 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; and

[0366] (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.

[0367] 16. The dsRNA activator according to any one of embodiments 1 to 12, wherein the dsRNA activator comprises a sense strand and an antisense strand, the nucleotide sequence of the sense strand comprising a nucleotide sequence selected from any one of the nucleotide sequences of the sense strand in Table 1, and / or the nucleotide sequence of the antisense strand comprising a nucleotide sequence selected from any one of the nucleotide sequences of the antisense strand in Table 1.

[0368] 17. The dsRNA activator according to any one of embodiments 1 to 16, wherein the dsRNA activator comprises a sense strand and an antisense strand, wherein the antisense strand and the sense strand respectively comprise SEQ ID NO:185 / SEQ ID NO:90, SEQ ID NO:146 / SEQ ID NO:51, SEQ ID NO:96 / SEQ ID NO:1, SEQ ID NO:97 / SEQ ID NO:2, SEQ ID NO:98 / SEQ ID NO:3, SEQ ID NO:99 / SEQ ID NO:4, SEQ ID NO:100 / SEQ ID NO:5, SEQ ID NO:101 / SEQ ID NO:6, SEQ ID NO:102 / SEQ ID NO:7, SEQ ID NO:103 / SEQ ID NO:8, SEQ ID NO:104 / SEQ ID NO:9, SEQ ID NO:105 / SEQ ID NO:10, SEQ ID NO:106 / SEQ ID NO:11, SEQ ID NO:107 / SEQ ID NO:12, SEQ ID NO:108 / SEQ ID NO:90, SEQ ID NO:105 / SEQ ID NO:10, SEQ ID NO:106 / SEQ ID NO:11, SEQ ID NO:107 / SEQ ID NO:12, SEQ ID NO:108 / SEQ ID NO:100, ... ID NO:13, SEQ ID NO:109 / SEQ ID NO:14, SEQ ID NO:110 / SEQ ID NO:15, SEQ ID NO:111 / SEQ ID NO:16, SEQ ID NO:112 / SEQ ID NO:17, SEQ ID NO:113 / SEQ ID NO:18, SEQ ID NO:114 / SEQ ID NO:19, SEQ ID NO:115 / SEQ ID NO:20, SEQ ID NO:116 / SEQ ID NO:21, SEQ ID NO:117 / SEQ ID NO:22, SEQ ID NO:118 / SEQ ID NO:23, SEQ ID NO:119 / SEQ ID NO:24, SEQ ID NO:120 / SEQ ID NO:25, SEQ ID NO:121 / SEQ ID NO:26, SEQ ID NO:122 / SEQ ID NO:27, SEQ ID NO:123 / SEQ ID NO:28, SEQ ID NO:124 / SEQ ID NO:29, SEQ ID NO:125 / SEQ ID NO:30, SEQ ID NO:126 / SEQ ID NO:31, SEQ ID NO:127 / SEQ ID NO:32, SEQ ID NO:128 / SEQ IDNO:33、SEQ ID NO:129 / SEQ ID NO:34、SEQ ID NO:130 / SEQ ID NO:35、SEQ ID NO:131 / SEQ ID NO:36、SEQ ID NO:132 / SEQ ID NO:37、SEQ ID NO:133 / SEQ ID NO:38、SEQ ID NO:134 / SEQ ID NO:39、SEQ ID NO:135 / SEQ ID NO:40、SEQ ID NO:136 / SEQ ID NO:41、SEQ ID NO:137 / SEQ ID NO:42、SEQ ID NO:138 / SEQ ID NO:43、SEQ ID NO:139 / SEQ ID NO:44、SEQ ID NO:140 / SEQ ID NO:45、SEQ ID NO:141 / SEQ ID NO:46、SEQ ID NO:142 / SEQ ID NO:47、SEQ ID NO:143 / SEQ ID NO:48、SEQ ID NO:144 / SEQ ID NO:49、SEQ ID NO:145 / SEQ ID NO:50、SEQ ID NO:147 / SEQ ID NO:52、SEQ ID NO:148 / SEQ ID NO:53、SEQ ID NO:149 / SEQ ID NO:54、SEQ ID NO:150 / SEQ ID NO:55、SEQ ID NO:151 / SEQ ID NO:56、SEQ ID NO:152 / SEQ ID NO:57、SEQ ID NO:153 / SEQ ID NO:58、SEQ ID NO:154 / SEQ ID NO:59、SEQ ID NO:155 / SEQ ID NO:60、SEQ ID NO:156 / SEQ ID NO:61、SEQ ID NO:157 / SEQ ID NO:62、SEQ ID NO:158 / SEQ ID NO:63、SEQ ID NO:159 / SEQ ID NO:64、SEQ ID NO:160 / SEQ ID NO:65、SEQ ID NO:161 / SEQ ID NO:66、SEQ ID NO:162 / SEQ ID NO:67、SEQ ID NO:163 / SEQ ID NO:68、SEQ ID NO:164 / SEQ ID NO:69、SEQ ID NO:165 / SEQ ID NO:70、SEQ ID NO:166 / SEQ IDNO:71, SEQ ID NO:167 / SEQ ID NO:72, SEQ ID NO:168 / SEQ ID NO:73, SEQ ID NO:169 / SEQ ID NO:74, SEQ ID NO:170 / SEQ ID NO:75, SEQ ID NO:171 / SEQ ID NO:76, SEQ ID NO:172 / SEQ ID NO:77, SEQ ID NO:173 / SEQ ID NO:78, SEQ ID NO:174 / SEQ ID NO:79, SEQ ID NO:175 / SEQ ID NO:80, SEQ ID NO:176 / SEQ ID NO:81, SEQ ID NO:177 / SEQ ID NO:82, SEQ ID NO:178 / SEQ ID NO:83, SEQ ID NO:179 / SEQ ID NO:84, SEQ ID NO:180 / SEQ ID NO:85, SEQ ID NO:181 / SEQ ID NO:86, SEQ ID NO:182 / SEQ ID NO:87, SEQ The nucleotide sequences shown in SEQ ID NO:183 / SEQ ID NO:88, SEQ ID NO:184 / SEQ ID NO:89, SEQ ID NO:186 / SEQ ID NO:91, SEQ ID NO:187 / SEQ ID NO:92, SEQ ID NO:188 / SEQ ID NO:93, SEQ ID NO:189 / SEQ ID NO:94 or SEQ ID NO:190 / SEQ ID NO:95; or the sequences shown in the nucleotide sequences shown.

[0369] 18. The dsRNA activator according to any one of embodiments 1 to 17, wherein the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand each comprise the nucleotide sequences shown in SEQ ID NO:90 / SEQ ID NO:185; or each comprises the nucleotide sequences shown.

[0370] 19. The dsRNA activator according to any one of embodiments 1 to 17, wherein the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand each comprise the nucleotide sequences shown in SEQ ID NO:51 / SEQ ID NO:146; or each comprises the nucleotide sequences shown.

[0371] 20. The dsRNA activator according to any one of embodiments 1 to 19, wherein the dsRNA activator comprises at least one modified nucleotide, optionally wherein 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.

[0372] 21. The dsRNA activator according to any one of embodiments 1 to 20, wherein all nucleotides of the sense strand are modified nucleotides; or 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.

[0373] 22. The dsRNA activator according to embodiment 20 or 21, 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'-methoxy 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 (2'-O-hexadecyl 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.

[0374] 23. The dsRNA activator according to any one of embodiments 1 to 22, wherein the antisense strand comprises a phosphate thioester bond, and / or the sense strand comprises a phosphate thioester bond.

[0375] Choose one of them

[0376] The thiophosphate nucleotide bond is located between the 1st and 3rd nucleotides at the 5' end of the sense strand, and the thiophosphate nucleotide bond is located between the 1st and 3rd nucleotides at the 5' end and the 1st and 3rd nucleotides at the 3' end of the antisense strand; or

[0377] The thiophosphate nucleotide inter-links are located between the 1st and 3rd nucleotides at the 5' end and the 1st and 3rd nucleotides at the 3' end of the sense strand, and the thiophosphate nucleotide inter-links are located between the 1st and 3rd nucleotides at the 5' end and the 1st and 3rd nucleotides at the 3' end of the antisense strand.

[0378] 24. The dsRNA activator according to any one of embodiments 1 to 23, wherein the nucleotides at positions 2, 6, 14 and 16 of the antisense strand, counting from the 5' end, are 2'-fluorinated nucleotides, and the nucleotides at positions 7-9 of the sense strand, counting from the 5' end, are 2'-fluorinated nucleotides.

[0379] 25. The dsRNA activator according to any one of embodiments 1 to 24, wherein the antisense strand has nucleotides 1, 3-5, 7-13, 15, and 17-21 from the 5' end that are 2'-methoxy modified nucleotides, and the sense strand has nucleotides 1-6 and 10-19 from the 5' end that are 2'-methoxy modified nucleotides; or the antisense strand has nucleotides 1, 3-5, 7-13, 15, and 17-21 from the 5' end that are 2'-methoxy modified nucleotides, and the sense strand has nucleotides 1-5 and 10-19 from the 5' end that are 2'-methoxy modified nucleotides.

[0380] 26. The dsRNA activator according to any one of embodiments 1 to 25, wherein the 5' nucleotide of the antisense strand has a 5' phosphate ester modification, such as 5'-vinylphosphonate (5'-VP) or 5'-(E)-vinylphosphonate (5'-(E)-VP).

