RNAi agent for inhibiting GPR75 gene expression as well as preparation method and application of RNAi agent

Abstract

The present invention relates to an RNAi agent for inhibiting GPR75 gene expression, and a preparation method and use thereof, the RNAi agent, or a pharmaceutically acceptable salt thereof, can inhibit GPR75 gene expression to treat GPR75 gene related diseases and disorders, such as obesity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, and more particularly, to an RNAi agent for inhibiting GPR75 gene and a preparation method and use thereof, which can be used for inhibiting GPR75 gene expression. BACKGROUND

[0002] G protein-coupled receptor 75 (GPR75) is a member of the G protein-coupled receptor family. It contains the features of most GPCRs, i.e. 7 transmembrane domains, an N-glycosylation site at the N-terminus and many serine and threonine phosphorylation sites at the C-terminus. Amino acid sequence analysis shows that GPR75 is most closely related to the recognized Caenorhabditis elegans neuropeptide Y receptor (24% homology), rat galanin receptor type 3 (25% homology) and pig growth hormone secretagogue receptor type 1b (25% homology) (Tarttelin et al. (1999) Biochem Biophys Res Commun. 260: 174-180). GPR75 is classified as a class A orphan receptor coupled to Gq, whose activation is associated with increased intracellular calcium and IP-1 accumulation. GPR75 is expressed in many tissues, and in the brain, GPR75 is expressed in the neocortex, entorhinal cortex, hippocampus, thalamus and hypothalamus.

[0003] Currently, there are many patent documents reporting that nucleic acids, small molecules and polypeptide molecules can inhibit the expression of G protein-coupled receptor 75, for example, WO2022076291, WO2023165108, WO2017156164, WO2023185821, etc., but no small molecule or nucleic acid has been approved for treating GPR75-related diseases. SUMMARY

[0004] In view of the problems existing in the prior art, the present application provides an RNAi agent for inhibiting GPR75 gene and a preparation method and use thereof, which has a good inhibitory effect on the expression of GPR75 gene.

[0005] In a first aspect, the present application provides an RNAi agent for inhibiting GPR75 gene expression, or a pharmaceutically acceptable salt thereof, wherein the RNAi agent comprises a sense strand and an antisense strand, the sense strand is at least partially complementary to the antisense strand, and the sense strand and the antisense strand are selected from the sequences shown in Table 1, wherein at least one nucleotide in the sense strand and / or the antisense strand is modified, and the modification is selected from a fatty acid chain.

[0006] The sense strand and the antisense strand can be partially, substantially or completely complementary to each other, for example, the sense strand and the antisense strand can be 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary.

[0007] The length of the nucleotides of the sense strand and the antisense strand can be the same or different, for example, the sense strand includes 19 nucleotides, the antisense strand includes 21 nucleotides, or the sense strand includes 19 nucleotides, the antisense strand also includes 19 nucleotides, or the sense strand includes 21 nucleotides, the antisense strand includes 19 nucleotides, or the sense strand includes 21 nucleotides, and the antisense strand includes 21 nucleotides.

[0008] As a preferred technical solution of the present application, the fatty acid chain is selected from: hexadecane sulfonamidophosphate nucleotide interlinking (C16muP),

[0009]

[0010] As a preferred technical solution of the present application, at least one nucleotide from the 3rd to the 10th at the 5' end of the sense strand is modified by a fatty acid chain, preferably the 5th, 6th or 7th nucleotide is modified by a fatty acid chain.

[0011] As a preferred technical solution of the present application, the 5th nucleotide at the 5' end of the sense strand is modified by 2'-O-hexadecyl (C16).

[0012] As a preferred technical solution of the present application, the first nucleotide at the 3' end of the sense strand is modified by a fatty acid chain.

[0013] As a preferred technical solution of the present application, the first nucleotide at the 3' end of the sense strand is modified by DTx-01-08, wherein the structure of DTx-01-08 is:

[0014] As a preferred technical solution of the present application, the nucleotides on the sense strand and / or the antisense strand can also be modified by other modifications, and the modified nucleotides are selected from:

[0015] alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluorinated nucleotides, deoxy nucleotides, 5'-methylphosphonate nucleotides, 5'-C-methylphosphonate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinyl phosphonate nucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), glycol nucleic acids (GNA); wherein the alkyl nucleotides are selected from methyl nucleotides, ethyl nucleotides; the glycol nucleic acids include (S)-glycol nucleic acids ((S)-GNA) and (R)-glycol nucleic acids ((R)-GNA).