[0381] 27. The dsRNA activator according to any one of embodiments 1 to 26, wherein the modified nucleotides in the sense and antisense strands have the following modification patterns:

[0382] antisense chain:

[0383] NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0384] and / or

[0385] Chain of Justice:

[0386] NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm,

[0387] The symbols mentioned herein are as defined herein.

[0388] Nf = any 2'-fluorine modified nucleotide

[0389] Nfs = any 2'-fluorine modified nucleoside-3' thiophosphate;

[0390] Nm = any 2'-methoxynucleotide;

[0391] Nms = any 2'-methoxynucleoside-3'-thiophosphate;

[0392] 's' indicates that the two nucleotides are linked by a phosphate thioester bond.

[0393] 28. The dsRNA activator according to any one of embodiments 1 to 26, wherein the modified nucleotides in the sense and antisense strands have the following modification patterns:

[0394] antisense chain:

[0395] VPNmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0396] and / or

[0397] Chain of Justice:

[0398] NmsNmsNmNmNmNNfNfNfNmNmNmNmNmNmNmNmNmsNmsNm,

[0399] in,

[0400] Nf = any 2'-fluorine modified nucleotide

[0401] Nfs = any 2'-fluorine modified nucleoside-3' thiophosphate;

[0402] Nm = any 2'-methoxynucleotide;

[0403] Nms = any 2'-methoxynucleoside-3'-thiophosphate;

[0404] N = any form of nucleotide;

[0405] s indicates that the two nucleotides are linked by a phosphate thioester bond; VP indicates that the 5' end nucleotide of the antisense strand has a 5'-vinyl phosphonate (5'-VP) modification, such as 5'-(E)-VP modification;

[0406] For example, the 5' nucleotide VPNms of the antisense strand can be VPUms, with the following structure:

[0407] Preferably, the nucleotide at position 6, counting from the 5' end of the positive strand, is further conjugated with an adipose chain.

[0408] 29. The dsRNA activator according to any one of embodiments 1 to 28, wherein the antisense strand comprises any modified nucleotide sequence of the antisense strands in Table 2, and / or the sense strand comprises any modified nucleotide sequence of the sense strands in Table 2; optionally, the dsRNA activator comprises a combination of modified antisense strands and sense strands as shown in Table 2.

[0409] 30. The dsRNA active agent according to any one of embodiments 1 to 29, wherein the sense strand and antisense strand respectively comprise SEQ ID NO:377 / SEQ ID NO:472, SEQ ID NO:338 / SEQ ID NO:433, SEQ ID NO:288 / SEQ ID NO:383, SEQ ID NO:289 / SEQ ID NO:384, SEQ ID NO:290 / SEQ ID NO:385, SEQ ID NO:291 / SEQ ID NO:386, SEQ ID NO:292 / SEQ ID NO:387, SEQ ID NO:293 / SEQ ID NO:388, SEQ ID NO:294 / SEQ ID NO:389, SEQ ID NO:295 / SEQ ID NO:390, SEQ ID NO:296 / SEQ ID NO:391, SEQ ID NO:297 / SEQ ID NO:392, SEQ ID NO:298 / SEQ ID NO:393, SEQ ID NO:299 / SEQ ID NO:394, SEQ ID NO:300 / SEQ ID NO:395, SEQ ID NO:301 / SEQ ID NO:396, SEQ ID NO:302 / SEQ ID NO:397, SEQ ID NO:303 / SEQ ID NO:398, SEQ ID NO:304 / SEQ ID NO:399, SEQ ID NO:305 / SEQ ID NO:400, SEQ ID NO:306 / SEQ ID NO:401, SEQ ID NO:307 / SEQ ID NO:402, SEQ ID NO:308 / SEQ ID NO:403, SEQ ID NO:309 / SEQ ID NO:404, SEQ ID NO:310 / SEQ ID NO:405, SEQ ID NO:311 / SEQ ID NO:406, SEQ ID NO:312 / SEQ ID NO:407, SEQ ID NO:313 / SEQ ID NO:408, SEQ ID NO:314 / SEQ ID NO:409, SEQ ID NO:315 / SEQ ID NO:410, SEQ ID NO:316 / SEQ ID NO:411, SEQ ID NO:317 / SEQ ID NO:412, SEQ ID NO:318 / SEQ ID NO:413, SEQ ID NO:319 / SEQ ID NO:414, SEQID NO:320 / SEQ ID NO:415、SEQ ID NO:321 / SEQ ID NO:416、SEQ ID NO:322 / SEQ ID NO:417、SEQ ID NO:323 / SEQ ID NO:418、SEQ ID NO:324 / SEQ ID NO:419、SEQ ID NO:325 / SEQ ID NO:420、SEQ ID NO:326 / SEQ ID NO:421、SEQ ID NO:327 / SEQ ID NO:422、SEQ ID NO:328 / SEQ ID NO:423、SEQ ID NO:329 / SEQ ID NO:424、SEQ ID NO:330 / SEQ ID NO:425、SEQ ID NO:331 / SEQ ID NO:426、SEQ ID NO:332 / SEQ ID NO:427、SEQ ID NO:333 / SEQ ID NO:428、SEQ ID NO:334 / SEQ ID NO:429、SEQ ID NO:335 / SEQ ID NO:430、SEQ ID NO:336 / SEQ ID NO:431、SEQ ID NO:337 / SEQ ID NO:432、SEQ ID NO:339 / SEQ ID NO:434、SEQ ID NO:340 / SEQ ID NO:435、SEQ ID NO:341 / SEQ ID NO:436、SEQ ID NO:342 / SEQ ID NO:437、SEQ ID NO:343 / SEQ ID NO:438、SEQ ID NO:344 / SEQ ID NO:439、SEQ ID NO:345 / SEQ ID NO:440、SEQ ID NO:346 / SEQ ID NO:441、SEQ ID NO:347 / SEQ ID NO:442、SEQ ID NO:348 / SEQ ID NO:443、SEQ ID NO:349 / SEQ ID NO:444、SEQ ID NO:350 / SEQ ID NO:445、SEQ ID NO:351 / SEQ ID NO:446、SEQ ID NO:352 / SEQ ID NO:447、SEQ ID NO:353 / SEQ ID NO:448、SEQ ID NO:354 / SEQ ID NO:449、SEQ ID NO:355 / SEQ ID NO:450、SEQ ID NO:356 / SEQ IDNO:451, SEQ ID NO:357 / SEQ ID NO:452, SEQ ID NO:358 / SEQ ID NO:453, SEQ ID NO:359 / SEQ ID NO:454, SEQ ID NO:360 / SEQ ID NO:455, SEQ ID NO:361 / SEQ ID NO:456, SEQ ID NO:362 / SEQ ID NO:457, SEQ ID NO:363 / SEQ ID NO:458, SEQ ID NO:364 / SEQ ID NO:459, SEQ ID NO:365 / SEQ ID NO:460, SEQ ID NO:366 / SEQ ID NO:461, SEQ ID NO:367 / SEQ ID NO:462, SEQ ID NO:368 / SEQ ID NO:463, SEQ ID NO:369 / SEQ ID NO:464、SEQ ID NO:370 / SEQ ID NO:465、SEQ ID NO:371 / SEQ ID The modified nucleotide sequences shown in SEQ ID NO:466, SEQ ID NO:372 / SEQ ID NO:467, SEQ ID NO:373 / SEQ ID NO:468, SEQ ID NO:374 / SEQ ID NO:469, SEQ ID NO:375 / SEQ ID NO:470, SEQ ID NO:376 / SEQ ID NO:471, SEQ ID NO:378 / SEQ ID NO:473, SEQ ID NO:379 / SEQ ID NO:474, SEQ ID NO:380 / SEQ ID NO:475, SEQ ID NO:381 / SEQ ID NO:476 or SEQ ID NO:382 / SEQ ID NO:477, or each of the modified nucleotide sequences shown.

[0410] Optionally, it also has one or more of the following modifications: the first nucleotide at the 5' end of the antisense strand is modified with 5'-vinylphosphonate (5'-VP), for example, 5'-(E)-VP; for example, VPUm or VPAm; the 6th nucleotide from the 5' end of the sense strand is conjugated with an aliphatic chain, such as a hydrocarbon group or hydrocarbon chain, which can serve as a ligand, for example, C 16 -C 22 Hydrocarbon chains, such as those containing C 16 -C 22Nucleoside monomers with saturated hydrocarbon chains, such as Uhd, Chd, Ahd, or Ghd; and / or phosphate thioester bonds between the first to third nucleotides starting from the 3' end of the sense chain.

[0411] 31. The dsRNA activator according to any one of embodiments 1 to 30, wherein the sense strand and antisense strand respectively comprise the nucleotide sequences shown in SEQ ID NO:338 / SEQ ID NO:433 or are respectively composed of the modified nucleotide sequences shown.

[0412] 32. The dsRNA activator according to any one of embodiments 1 to 30, wherein the sense strand and antisense strand respectively comprise the nucleotide sequences shown in SEQ ID NO:377 / SEQ ID NO:472 or are respectively composed of the modified nucleotide sequences shown.

[0413] 33. The dsRNA activator according to any one of embodiments 1 to 32, which is conjugated to a ligand capable of targeted delivery to a target tissue or target cell, preferably, the target tissue being tissue of the central nervous system, preferably brain tissue or neurons, more preferably the cortex or cerebellum, most preferably tissue of the cerebral cortex, hypothalamus, hippocampus and other brain regions; spinal tissue, preferably cervical, lumbar and thoracic vertebrae; muscle tissue, preferably skeletal muscle tissue or cardiac tissue; ocular tissue or adipose tissue; or the target cell being a cell derived from the target tissue.

[0414] 34. The dsRNA activator according to embodiment 33, wherein the target tissue is selected from tissues of the central nervous system, preferably brain tissue or neurons; or muscle tissue, preferably skeletal muscle tissue or myocardial tissue; or the target cells are selected from cells of the central nervous system, preferably brain cells or neuronal cells; or muscle cells, preferably skeletal muscle cells or myocardial cells.

[0415] 35. The dsRNA activator according to any one of embodiments 1 to 34, wherein one or two chains of the dsRNA activator are conjugated to a C16-C22 hydrocarbon chain, for example, one or more C16-C22 hydrocarbon chains (e.g., saturated or unsaturated) are conjugated to one or more internal positions of at least one chain of the dsRNA activator, for example, one or more C16 hydrocarbon chains (e.g., saturated or unsaturated) are conjugated to one or more internal positions of at least one chain of the dsRNA activator.

[0416] 36. The dsRNA activator according to embodiment 35, wherein the C16-C22 hydrocarbon chain (e.g., saturated or unsaturated) is conjugated to the 6th nucleotide from the 5' end of the positive strand of the dsRNA activator.

[0417] 37. The dsRNA activator according to embodiment 36, wherein the modified nucleotides in the sense and antisense strands have the following pattern:

[0418] antisense chain:

[0419] VPNmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0420] and

[0421] Chain of Justice:

[0422] NmsNmsNmNmNmNhdNfNfNfNmNmNmNmNmNmNmNmNmsNmsNm,

[0423] in,

[0424] Nf = any 2'-fluorine modified nucleotide

[0425] Nfs = any 2'-fluorine modified nucleoside-3' thiophosphate;

[0426] Nm = any 2'-methoxynucleotide;

[0427] Nms = any 2'-methoxynucleoside-3'-thiophosphate;

[0428] Nhd = any nucleotide conjugated with 2′-O-hexadecyl;

[0429] s indicates that the two nucleotides are linked by a phosphate thioester bond; VP indicates that the 5' end nucleotide of the antisense strand has a 5'-vinyl phosphonate (5'-VP) modification, such as 5'-(E)-VP modification;

[0430] For example, the 5' nucleotide VPNms of the antisense strand can be VPUms, with the following structure:

[0431] 38. The dsRNA activator according to embodiment 36, wherein the sense strand and antisense strand respectively comprise the nucleotide sequences shown in SEQ ID NO:478 / SEQ ID NO:480 or are respectively composed of the modified nucleotide sequences shown.

[0432] 39. The dsRNA activator according to any one of embodiments 1 to 38, wherein the dsRNA activator is in the form of a salt, a mixed salt, or a free acid.

[0433] 40. A cell containing a dsRNA activator according to any one of embodiments 1 to 39.

[0434] 41. A pharmaceutical composition comprising a dsRNA activator according to any one of embodiments 1 to 39 and a pharmaceutically acceptable carrier.

[0435] 42. The pharmaceutical composition according to embodiment 41, wherein the dsRNA active agent is in a non-buffered solution, for example, saline or water; or

[0436] 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).

[0437] 43. A pharmaceutical combination comprising a dsRNA activator according to any one of embodiments 1 to 39 and one or more other therapeutic agents, preferably, said other therapeutic agents being selected from any therapeutic agents effective in preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), covering a variety of therapeutic agents for weight management, for reducing weight or body fat, or for preventing or treating obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, covering obesity) or lipid metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions).

[0438] 44. A method for inhibiting the expression of the GPR75 gene in cells, the method comprising contacting the cells with a dsRNA activator according to any one of embodiments 1 to 39, or a pharmaceutical composition according to embodiment 41 or 42, or a pharmaceutical composition according to embodiment 43, thereby inhibiting the expression of the GPR75 gene in the cells.

[0439] 45. The method according to embodiment 44, wherein the cells are in a subject, and optionally the subject is a mammal, such as a human.

[0440] 46. ​​The method according to embodiment 45, wherein the subject suffers from a GPR75-related disease and / or condition.

[0441] 47. The method according to embodiment 46, wherein the GPR75-related disease and / or condition is obesity or lipid metabolism-related disease; preferably, the obesity is selected from overweight, obese, severely obese or extremely obese, covering obesity.

[0442] 48. The method according to any one of embodiments 45-47, wherein the subject needs to undergo weight management, such as weight reduction, body fat reduction or control of weight gain, and / or improvement of obesity-related metabolic status.

[0443] 49. The method according to any one of embodiments 44 to 48, wherein contacting the cells with the dsRNA activator inhibits GPR75 expression by at least 15%, 20%, 25%, 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%, or about 92%.

[0444] 50. A method for preventing or treating GPR75-related diseases and / or conditions in a subject, comprising administering to the subject a dsRNA active agent of any one of embodiments 1-39 or a pharmaceutical composition of embodiment 41 or 42 or a pharmaceutical combination of embodiment 43.

[0445] 51. The method of implementation scheme 50, wherein the GPR75-related disease and / or condition is obesity or lipid metabolism-related disease; preferably, the obesity is selected from overweight, obese, severely obese or extremely obese, covering obesity.

[0446] 52. A method for weight management in a subject, such as reducing weight, reducing body fat, or controlling weight gain, and / or improving obesity-related metabolic status, comprising administering to the subject a dsRNA active agent of any one of embodiments 1-39 or a pharmaceutical composition of embodiment 41 or 42 or a pharmaceutical composition of embodiment 43.

[0447] 53. The method of implementation scheme 52, wherein the subject has obesity or lipid metabolism-related diseases; preferably, the obesity is selected from overweight, obese, severely obese or extremely obese, covering obesity.

[0448] 54. The method of any one of embodiments 50-53, wherein administration of the dsRNA activator to the subject results in a decrease in GPR75 concentration or content (e.g., in target tissues or target cells), or a decrease in GPR75 protein accumulation or content in the subject (e.g., in target tissues or target cells); or a decrease in the subject's weight, body fat, control of weight gain, and / or improvement in obesity-related metabolic status.

[0449] 55. The method of embodiment 54, wherein the target tissue is tissue of the central nervous system, preferably brain tissue or neurons, more preferably cortex or cerebellum, most preferably tissue of the cerebral cortex, hypothalamus, hippocampus and other brain regions; spinal tissue, preferably cervical, lumbar and thoracic vertebrae; muscle tissue, preferably skeletal muscle tissue or myocardial tissue; eye tissue or adipose tissue; or the target cells are cells derived from the target tissue.

[0450] 56. The method of embodiment 54 or 55, wherein the target tissue is selected from tissues of the central nervous system, preferably brain tissue or neurons; or muscle tissue, preferably skeletal muscle tissue or myocardial tissue; or the target cells are selected from cells of the central nervous system, preferably brain cells or neuronal cells; or muscle cells, preferably skeletal muscle cells or myocardial cells.

[0451] 57. The method of any one of embodiments 44 to 56, further comprising administering one or more other therapeutic agents to a subject, preferably, said other therapeutic agents being selected from any therapeutic agents effective in preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), covering a variety of therapeutic agents for weight management, for reducing weight or body fat, or for preventing or treating obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, covering obesity) or lipid metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions).

[0452] 58. The method according to any one of embodiments 44 to 57, further comprising determining the level of GPR75 in a sample from the subject.

[0453] 59. Use of the dsRNA activator of any one of embodiments 1-39, or the pharmaceutical composition of embodiment 41 or 42, or the pharmaceutical composition of embodiment 43, in the preparation of a medicament, wherein the medicament is used for the prevention or treatment of GPR75-related diseases and / or conditions.

[0454] 60. Use of embodiment 59, wherein the GPR75-related disease and / or condition is obesity or lipid metabolism-related disease; preferably, the obesity is selected from overweight, obese, severely obese or extremely obese, covering obesity.

[0455] 61. Use of the dsRNA activator of any one of embodiments 1-39, or the pharmaceutical composition of embodiment 41 or 42, or the pharmaceutical composition of embodiment 43 in the preparation of a medicament, wherein the medicament is used for weight management, such as reducing weight, reducing body fat, or controlling weight gain, and / or improving obesity-related metabolic conditions.

[0456] 62. The use described in embodiment 61, wherein the subject has obesity or a lipid metabolism-related disease; preferably, the obesity is selected from overweight, obese, severely obese or extremely obese, covering obesity.

[0457] 63. The use according to any one of embodiments 59-62, wherein administration of the dsRNA activator to the subject results in a decrease in GPR75 concentration or content (e.g. in target tissues or target cells), or a decrease in GPR75 protein accumulation or content in the subject (e.g. in target tissues or target cells); or a decrease in the subject's weight, body fat, control of weight gain, and / or improvement of obesity-related metabolic status.

[0458] 64. The use described in embodiment 63, wherein the target tissue is tissue of the central nervous system, preferably brain tissue or neurons, more preferably cortex or cerebellum, most preferably tissue of the cerebral cortex, hypothalamus, hippocampus and other brain regions; spinal tissue, preferably cervical, lumbar and thoracic vertebrae; muscle tissue, preferably skeletal muscle tissue or myocardial tissue; eye tissue or adipose tissue; or the target cells are cells derived from the target tissue.