[0016] In one embodiment, the sense strand and the antisense strand of the RNAi agent both comprise at least one modified nucleotide, in one specific embodiment, the sense strand comprises at least one modified nucleotide, and the nucleotides of the antisense strand are unmodified, or the nucleotides of the sense strand are unmodified, and the antisense strand comprises at least one modified nucleotide. In one specific embodiment, each nucleotide of the sense strand is modified, and each nucleotide of the antisense strand is also modified.

[0017] In one embodiment, the modified RNAi agent is selected from the sequences shown in Table 2.

[0018] In a second aspect, the present application provides a pharmaceutical composition for inhibiting the expression of GPR75 receptor, comprising the RNAi agent as described above, or a pharmaceutically acceptable salt thereof.

[0019] In a third aspect, the present application also provides use of the RNAi agent as described above, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described above, in the manufacture of a medicament for treating a GPR75 receptor related disease, disorder or symptom.

[0020] In one embodiment, the disease is selected from obesity and the like.

[0021] In a fourth aspect, the present application also provides an RNAi agent for reducing the expression of a target gene in a subject, comprising administering to the subject an RNAi agent comprising: a sense strand and an antisense strand, the antisense strand being complementary to a target gene, the sense strand being at least partially complementary to the antisense strand, and one or more fatty acid chains conjugated to an internal position of the sense strand and / or the antisense strand.

[0022] In one embodiment, the target gene is a GPR75 gene, and the gene sequence thereof is selected from GenBank NM_006794.4.

[0023] In one embodiment, the administration is extrahepatic administration.

[0024] As a preferred technical solution of the present application, the administration is intrathecal administration.

[0025] As a preferred technical solution of the present application, the RNAi agent can reduce the expression of the target gene in brain or spinal tissue.

[0026] As a preferred technical solution of the present application, the brain or spinal tissue is selected from the group consisting of cortex, cerebellum, cervical vertebra, lumbar vertebra, and thoracic vertebra.

[0027] The RNAi agent for inhibiting the expression of GPR75 gene, or a pharmaceutically acceptable salt thereof provided by the present application has good inhibitory activity on GPR75 gene, and can be used for preventing and / or treating diseases related to GPR75 gene. DETAILED DESCRIPTION

[0028] The present application will be further described in detail below with reference to examples, but the embodiments of the present application are not limited thereto.

[0029] The term "comprising" as used herein is used to mean "including, but not limited to," and is to be construed in the manner set out in the phrase "comprising." Unless specifically stated otherwise.

[0030] The term "or" as used herein is used to mean "and / or," and is to be construed in the manner set out in the phrase "and / or" unless otherwise specifically stated.

[0031] The terms "sequence" and "nucleotide sequence" as used herein mean the order or succession of nucleobases or nucleotides, described in alphabetical order using standard nomenclature.

[0032] In the present application, the term "RNAi agent" refers 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" orientation with respect to the target RNA.

[0033] In the present application, "complementary" has the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases on the other strand in a complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair includes one purine and one pyrimidine. When adenine on one strand always pairs with uracil on the other strand, and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand.

[0034] The term "antisense strand" generally refers to the strand of an RNAi agent that includes a region of substantial complementarity to a target sequence. As used herein, the term "region of complementarity" generally refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., a target sequence). When the region of complementarity is not perfectly complementary to the target sequence, the mismatches can be internal or at the terminal regions of the molecule. Generally, the most tolerated mismatches are at the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' terminus.

[0035] The term "sense strand" generally refers to the strand of an RNAi agent that includes a region of substantial complementarity to a region that is an antisense strand as defined herein. The "sense" strand is sometimes referred to as the "sense" strand, the "passenger" strand, or the "anti-guide" strand. By virtue of their sequences, the antisense strand targets the desired mRNA, while the sense strand targets a different target. Thus, if the antisense strand is incorporated into RISC, the correct target is targeted. Incorporation of the sense strand can result in off-target effects. These off-target effects can be limited by using modifications on the sense strand or using a 5' end cap. In the present invention, modified nucleotides include, but are not limited to: alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5'-methylphosphonate nucleotides, 5'-C-methylphosphonate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinylphosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), inverted abasic deoxyribose residues (invAb), glycol nucleic acids (GNA), fatty acid chain modifications.