[0459] 65. The use described in embodiment 63 or 64, wherein the target tissue is selected from tissues of the central nervous system, preferably brain tissue or neurons; or muscle tissue, preferably skeletal muscle tissue or myocardial tissue; or the target cells are selected from cells of the central nervous system, preferably brain cells or neuronal cells; or muscle cells, preferably skeletal muscle cells or myocardial cells.

[0460] 66. Use of any one of embodiments 59 to 65, wherein the drug is administered in combination with one or more other therapeutic agents, preferably, the other therapeutic agents being selected from any therapeutic agents effective in preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), covering a variety of therapeutic agents for weight management, for reducing weight or body fat, or for preventing or treating obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, covering obesity) or lipid metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions).

[0461] 67. The method according to any one of embodiments 59 to 66, wherein the prevention or treatment of administering the drug further includes determining the level of GPR75 in a sample from the subject.

[0462] 68. The dsRNA activator of any one of embodiments 1-39, or the pharmaceutical composition of embodiment 41 or 42, or the pharmaceutical composition of embodiment 43, for the prevention or treatment of GPR75-related diseases and / or conditions.

[0463] 69. A dsRNA active agent or pharmaceutical composition or combination of drugs for use in embodiment 68, wherein the GPR75-related disease and / or condition is obesity or lipid metabolism-related disease; preferably, the obesity is selected from overweight, obese, severely obese or extremely obese, covering obesity.

[0464] 70. A method for weight management in a subject, such as weight reduction, body fat reduction or control of weight gain, and / or improvement of obesity-related metabolic status, using any dsRNA active agent of any of embodiments 1-39, or a pharmaceutical composition of embodiment 41 or 42, or a pharmaceutical composition of embodiment 43.

[0465] 71. A dsRNA active agent or pharmaceutical composition or combination of drugs for use in embodiment 70, wherein the subject has obesity or lipid metabolism-related diseases; preferably, the obesity is selected from overweight, obese, severely obese or extremely obese, covering obesity.

[0466] 72. A dsRNA active agent or pharmaceutical composition or combination of drugs for use in any of embodiments 68-71, wherein administration of said dsRNA active agent to a subject results in a decrease in GPR75 concentration or content (e.g., in target tissues or target cells), or results in a decrease in GPR75 protein accumulation or content in the subject (e.g., in target tissues or target cells); or results in a decrease in weight, a decrease in body fat, control of weight gain, and / or improvement in obesity-related metabolic status in the subject.

[0467] 73. A dsRNA active agent or pharmaceutical composition or combination of drugs for use in embodiment 72, wherein the target tissue is tissue of the central nervous system, preferably brain tissue or neurons, more preferably the cortex or cerebellum, most preferably tissue of the cerebral cortex, hypothalamus, hippocampus and other brain regions; spinal tissue, preferably cervical, lumbar and thoracic vertebrae; muscle tissue, preferably skeletal muscle tissue or cardiac tissue; ocular tissue or adipose tissue; or the target cells are cells derived from the target tissue.

[0468] 74. A dsRNA active agent or pharmaceutical composition or combination of drugs for use in embodiment 72 or 73, wherein the target tissue is selected from tissues of the central nervous system, preferably brain tissue or neurons; or muscle tissue, preferably skeletal muscle tissue or myocardial tissue; or the target cells are selected from cells of the central nervous system, preferably brain cells or neuronal cells; or muscle cells, preferably skeletal muscle cells or myocardial cells.

[0469] 75. A dsRNA active agent or pharmaceutical composition or combination of drugs for use in any of embodiments 68-74, wherein the drug is administered in combination with one or more other therapeutic agents, preferably selected from any therapeutic agents effective in preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), covering a variety of therapeutic agents for weight management, for reducing weight or body fat, or for preventing or treating obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, covering obesity) or lipid metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions).

[0470] 76. A dsRNA active agent or pharmaceutical composition or combination of drugs for use in any of embodiments 68-75, wherein the prevention or treatment of administration of said dsRNA active agent or pharmaceutical composition or combination of drugs further includes determining the level of GPR75 in a sample from said subject.

[0471] 77. A kit comprising the dsRNA activator of any one of embodiments 1-39, or the pharmaceutical composition of embodiment 41 or 42, or the pharmaceutical composition of embodiment 43.

[0472] 78. A vial or syringe comprising a dsRNA active agent of any one of embodiments 1-39, or a pharmaceutical composition of embodiment 41 or 42, or a pharmaceutical composition of embodiment 43.

[0473] 79. An RNA-induced silencing complex (RISC) comprising the antisense strand of any one of the dsRNA activators according to any one of embodiments 1 to 39. Example

[0474] Example 1: siRNA Synthesis

[0475] RNA solid-phase synthesis is a commonly used technique for synthesizing RNA molecules, allowing for the stepwise construction of RNA chains on a solid support. This method is characterized by high throughput, high efficiency, and automation, and is widely used in biotechnology and research fields.

[0476] The following describes the synthesis of the GPR75 RNAi agent duplexes shown in Table 2 according to the following method: All RNA and 2'-modified phosphoramidene were purchased from Shanghai Zhaowei Technology Development Co., Ltd. Specifically, the following 2'-O-methylphosphoramidenes were used: (5'-O-dimethoxytriphenylmethyl-N6-(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidene, (5'-O-dimethoxytriphenylmethyl-N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidene, (5'-O-dimethoxytriphenylmethyl-N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidene, (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.

[0477] 1. Synthesis of the Justice Chain (SS Chain)

[0478] The oligonucleotide synthesis method employed a solid-phase phosphoramide approach, using a blank CPG solid support as the starting cycle. Nucleoside monomers (including VPUms, Ahd, and Uhd) or nucleotide analog monomers were sequentially ligated from the 3'-5' direction according to the nucleotide arrangement of the positive strand. Each ligation of a nucleoside monomer or nucleotide analog monomer involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for oligonucleotides at a scale of 5 μmol are as follows:

[0479] 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).

[0480] 2. Synthesis of the antisense chain (AS chain)

[0481] The solid-phase phosphoramide synthesis method utilizes a blank CPG solid-phase support as the starting cycle, sequentially linking nucleoside monomers (including nucleoside monomers VPUms, Ahd, and Uhd) or nucleotide analog monomers from the 3'-5' direction according to the antisense strand nucleotide arrangement sequence. Each linking 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.

[0482] 3. Purification and Annealing of Oligonucleotides

[0483] 3.1 Ammonolysis

[0484] 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.

[0485] 3.2 Purification

[0486] 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.

[0487] 3.3 Annealing

[0488] 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.

[0489] Table 1: Sensitive and antisense strands of unmodified GPR75 dsRNA (siRNA)

[0490] Table 2: Sensitive and antisense strands of GPR75-modified dsRNA (siRNA)

[0491] *The sequence list shows the unmodified bare sequences. For the sequences corresponding to the SEQ ID NO, please refer to this table.

[0492] Table 3: Sensitive and antisense strands of GPR75 dsRNA (siRNA) with delivery modification (conjugated with fatty chains) (in vivo verification)

[0493] *The sequence listing shows the unmodified and unconjugated bare sequences. For the sequences corresponding to the SEQ ID NO, please refer to this table.

[0494] The structural formulas and preparation methods of VPUms and Nhd, such as Ahd and Uhd, are shown in WO2019217459A1:

[0495] The structure of VPUms is as follows:

[0496] The structural formulas for Nhd, such as Ahd and Uhd, are as follows:

[0497] Example 2: Construction of a human GPR75-overexpressing HEK293T cell line

[0498] To screen and verify the knockdown effect of GPR75 siRNA on GPR75 mRNA, we constructed a HEK293T cell line overexpressing the human GPR75 gene and transfected it with different concentrations of GPR75 siRNA. The remaining level of GPR75 mRNA was detected by real-time PCR.

[0499] 1. Packaging of human GPR75 lentivirus: HEK293T cells (Nanjing Kebai, Cat#CBP60439) were cultured in DMEM medium (ATCC, Cat#30-2002) containing 10% fetal bovine serum at 37°C and 5% CO2. After reaching 80% cell coverage, transfection was performed, and the medium was replaced with serum-free DMEM medium. The CDS region sequence of human GPR75 (NM_006794.4) was inserted into a lentiviral vector to construct a plasmid (Witty Gene, order number 80-1625439648). Transfection was then performed using PEI (polyplus, Cat#115-100) reagent, as follows: 14 μg psPAX2 (Witty Gene, order number 80-514048504), 7 μg pDM2.G (Witty Gene, order number 80-448575734), and 14 μg human GPR75 lentiviral plasmid were added to 500 μl Opti-MEM medium (GIBCO, Cat#31985070) to prepare a plasmid mixture. 105 μl PEI reagent was added to 500 μl Opti-MEM medium to prepare a PEI solution. PEI solution was slowly added to the plasmid mixture at a 1:1 ratio and incubated at room temperature for 20 min. This mixture was then added to HEK293T cells that had been replaced with new medium. After 6 hours of transfection, DMEM medium containing 4% fetal bovine serum was added, and the cells were cultured for another 48 hours. The supernatant was then collected. The supernatant was centrifuged at 500g for 10 min to remove the cell pellet, and filtered through a 0.45 μM PES membrane (Sartorius, Cat#16533K) into 50 mL centrifuge tubes for virus concentration. The specific method for virus concentration was as follows: Lenti-X™ Concentrator (TaKaRa, Cat#631232) was added at a 3:1 ratio, and the mixture was thoroughly mixed by inversion and incubated overnight at 4°C. The virus and Lenti-X concentrator mixture was centrifuged at 1500g for 45 min at 4°C, the supernatant was removed, and the cells were resuspended in 300 μl of serum-free DMEM. The mixture was then aliquoted and stored at -80°C.