[0036] wherein the fatty acid chain modification is selected from the group consisting of C 10 -C 25 lipophilic ligand, the lipophilic ligand being a C 10 -C 25 conjugated to the 2'-position of a nucleotide or modified nucleotide within the sense strand or the antisense strand, for example: 2'-0-hexadecyl (C16) modification 2'-0-docosyl (C22) modification or, C 10 -C 25 a linker conjugated to the 2'-position of a nucleotide within the sense strand or the antisense strand, for example,

[0037] The fatty acid chain can also be a hexadecylsulfonyl phosphoramidate internucleoside linkage (C16muP), for example: The structure of DTx-01-08 is:

[0038] 2'-O-methoxy modified nucleotides, such as, 2'-ethyl nucleotides, 2'-O-methoxy modified nucleotides, such as, 2'-methoxyethyl nucleotides, such as, 2'-fluoro nucleotides, such as, 5'-C-methylphosphonate nucleotides, such as

[0039] vinylphosphonate nucleotides (VP), such as, phosphorothioate nucleotides (S), such as, phosphate nucleotides (p), with the structure: 2'-deoxyribonucleotides, such as: inverted abasic deoxyribose residues (invAb), such as: glycol nucleic acids (GNA), including (S)-glycol nucleic acids ((S)-GNA) such as: and (R)-glycol nucleic acids ((R)-GNA), such as:

[0040] wherein Base (B) represents a base, Me represents a methyl group, and Et represents an ethyl group.

[0041] wherein the term "C10-C25 modification" or "C10-C25 ligand" means that the 2'-position of the nucleotide or modified nucleotide is connected with a C10-C25 containing hydrocarbon chain that is saturated or unsaturated, or that the nucleotide is connected with a C10-C25 containing hydrocarbon chain that is saturated or unsaturated on the internucleotide linkage, and can be specifically referred to in patent documents CN201980045507.X, CN202080093231.5, CN202180059287.3, WO2023064530A1, etc., which can be added to the present application by way of introduction.

[0042] The term "locked nucleic acid" is a nucleotide having a modified ribose moiety, wherein the ribose moiety includes an extra bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in the 3 '-endo conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and to reduce off-target effects (Elmen, J. et al. (2005) Nucleic Acids Research 33(1): 439-447; Mook, OR. et al. (2007) Mol Cancer Ther 6(3): 833-843; Grunweller, A. et al. (2003) Nucleic Acids Research 31(12): 3185-3193).

[0043] Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are incorporated herein by reference.

[0044] Locked nucleic acid structures are as follows:

[0045]

[0046] In certain embodiments, the sugar surrogate comprises a ring having more than 5 atoms and more than 1 heteroatom. For example, nucleosides comprising a morpholino sugar moiety and their use in oligomeric compounds have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0047] The term "morpholino" means a sugar surrogate having the following formula:

[0048]

[0049] In certain embodiments, the morpholino group can be modified, e.g., by adding or altering various substituents according to the above morpholino structure. Such sugar surrogates are referred to herein as "modified morpholino groups."

[0050] In the present disclosure, capital letters C, G, U, A represent the base composition of nucleotides, unless otherwise specified. Lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; LNA represents that the nucleotide adjacent to the right is a locked nucleic acid (LNA) modification; GNA represents that the nucleotide adjacent to the left is a GNA modification; lowercase letter s represents that the two nucleotides between the letters are connected by a phosphorothioate group; VP represents that the nucleotide adjacent to the right of the letter VP is a vinyl phosphonate-modified nucleotide. invAb represents an inverted abasic deoxyribonucleotide; dN represents any deoxyribonucleotide; dA represents a deoxyadenine nucleotide; dT represents a deoxythymine nucleotide; dU represents a deoxyuracil nucleotide; dC represents a deoxycytosine nucleotide; and dG represents a deoxyguanine nucleotide.

[0051] It is emphasized that the "modification" of the nucleotides described in the present disclosure includes, but is not limited to, the examples described above, and the nucleotides can also be replaced by other nucleotides, such as (S)-glycerol nucleic acid, etc.

[0052] The pharmaceutical compositions of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal, and / or parenteral administration. The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of a compound that produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of the dosage unit, preferably from about 5% to about 70%, and most preferably from about 10% to about 30% by weight of the active ingredient.

[0053] The term "preventing and / or treating" includes not only preventing and / or treating a disease, but also generally includes preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or slowing the onset of one or more symptoms associated with a disease, reducing and / or alleviating one or more symptoms associated with a disease, reducing the severity and / or duration of a disease and / or any symptoms associated therewith and / or preventing further increases in the severity of a disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by a disease, and generally any pharmacological action that is beneficial to the patient being treated. The nucleic acids or pharmaceutical compositions of the present application form viable therapeutic agents without requiring that complete cure or eradication of any symptom or manifestation of a disease be achieved. As recognized in the relevant art, a drug used as a therapeutic agent can reduce the severity of a given disease state, but need not eliminate every manifestation of a disease to be considered a useful therapeutic agent. Similarly, a therapeutic agent prophylactically administered constitutes a viable prophylactic agent without being completely effective in preventing the onset of a disorder. It is sufficient to simply reduce the impact of a disease in a subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another therapy, or by producing another beneficial effect), or to reduce the likelihood of a disease from occurring or worsening.

[0054] The terms "disease" or "disorder" can be used interchangeably and generally refer to any deviation from the normal state of a subject, for example, any change in the state of the body or of some of its organs, impairs or interferes with the performance of the functions, and / or causes symptoms such as discomfort, dysfunction, pain, or even death in a person affected or exposed to it. A disease or disorder can also be referred to as a distemper, an ailing, an ailment, a malady, a disorder, a sickness, an illness, a complaint, an inderdisposion, or an affectation.

[0055] The term "inhibiting" is used interchangeably with "reducing", "silencing", "down- regulating", "arresting" and other similar terms and includes inhibition at any level.

[0056] The term "G protein-coupled receptor 75" ("GPR75") refers to the well-known gene and polypeptide also known in the art as "putative G protein-coupled receptor 75", "WI-31133", "GPRchr2", and "WI31133". GPR75 binds to 20-HETE and interferes with insulin signaling leading to obesity. Among others, the term "GPR75-related disease, disorder" refers to a body weight disorder, such as obesity.

[0057] The term "GPR75" includes human GPR75, the amino acid and nucleotide sequences of which can be found in, for example, GenBank Accession No. NM_006794.4; mouse GPR75, the amino acid and nucleotide sequences of which can be found in, for example, GenBank Accession No. NM_175490.4; and rat GPR75, the amino acid and nucleotide sequences of which can be found in, for example, GenBank Accession No: NM_001109096.1.

[0058] The term "GPR75" also includes Macaca mulatta GPR75, the amino acid and nucleotide sequences of which can be found in, for example, GenBank Accession No. NM_001204509.2. Other examples of GPR75 mRNA sequences can be readily obtained using, for example, the GenBank, UniProt, OMIM, and Macaca Genome Project websites. The sequences of the above-mentioned NM_006794.4, NM_175490.4, NM_001109096.1, NM_001204509.2 can be found in the sequence listing of patent document CN202180077184.X.

[0059] The phrase "inhibiting expression of a GPR75 gene" in the present application includes inhibiting expression of any GPR75 gene (e.g., a mouse GPR75 gene, a rat GPR75 gene, a monkey GPR75 gene, or a human GPR75 gene, as well as variants or mutants encoding a GPR75 gene.

[0060] "Inhibiting expression of a GPR75 gene" includes inhibition of a GPR75 gene at any level, e.g., at least partially preventing expression of a GPR75 gene, e.g., inhibiting by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0061] The term "pharmaceutically acceptable salt" refers to a salt of a compound (RNAi agent) of the present application, prepared from a compound of the present application having specific substituents with a pharmaceutically acceptable acid or base, and the above-mentioned salt includes, but is not limited to, sodium salt, potassium salt, calcium salt, magnesium salt, iron salt, ammonium salt, lithium salt, hydrochloride, trifluoroacetate, oxalate, maleate, benzoate, salicylate, and the like.

[0062] The term "pharmaceutically acceptable carrier" refers to any formulation vehicle or medium that can deliver an effective amount of an active substance of the present application, does not interfere with the biological activity of the active substance, and is nontoxic to the host or patient. Representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, and the like. These bases include suspending agents, viscosity increasing agents, transdermal enhancers, and the like. Their formulation is well known to those skilled in the art of cosmetics or topical pharmaceuticals. Additional information on carriers can be found in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0063] The term "pharmaceutically acceptable excipient" is a substance that is intentionally included in a drug delivery system other than the active pharmaceutical ingredient (API, therapeutic product, e.g., GPR75 gene). The excipient does not exert or is not intended to exert a therapeutic effect at the intended dose. Excipients can serve the following purposes: a) aid in the handling of the drug delivery system during manufacture, b) protect, support or enhance the stability, bioavailability or patient acceptability of the API; c) aid in product identification; and / or d) enhance any other property of the API with respect to overall safety, effectiveness or delivery during storage or use.

[0064] Among excipients are (but are not limited to): absorption enhancers, anti-adherents, antifoaming agents, antioxidants, binders, buffering agents, carriers, coating agents, coloring agents, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, effervescent agents, fillers, flavoring agents, glidants, humectants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, waterproofing agents, wetting agents, lubricants such as sodium lauryl sulfate and magnesium stearate, flavorings and fragrances.

[0065] The term "active ingredient", "therapeutic agent", "active substance" or "active agent" refers to a chemical entity that is effective in treating a target disorder, disease or condition.