[0500] 2. Lentiviral Infection: HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum until the cell coverage reached 60-70% before infection. 50 μl of concentrated lentivirus was mixed with 1.2 ml of DMEM medium containing 10% fetal bovine serum. The original HEK293T cell culture medium was then replaced with DMEM medium containing lentivirus, and 5 μg / ml polybrene (Solepro, Cat#H8761) was added to improve infection efficiency. After 6 hours of lentivirus infection, 1 ml of DMEM medium containing 10% fetal bovine serum was added, and the cells were cultured for another 24 hours. The medium was then replaced with fresh DMEM medium containing 10% fetal bovine serum, and cultured for another 24 hours. Finally, 1 μg / ml puromycin (Gibco, Cat#A11138-02) was added for stress selection to obtain HEK293T cell lines overexpressing the human GPR75 gene.

[0501] Example 3: Inhibition of human GPR75 mRNA levels in HEK293T cells overexpressing the human GPR75 gene by 10 nM siRNA

[0502] To verify the function of the synthesized GPR75 siRNA, we first transfected human GPR75-overexpressing HEK293T cells with a high concentration of 10 nM GPR75 siRNA for preliminary screening, and then selected the siRNA sequence with the best knockdown effect for further verification and screening.

[0503] HEK293T cells overexpressing human GPR75 were transfected with 10 nM siRNA:

[0504] HEK293T cells overexpressing GPR75 were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum.

[0505] In Cat#12430054, cultured at 37°C and 5% CO2. siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat#13778150), as follows:

[0506] The siRNA was prepared into a 500 nM working solution using DEPC water. Solution A was prepared, with each aliquot containing 2 μl of siRNA working solution and 8 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. Solutions A and B were mixed and incubated in 96-well plates at room temperature for 20 min. Then, 80 μl of HEK293-hGPR75 cells were added, with 30,000 cells per well, resulting in a final siRNA concentration of 10 nM. 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 the GPR75 mRNA level and the GAPDH internal control mRNA level. GPR75 mRNA levels were corrected for based on GAPDH internal control mRNA levels. Human GPR75 mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of remaining GPR75 mRNA expression relative to cells treated with negative siRNA control. The calculation formula is as follows:

[0507] Ct = Ct(target gene) - Ct(internal reference gene)

[0508] △△Ct=△Ct(drug-treated group)-△Ct(negative siRNA control group PBS)

[0509] GPR75 mRNA relative expression level = 2 -△△Ct ×100%

[0510] The test results are summarized in Table 4.

[0511] Table 4 Results of in vitro 10 nM screening tests of the compounds disclosed herein

[0512] AD1424520, AD1424911, AD1423792, and AD1425165 all originate from WO2022076291A1.

[0513] Example 4: Inhibitory effect of 1 nM and 0.1 nM siRNA on human GPR75 mRNA levels in HEK293T cells overexpressing the human GPR75 gene.

[0514] To further validate and screen siRNA molecules that can be stably knocked down, 28 siRNA sequences were further validated and screened at low concentrations of siRNA, namely 1 nM and 0.1 nM. HEK293 cells overexpressing human GPR75 were transfected with 1 nM and 0.1 nM siRNA:

[0515] HEK293T cells overexpressing GPR75 were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum.

[0516] In Cat#12430054, cultured at 37°C and 5% CO2. siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat#13778150), as follows:

[0517] siRNA was prepared into 50 nM and 5 nM working solutions using DEPC water. Solution A was prepared, with each aliquot containing 2 μl of siRNA working solution and 8 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. Solutions A and B were mixed and incubated in 96-well plates at room temperature for 20 min. Then, 80 μl of HEK293T-hGPR75 cells were added, with 30,000 cells per well, resulting in final siRNA concentrations of 1 nM and 0.1 nM, respectively. 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 GPR75 mRNA levels and GAPDH internal control mRNA levels. GPR75 mRNA levels were corrected for based on GAPDH internal control mRNA levels. Human GPR75 mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of remaining GPR75 mRNA expression relative to cells treated with negative siRNA control. The calculation formula is as follows:

[0518] Ct = Ct(target gene) - Ct(internal reference gene)

[0519] △△Ct=△Ct(drug-treated group)-△Ct(negative siRNA control group PBS)

[0520] GPR75 mRNA relative expression level = 2 -△△Ct ×100%

[0521] The test results are summarized in Table 5.

[0522] Table 5. Results of in vitro screening tests of the disclosed compounds at 1 nM and 0.1 nM.

[0523] Example 5: Dosage curve experiment of GPR75 siRNA in HEK293T cells overexpressing human GPR75.

[0524] To further validate and screen siRNA molecules that can be stably knocked down, we selected 7 siRNA sequences based on the screening results of 1 nM and 0.1 nM and conducted dose-curving experiments and in vivo validation experiments.

[0525] Dosage curve of siRNA transfection in HEK293 cells overexpressing human GPR75:

[0526] HEK293T cells overexpressing GPR75 were cultured in DMEM medium (Gibco, Cat#12430054) containing 10% fetal bovine serum at 37°C and 5% CO2. siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat#13778150) as follows:

[0527] siRNA was prepared into working solutions with different concentration gradients using DEPC water (2500, 1000, 500, 50, 5, 0.5, 0.05, 0.005, 0.0005, 0.00005, 0.000005, and 0.0000005 nM). Solution A was prepared, with each aliquot containing 2 μl of siRNA working solution and 8 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 HEK293T-hGPR75 cells were added, with 30,000 cells per well. The final siRNA concentrations were 50, 20, 10, 1, 0.1, 0.001, 0.0001, 0.00001, 0.000001, 0.0000001, and 0.00000001 nM. Twenty-four hours after transfection, cellular RNA was extracted using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat#CL132). Reverse transcription and qPCR were performed according to the kit instructions to determine GPR75 mRNA levels and GAPDH internal control mRNA levels. GPR75 mRNA levels were corrected based on the GAPDH internal control mRNA levels. Human GPR75 mRNA expression levels were calculated using the ΔΔCt relative quantification method, expressed as the percentage of remaining GPR75 mRNA expression in cells treated with negative siRNA control group. The calculation formula is as follows:

[0528] Ct = Ct(target gene) - Ct(internal reference gene)

[0529] △△Ct=△Ct(drug-treated group)-△Ct(negative siRNA control group PBS)

[0530] GPR75 mRNA relative expression level = 2 -△△Ct ×100%

[0531] The test results are shown in Table 6 and Figure 1.

[0532] Among these, the CDS region is generally more conserved across different transcripts compared to the 3'UTR region. siRNA drugs targeting this region tend to exhibit more stable efficacy across different populations, while targeting the UTR region may result in varying therapeutic effects. Therefore, siRNA drug development tends to focus on the CDS region. While XD001005 targets the 3'UTR region of the GPR75 gene transcript, its IC50 in in vitro dose-response functional experiments was the best among all sequences, comparable to the Alnylam bench mark. XD000957 targets the CDS region of the GPR75 gene transcript, potentially offering more stable efficacy across different populations. Furthermore, XD000957 at low doses (<10) -3 Its knockdown performance is also better than benchmark when it reaches nM.

[0533] Table 6. Dose curve results of in vitro screening of the compounds disclosed herein at different concentrations.

[0534] Example 6: Knockdown efficiency of GPR75 siRNA on GPR75 gene in brain tissue of humanized mice

[0535] To evaluate the effect of GPR75-targeting siRNA on reducing GPR75 mRNA levels in vivo, on day 0, GPR75 humanized mice (Biocytogen) were injected intraventricularly with a single dose of 150 μg / mouse of double-stranded XD001005.1 or a positive control. At the same time, one group of negative control mice (6 mice) were given a normal diet (chow diet, CD), while the other experimental groups (8 mice / group) were given a 60% high-fat diet (high fat diet, HFD, Research Diets, Cat. No: D12492).

[0536] Animals were sacrificed on day 38 after administration, and samples were collected from the cerebral cortex, hypothalamus, hippocampus, and other brain regions. RNA was extracted from different sites using Freezol reagent (R711-02, Vazyme) and a universal total RNA extraction kit using magnetic beads (ROA3303-01, Vazyme), and the level of GPR75 mRNA was quantified by RT-qPCR (Q226-01, Vazyme).

[0537] The percentage of remaining GPR75 mRNA expression relative to the PBS + normal diet (CD) group mice is expressed as follows:

[0538] Ct = Ct(target gene) - Ct(internal reference gene)

[0539] △△Ct=△Ct(drug-treated group)-△Ct(PBS+CD group)

[0540] GPR75 mRNA relative expression level = 2 -△△Ct ×100%

[0541] As shown in Figure 2, administration of XD001005.1 resulted in a significant reduction in GPR75 expression in the hypothalamus and other brain tissues, ranging from 0.72 to 0.86 times the expression level detected in control siRNA-administered mice.

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, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of a target sequence, and the complementary region contains a mismatch of no more than 3, 2, or 1 nucleotide with the at least 15 consecutive nucleotides of the target sequence, wherein the target sequence is selected from... (i) The target sequence corresponding to positions 1349-1369 or 1984-2004 of GPR75 mRNA; (ii) The target sequence shown in SEQ ID NO:280 or SEQ ID NO:241; (iii) The target sequence shown in any one of SEQ ID NO: 191-285; or (iv) The target sequences corresponding to the positions in GPR75 mRNA, such as NM_006794.4, shown in Table 1; Optionally, the dsRNA activator is used to inhibit the expression of a gene encoding GPR75.

2. The dsRNA activator of claim 1, 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 the mRNA of GPR75, and comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differing by no more than 3, 2, or 1 nucleotide from any antisense nucleotide sequence in the antisense strands in Table 1 or any antisense nucleotide sequence shown in SEQ ID NO:96-190.

3. 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 GPR75 mRNA, and comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differing by no more than 3, 2, or 1 nucleotide from any antisense nucleotide sequence in the antisense strands listed in Table 1 or any antisense nucleotide sequence shown in SEQ ID NO:96-190; Optionally, the dsRNA activator is used to inhibit the expression of a gene encoding GPR75.

4. The dsRNA activator according to any one of claims 1 to 3, wherein the nucleotide sequence of the antisense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ from the nucleotide sequence shown in SEQ ID NO:185 or SEQ ID NO:146 by no more than 3, 2 or 1 nucleotides.

5. The dsRNA activator according to any one of claims 1 to 4, wherein the antisense strand comprises a complementary region of 15 to 30 nucleotides or 18 to 23 nucleotides in length to the target sequence, for example, the complementary region is 15, 16, 17, 18, 19, 20 or 21 nucleotides in length.

6. The dsRNA activator according to any one of claims 1 to 5, wherein the complementary region comprises at least nucleotides 2-16, 2-17, 2-18, 2-19, 2-20, or 2-21, for example, nucleotides 2-19, 2-20, or 2-21, starting from the 5' end of the antisense strand.

7. The dsRNA activator according to any one of claims 1 to 6, wherein the antisense strand has the same number of nucleotides as the target sequence and is completely complementary to the target sequence except for the first nucleotide at the 5' end, or the antisense strand is completely complementary to the target sequence.

8. The dsRNA activator according to any one of claims 1 to 7, wherein the first nucleotide at the 5' end of the antisense strand is A or U, for example, U.

9. The dsRNA activator according to any one of claims 1 to 8, 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, 2 or 1 nucleotides from any nucleotide sequence of the sense strand in Table 1 or any sense nucleotide sequence shown in SEQ ID NO:1-95.

10. The dsRNA activator according to any one of claims 1 to 8, 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:51 or SEQ ID NO:90 by no more than 3 nucleotides.

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

12. The dsRNA activator according to any one of claims 1 to 11, wherein the length of the fully complementary double-stranded region is between 15 and 25 nucleotide pairs, 16 and 24 nucleotide pairs, 17 and 23 nucleotide pairs, or 18 and 22 nucleotide pairs, for example, the length of the fully complementary double-stranded region is 15, 16, 17, 18, or 19 nucleotide pairs, for example, 19 nucleotide pairs.

13. The dsRNA activator according to any one of claims 1 to 12, 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.

14. The dsRNA activator according to any one of claims 1 to 13, 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.

15. The dsRNA activator according to any one of claims 1 to 14, 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 GPR75 mRNA in the region excluding 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; 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.

16. 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 nucleotide sequence of the sense strand comprising a nucleotide sequence selected from any one of the nucleotide sequences of the sense strand in Table 1, and / or, the nucleotide sequence of the antisense strand comprising a nucleotide sequence selected from any one of the nucleotide sequences of the antisense strand in Table 1.

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

18. The dsRNA activator according to any one of claims 1 to 17, wherein the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand each comprise the nucleotide sequences shown in SEQ ID NO:90 / SEQ ID NO:185; or each comprises the nucleotide sequences shown.

19. The dsRNA activator according to any one of claims 1 to 17, wherein the dsRNA activator comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand each comprise the nucleotide sequences shown in SEQ ID NO:51 / SEQ ID NO:146; or each comprises the nucleotide sequences shown.

20. The dsRNA activator according to any one of claims 1 to 19, wherein the dsRNA activator comprises at least one modified nucleotide, optionally wherein 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.

21. The dsRNA activator according to any one of claims 1 to 20, wherein all nucleotides of the sense strand are modified nucleotides; or 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.

22. The dsRNA activator according to claim 20 or 21, 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'-methoxy 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 (2'-O-hexadecyl 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.

23. The dsRNA activator according to any one of claims 1 to 22, wherein the antisense strand comprises a phosphate thioester bond, and / or the sense strand comprises a phosphate thioester bond. Choose one of them The thiophosphate nucleotide bond is located between the 1st and 3rd nucleotides at the 5' end of the sense strand, and the thiophosphate nucleotide bond is located between the 1st and 3rd nucleotides at the 5' end and the 1st and 3rd nucleotides at the 3' end of the antisense strand; or The thiophosphate nucleotide inter-links are located between the 1st and 3rd nucleotides at the 5' end and the 1st and 3rd nucleotides at the 3' end of the sense strand, and the thiophosphate nucleotide inter-links are located between the 1st and 3rd nucleotides at the 5' end and the 1st and 3rd nucleotides at the 3' end of the antisense strand.

24. The dsRNA activator according to any one of claims 1 to 23, wherein the nucleotides at positions 2, 6, 14 and 16 of the antisense strand, counting from the 5' end, are 2'-fluorinated nucleotides, and the nucleotides at positions 7-9 of the sense strand, counting from the 5' end, are 2'-fluorinated nucleotides.

25. The dsRNA activator according to any one of claims 1 to 24, wherein the antisense strand has nucleotides 1, 3-5, 7-13, 15, and 17-21 from the 5' end that are 2'-methoxy modified nucleotides, and the sense strand has nucleotides 1-6 and 10-19 from the 5' end that are 2'-methoxy modified nucleotides; or the antisense strand has nucleotides 1, 3-5, 7-13, 15, and 17-21 from the 5' end that are 2'-methoxy modified nucleotides, and the sense strand has nucleotides 1-5 and 10-19 from the 5' end that are 2'-methoxy modified nucleotides.

26. The dsRNA activator according to any one of claims 1 to 25, wherein the 5' nucleotide of the antisense strand has a 5' phosphate ester modification, such as 5'-vinylphosphonate (5'-VP) or 5'-(E)-vinylphosphonate (5'-(E)-VP).

27. The dsRNA activator according to any one of claims 1 to 26, wherein the modified nucleotides in the sense and antisense strands have the following modification pattern: antisense chain: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm; and / or Chain of Justice: NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm, The symbols mentioned herein are as defined herein. Nf = any 2'-fluorine modified nucleotide Nfs = any 2'-fluorine modified nucleoside-3' thiophosphate; Nm = any 2'-methoxynucleotide; Nms = any 2'-methoxynucleoside-3'-thiophosphate; 's' indicates that the two nucleotides are linked by a phosphate thioester bond.

28. The dsRNA activator according to any one of claims 1 to 26, wherein the modified nucleotides in the sense and antisense strands have the following modification pattern: antisense chain: VPNmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm; and / or Chain of Justice: NmsNmsNmNmNmNNfNfNfNmNmNmNmNmNmNmNmNmsNmsNm, in, Nf = any nucleotide modified with 2'-fluorine; Nfs = any 2'-fluorine modified nucleoside-3' thiophosphate; Nm = any 2'-methoxynucleotide; Nms = any 2'-methoxynucleoside-3'-thiophosphate; N = any form of nucleotide; s indicates that the two nucleotides are linked by a phosphate thioester bond; VP indicates that the 5' end nucleotide of the antisense strand has a 5'-vinyl phosphonate (5'-VP) modification, such as 5'-(E)-VP modification; For example, the 5' end nucleotide VPNms of the antisense strand can be VPUms, which has the following structure: Preferably, the nucleotide at position 6, counting from the 5' end of the positive strand, is further conjugated with an adipose chain.

29. The dsRNA activator according to any one of claims 1 to 28, wherein the antisense strand comprises any modified nucleotide sequence of the antisense strands in Table 2, and / or the sense strand comprises any modified nucleotide sequence of the sense strands in Table 2; optionally, the dsRNA activator comprises a combination of modified antisense strands and sense strands as shown in Table 2.

30. The dsRNA activator according to any one of claims 1 to 29, wherein the sense strand and antisense strand respectively comprise SEQ ID NO:377 / SEQ ID NO:472, SEQ ID NO:338 / SEQ ID NO:433, SEQ ID NO:288 / SEQ ID NO:383, SEQ ID NO:289 / SEQ ID NO:384, SEQ ID NO:290 / SEQ ID NO:385, SEQ ID NO:291 / SEQ ID NO:386, SEQ ID NO:292 / SEQ ID NO:387, SEQ ID NO:293 / SEQ ID NO:388, SEQ ID NO:294 / SEQ ID NO:389, SEQ ID NO:295 / SEQ ID NO:390, SEQ ID NO:296 / SEQ ID NO:391, SEQ ID NO:297 / SEQ ID NO:392, SEQ ID NO:298 / SEQ ID NO:393, SEQ ID NO:299 / SEQ ID NO:394, SEQ ID NO:300 / SEQ ID NO:395, SEQ ID NO:301 / SEQ ID NO:396, SEQ ID NO:302 / SEQ ID NO:397, SEQ ID NO:303 / SEQ ID NO:398, SEQ ID NO:304 / SEQ ID NO:399, SEQ ID NO:305 / SEQ ID NO:400, SEQ ID NO:306 / SEQ ID NO:401, SEQ ID NO:307 / SEQ ID NO:402, SEQ ID NO:308 / SEQ ID NO:403, SEQ ID NO:309 / SEQ ID NO:404, SEQ ID NO:310 / SEQ ID NO:405, SEQ ID NO:311 / SEQ ID NO:406, SEQ ID NO:312 / SEQ ID NO:407, SEQ ID NO:313 / SEQ ID NO:408, SEQ ID NO:314 / SEQ ID NO:409, SEQ ID NO:315 / SEQ ID NO:410, SEQ ID NO:316 / SEQ ID NO:411, SEQ ID NO:317 / SEQ ID NO:412, SEQ ID NO:318 / SEQ ID NO:413, SEQ ID NO:319 / SEQ ID NO:414, SEQID NO:320 / SEQ ID NO:415、SEQ ID NO:321 / SEQ ID NO:416、SEQ ID NO:322 / SEQ ID NO:417、SEQ ID NO:323 / SEQ ID NO:418、SEQ ID NO:324 / SEQ ID NO:419、SEQ ID NO:325 / SEQ ID NO:420、SEQ ID NO:326 / SEQ ID NO:421、SEQ ID NO:327 / SEQ ID NO:422、SEQ ID NO:328 / SEQ ID NO:423、SEQ ID NO:329 / SEQ ID NO:424、SEQ ID NO:330 / SEQ ID NO:425、SEQ ID NO:331 / SEQ ID NO:426、SEQ ID NO:332 / SEQ ID NO:427、SEQ ID NO:333 / SEQ ID NO:428、SEQ ID NO:334 / SEQ ID NO:429、SEQ ID NO:335 / SEQ ID NO:430、SEQ ID NO:336 / SEQ ID NO:431、SEQ ID NO:337 / SEQ ID NO:432、SEQ ID NO:339 / SEQ ID NO:434、SEQ ID NO:340 / SEQ ID NO:435、SEQ ID NO:341 / SEQ ID NO:436、SEQ ID NO:342 / SEQ ID NO:437、SEQ ID NO:343 / SEQ ID NO:438、SEQ ID NO:344 / SEQ ID NO:439、SEQ ID NO:345 / SEQ ID NO:440、SEQ ID NO:346 / SEQ ID NO:441、SEQ ID NO:347 / SEQ ID NO:442、SEQ ID NO:348 / SEQ ID NO:443、SEQ ID NO:349 / SEQ ID NO:444、SEQ ID NO:350 / SEQ ID NO:445、SEQ ID NO:351 / SEQ ID NO:446、SEQ ID NO:352 / SEQ ID NO:447、SEQ ID NO:353 / SEQ ID NO:448、SEQ ID NO:354 / SEQ ID NO:449、SEQ ID NO:355 / SEQ ID NO:450、SEQ ID NO:356 / SEQ IDNO:451, SEQ ID NO:357 / SEQ ID NO:452, SEQ ID NO:358 / SEQ ID NO:453, SEQ ID NO:359 / SEQ ID NO:454, SEQ ID NO:360 / SEQ ID NO:455, SEQ ID NO:361 / SEQ ID NO:456, SEQ ID NO:362 / SEQ ID NO:457, SEQ ID NO:363 / SEQ ID NO:458, SEQ ID NO:364 / SEQ ID NO:459, SEQ ID NO:365 / SEQ ID NO:460, SEQ ID NO:366 / SEQ ID NO:461, SEQ ID NO:367 / SEQ ID NO:462, SEQ ID NO:368 / SEQ ID NO:463, SEQ ID NO:369 / SEQ ID NO:464, SEQ ID NO:370 / SEQ ID NO:465, SEQ ID NO:371 / SEQ ID The modified nucleotide sequences shown in SEQ ID NO:466, SEQ ID NO:372 / SEQ ID NO:467, SEQ ID NO:373 / SEQ ID NO:468, SEQ ID NO:374 / SEQ ID NO:469, SEQ ID NO:375 / SEQ ID NO:470, SEQ ID NO:376 / SEQ ID NO:471, SEQ ID NO:378 / SEQ ID NO:473, SEQ ID NO:379 / SEQ ID NO:474, SEQ ID NO:380 / SEQ ID NO:475, SEQ ID NO:381 / SEQ ID NO:476 or SEQ ID NO:382 / SEQ ID NO:477, or each of the modified nucleotide sequences shown. Optionally, it also has one or more of the following modifications: the first nucleotide at the 5' end of the antisense strand is modified with 5'-vinylphosphonate (5'-VP), for example, 5'-(E)-VP; for example, VPUm or VPAm; the 6th nucleotide from the 5' end of the sense strand is conjugated with an aliphatic chain, such as a hydrocarbon group or hydrocarbon chain, which can serve as a ligand, for example, C 16 -C 22 Hydrocarbon chains, such as those containing C 16 -C 22 Nucleoside monomers with saturated hydrocarbon chains, such as Uhd, Chd, Ahd, or Ghd; and / or phosphate thioester bonds between the first to third nucleotides starting from the 3' end of the sense chain.

31. The dsRNA activator according to any one of claims 1 to 30, wherein the sense strand and the antisense strand respectively comprise the nucleotide sequences shown in SEQ ID NO:338 / SEQ ID NO:433 or are respectively composed of the modified nucleotide sequences shown.

32. The dsRNA activator according to any one of claims 1 to 30, wherein the sense strand and antisense strand respectively comprise the nucleotide sequences shown in SEQ ID NO:377 / SEQ ID NO:472 or are respectively composed of the modified nucleotide sequences shown.

33. The dsRNA activator according to any one of claims 1 to 32, wherein it is conjugated to a ligand capable of targeted delivery to a target tissue or target cell, preferably, the target tissue being tissue of the central nervous system, preferably brain tissue or neurons, more preferably the cortex or cerebellum, most preferably tissue of the cerebral cortex, hypothalamus, hippocampus and other brain regions; spinal tissue, preferably cervical, lumbar and thoracic vertebrae; muscle tissue, preferably skeletal muscle tissue or cardiac tissue; ocular tissue or adipose tissue; or the target cell being a cell derived from the target tissue.

34. The dsRNA activator according to claim 33, wherein the target tissue is selected from tissues of the central nervous system, preferably brain tissue or neurons; or muscle tissue, preferably skeletal muscle tissue or myocardial tissue; or the target cells are selected from cells of the central nervous system, preferably brain cells or neuronal cells; or muscle cells, preferably skeletal muscle cells or myocardial cells.

35. The dsRNA activator according to any one of claims 1 to 34, wherein one or two chains of the dsRNA activator are conjugated to a C16-C22 hydrocarbon chain, for example, one or more C16-C22 hydrocarbon chains (e.g., saturated or unsaturated) are conjugated to one or more internal positions of at least one chain of the dsRNA activator, for example, one or more C16 hydrocarbon chains (e.g., saturated or unsaturated) are conjugated to one or more internal positions of at least one chain of the dsRNA activator.

36. The dsRNA activator of claim 35, wherein the C16-C22 hydrocarbon chain (e.g., saturated or unsaturated) is conjugated to the 6th nucleotide from the 5' end of the positive strand of the dsRNA activator.

37. The dsRNA activator according to claim 36, wherein the modified nucleotides in the sense and antisense strands have the following pattern: antisense chain: VPNmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm; and Chain of Justice: NmsNmsNmNmNmNhdNfNfNfNmNmNmNmNmNmNmNmNmsNmsNm, in, Nf = any 2'-fluorine modified nucleotide Nfs = any 2'-fluorine modified nucleoside-3' thiophosphate; Nm = any 2'-methoxynucleotide; Nms = any 2'-methoxynucleoside-3'-thiophosphate; Nhd = any nucleotide conjugated with 2′-O-hexadecyl; s indicates that the two nucleotides are linked by a phosphate thioester bond; VP indicates that the 5' end nucleotide of the antisense strand has a 5'-vinyl phosphonate (5'-VP) modification, such as 5'-(E)-VP modification; For example, the 5' end nucleotide VPNms of the antisense strand can be VPUms, which has the following structure:

38. The dsRNA activator according to claim 36, wherein the sense strand and antisense strand respectively comprise the nucleotide sequences shown in SEQ ID NO:478 / SEQ ID NO:480 or are respectively composed of the modified nucleotide sequences shown.

39. The dsRNA activator according to any one of claims 1 to 38, wherein the dsRNA activator is in the form of a salt, a mixed salt, or a free acid.

40. A cell containing a dsRNA activator according to any one of claims 1 to 39.

41. A pharmaceutical composition comprising a dsRNA activator according to any one of claims 1 to 39 and a pharmaceutically acceptable carrier.

42. The pharmaceutical composition of claim 41, wherein the dsRNA activator is in a non-buffered 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).

43. A pharmaceutical combination comprising a dsRNA activator according to any one of claims 1 to 39 and one or more other therapeutic agents, preferably, said other therapeutic agents being selected from any therapeutic agents effective in preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), covering a variety of therapeutic agents for weight management, for reducing weight or body fat, or for preventing or treating obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, covering obesity) or lipid metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions).

44. A method for inhibiting the expression of the GPR75 gene in cells, the method comprising contacting the cells with a dsRNA activator according to any one of claims 1 to 39, or a pharmaceutical composition according to claim 41 or 42, or a pharmaceutical composition according to claim 43, thereby inhibiting the expression of the GPR75 gene in the cells.

45. The method of claim 44, wherein the cells are in a subject, optionally the subject being a mammal, such as a human.

46. ​​The method of claim 45, wherein the subject suffers from a GPR75-related disease and / or condition.

47. The method of claim 46, wherein the GPR75-related disease and / or condition is obesity or a lipid metabolism-related disease; preferably, the obesity is selected from overweight, obese, severely obese, or extremely obese, and covers obesity.

48. The method according to any one of claims 45-47, wherein the subject needs to undergo weight management, such as reducing weight, reducing body fat or controlling weight gain, and / or improving obesity-related metabolic status.

49. The method according to any one of claims 44 to 48, wherein contacting the cells with the dsRNA activator inhibits GPR75 expression by at least 15%, 20%, 25%, 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%, or about 92%.

50. A method for preventing or treating GPR75-related diseases and / or conditions in a subject, comprising administering to the subject a dsRNA active agent of any one of claims 1-39, or a pharmaceutical composition of claim 41 or 42, or a pharmaceutical composition of claim 43.

51. The method of claim 50, wherein the GPR75-related disease and / or condition is obesity or a lipid metabolism-related disease; preferably, the obesity is selected from overweight, obese, severely obese, or extremely obese, encompassing obesity.

52. A method for weight management in a subject, such as reducing weight, reducing body fat, or controlling weight gain, and / or improving obesity-related metabolic status, comprising administering to the subject a dsRNA active agent of any one of claims 1-39, or a pharmaceutical composition of claim 41 or 42, or a pharmaceutical composition of claim 43.

53. The method of claim 52, wherein the subject has obesity or a lipid metabolism-related disease; preferably, the obesity is selected from overweight, obese, severely obese or extremely obese, covering obesity.

54. The method of any one of claims 50-53, wherein administration of the dsRNA activator to the subject results in a decrease in GPR75 concentration or content (e.g., in target tissues or target cells), or a decrease in GPR75 protein accumulation or content in the subject (e.g., in target tissues or target cells); or a decrease in the subject's weight, body fat, control of weight gain, and / or improvement in obesity-related metabolic status.

55. The method of claim 54, wherein the target tissue is tissue of the central nervous system, preferably brain tissue or neurons, more preferably cortex or cerebellum, most preferably tissue of the cerebral cortex, hypothalamus, hippocampus and other brain regions; spinal tissue, preferably cervical, lumbar and thoracic vertebrae; muscle tissue, preferably skeletal muscle tissue or myocardial tissue; eye tissue or adipose tissue; or the target cells are cells derived from the target tissue.

56. The method of claim 54 or 55, wherein the target tissue is selected from tissues of the central nervous system, preferably brain tissue or neurons; or muscle tissue, preferably skeletal muscle tissue or myocardial tissue; or the target cells are selected from cells of the central nervous system, preferably brain cells or neuronal cells; or muscle cells, preferably skeletal muscle cells or myocardial cells.

57. The method of any one of claims 44 to 56, further comprising administering to the subject one or more other therapeutic agents, preferably selected from any therapeutic agents effective in preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), covering a variety of therapeutic agents for weight management, for reducing weight or body fat, or for preventing or treating obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, covering obesity) or lipid metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions).

58. The method according to any one of claims 44 to 57, further comprising determining the level of GPR75 in a sample from the subject.

59. Use of the dsRNA activator of any one of claims 1-39, or the pharmaceutical composition of claim 41 or 42, or the pharmaceutical composition of claim 43, in the preparation of a medicament, wherein the medicament is used for the prevention or treatment of GPR75-related diseases and / or conditions.

60. The use of claim 59, wherein the GPR75-related disease and / or condition is obesity or a lipid metabolism-related disease; preferably, the obesity is selected from overweight, obese, severely obese, or extremely obese, and covers obesity.

61. Use of the dsRNA active agent of any one of claims 1-39, or the pharmaceutical composition of claim 41 or 42, or the pharmaceutical composition of claim 43, in the preparation of a medicament, wherein the medicament is used for weight management, such as reducing weight, reducing body fat, or controlling weight gain, and / or improving obesity-related metabolic conditions.

62. The use according to claim 61, wherein the subject has obesity or a lipid metabolism-related disease; preferably, the obesity is selected from overweight, obese, severely obese or extremely obese, covering obesity.

63. The use according to any one of claims 59-62, wherein administration of the dsRNA activator to the subject results in a decrease in GPR75 concentration or content (e.g., in target tissues or target cells), or a decrease in GPR75 protein accumulation or content in the subject (e.g., in target tissues or target cells); or a decrease in the subject's weight, body fat, control of weight gain, and / or improvement of obesity-related metabolic status.

64. The use according to claim 63, wherein the target tissue is tissue of the central nervous system, preferably brain tissue or neurons, more preferably cortex or cerebellum, most preferably tissue of the cerebral cortex, hypothalamus, hippocampus and other brain regions; spinal tissue, preferably cervical, lumbar and thoracic vertebrae; muscle tissue, preferably skeletal muscle tissue or cardiac tissue; eye tissue or adipose tissue; or the target cells are cells derived from the target tissue.

65. The use according to claim 63 or 64, wherein the target tissue is selected from tissues of the central nervous system, preferably brain tissue or neurons; or muscle tissue, preferably skeletal muscle tissue or myocardial tissue; or the target cells are selected from cells of the central nervous system, preferably brain cells or neuronal cells; or muscle cells, preferably skeletal muscle cells or myocardial cells.

66. The use of any one of claims 59 to 65, wherein the drug is administered in combination with one or more other therapeutic agents, preferably, the other therapeutic agents being selected from any therapeutic agents effective in preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), covering a variety of therapeutic agents for weight management, for reducing weight or body fat, or for preventing or treating obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, covering obesity) or lipid metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions).

67. The method according to any one of claims 59 to 66, wherein the prevention or treatment of administering the drug further comprises determining the level of GPR75 in a sample from the subject.

68. The dsRNA active agent of any one of claims 1-39, or the pharmaceutical composition of claim 41 or 42, or the pharmaceutical composition of claim 43, for the prevention or treatment of GPR75-related diseases and / or conditions.

69. A dsRNA active agent or pharmaceutical composition or combination of drugs for the use of claim 68, wherein the GPR75-related disease and / or condition is obesity or a lipid metabolism-related disease; preferably, the obesity is selected from overweight, obese, severely obese or extremely obese, covering obesity.

70. A method for weight management in a subject, such as weight loss, weight reduction, or control of weight gain, and / or improvement of obesity-related metabolic status, using the dsRNA active agent of any one of claims 1-39, or the pharmaceutical composition of claim 41 or 42, or the pharmaceutical composition of claim 43.

71. A dsRNA active agent or pharmaceutical composition or combination of drugs for use in claim 70, wherein the subject has obesity or a lipid metabolism-related disease; preferably, the obesity is selected from overweight, obese, severely obese or extremely obese, covering obesity.

72. A dsRNA active agent or pharmaceutical composition or combination thereof for use in any one of claims 68-71, wherein administration of the dsRNA active agent to a subject results in a decrease in GPR75 concentration or content (e.g., in target tissues or target cells), or results in a decrease in GPR75 protein accumulation or content in the subject (e.g., in target tissues or target cells); or results in a decrease in weight, a decrease in body fat, control of weight gain, and / or improvement in obesity-related metabolic status in the subject.

73. A dsRNA active agent or pharmaceutical composition or combination of drugs for the use of claim 72, wherein the target tissue is tissue of the central nervous system, preferably brain tissue or neurons, more preferably the cortex or cerebellum, most preferably tissue of the cerebral cortex, hypothalamus, hippocampus and other brain regions; spinal tissue, preferably cervical, lumbar and thoracic vertebrae; muscle tissue, preferably skeletal muscle tissue or cardiac muscle tissue; ocular tissue or adipose tissue; or the target cells are cells derived from the target tissue.

74. A dsRNA active agent or pharmaceutical composition or combination of drugs for use in claim 72 or 73, wherein the target tissue is selected from tissues of the central nervous system, preferably brain tissue or neurons; or muscle tissue, preferably skeletal muscle tissue or myocardial tissue; or the target cells are selected from cells of the central nervous system, preferably brain cells or neuronal cells; or muscle cells, preferably skeletal muscle cells or myocardial cells.

75. A dsRNA active agent or pharmaceutical composition or combination of drugs for use in any one of claims 68-74, wherein the drug is administered in combination with one or more other therapeutic agents, preferably selected from any therapeutic agents effective in preventing or treating GPR75-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the GPR75 gene), covering a variety of therapeutic agents for weight management, for reducing weight or body fat, or for preventing or treating obesity (e.g., overweight, obesity, severe obesity, or extreme obesity, covering obesity) or lipid metabolism-related diseases (e.g., reducing weight gain and improving obesity-related metabolic conditions).

76. A dsRNA active agent or pharmaceutical composition or combination of drugs for use in any one of claims 68-75, wherein the prevention or treatment of administration of said dsRNA active agent or pharmaceutical composition or combination of drugs further comprises determining the level of GPR75 in a sample from said subject.

77. A kit comprising the dsRNA activator of any one of claims 1-39, or the pharmaceutical composition of claim 41 or 42, or the pharmaceutical composition of claim 43.

78. A vial or syringe comprising the dsRNA active agent of any one of claims 1-39, or the pharmaceutical composition of claim 41 or 42, or the pharmaceutical composition of claim 43.

79. 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 39.

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