[0066] Example 1 Synthesis of RNAi agents

[0067] RNAi agents were prepared using an OligoMaker ApS 192 RNA synthesizer (Denmark), and the specific synthesis route can refer to the patent document CN202180077184.X, and the RNAi agent sequences are shown in Table 1.

[0068] Table 1 is the RNAi agent

[0069]

[0070]

[0071] Synthesis of modified RNAi agents of Example 2

[0072] The modified RNAi agents were prepared by using OligoMaker ApS 192 RNA synthesizer (Denmark), and the specific synthesis route can refer to the patent document CN202180077184.X, and the sequence of the modified RNAi agent is shown in Table 2.

[0073] Table 2 is a modified RNAi sequence table

[0074]

[0075] Wherein, A = adenosine-3'-phosphate; U = uridine-3'-phosphate; C = cytidine-3'-phosphate; G = guanosine-3'-phosphate; Am = 2'-O-methyl adenosine-3'-phosphate; Um = 2'-O-methyl uridine-3'-phosphate; Cm = 2'-O-methyl cytidine-3'-phosphate; Gm = 2'-O-methyl guanosine-3'-phosphate; Tm = 2'-O-methyl thymidine-3'-phosphate; Ams = 2'-O-methyl adenosine-3'-phosphorothioate; Ums = 2'-O-methyl uridine-3'-phosphorothioate; Cms = 2'-O-methyl cytidine-3'-phosphorothioate; Gms = 2'-O-methyl guanosine-3'-phosphorothioate; Af = 2'-fluoro adenosine-3'-phosphate; Uf = 2'-fluoro uridine-3'-phosphate; Cf = 2'-fluoro cytidine-3'-phosphate; Gf = 2'-fluoro guanosine-3'-phosphate; dA = 2'-deoxy adenosine-3'-phosphate; dT = 2'-deoxy thymidine-3'-phosphate; dG = 2'-deoxy guanosine-3'-phosphate; Afs = 2'-fluoro adenosine-3'-phosphorothioate; Ufs = 2'-fluoro uridine-3'-phosphorothioate; Cfs = 2'-fluoro cytidine-3'-phosphorothioate; Gfs = 2'-fluoro guanosine-3'-phosphorothioate; m = 2'-O-methyl; f = 2'-fluoro; s = phosphorothioate linkage; Uhd is 2'-O-hexadecyl-uridine-3'-phosphate; Ahd is 2'-O-hexadecyl-adenosine-3'-phosphate; Chd is 2'-O-hexadecyl-cytidine-3'-phosphate; Ghd is 2'-O-hexadecyl-guanosine-3'-phosphate; the structure of DTx-01-08 is:

[0076] DTx-01-08 is connected to the nucleotide by phosphate or phosphorothioate; the structure of VP is: The structure of gna is:

[0077] In vitro testing of RNAi agents in HuH7 cells

[0078] Human GPR75 cDNA (containing 5' UTR and 3' UTR sequences, GenBank NM_006794.4) was cloned into a reporter-based screening plasmid, psiCHECK2 (Promega-C8021), to generate Renilla luciferase / GPR75 fusion mRNA. HuH7 cells were cultured in DMEM (Gibco-10313021) containing 10% fetal bovine serum (ExCell Bio-FSP500), 1% glutamine (Gibco-35050061), 1% non-essential amino acids (Gibco-11140050), 1% penicillin-streptomycin (HyClone-SV30010). GPR75-psiCHECK2 plasmid, RNAi agents and Lipo2000 (Invitrogen-11668019) transfection reagent were diluted with Opti-MEM (Gibico-11058021) and added to HuH7 cell suspension at a cell density of 1 x 10 5 / ml, and plated in 96-well plates so that the final concentration of RNAi agents was 1 nM, 0.1 nM, 0.01 nM or 0.02 nM. After 24 hours of incubation, the relative level of Renilla luciferase normalized to the level of constitutively expressed firefly luciferase also present in the psiCHECK2 plasmid was measured using the Dual Luciferase Reporter Assay (Promega-E2920).

[0079] The inhibition rate of GPR75 gene mediated by the RNAi agent was calculated according to the following formula.

[0080] The GPR75 gene inhibition rate (%) of the sample = (1 - the average of the relative level of Renilla luciferase of the sample / the relative level of Renilla luciferase of the control group) x 100. The test results are shown in Table 3.

[0081] Table 3 is the in vitro activity data of the RNAi agent

[0082]

[0083] wherein the inhibition rate A is greater than 60%.

[0084] As can be seen from Table 3, the RNAi agent of the present application has good inhibitory activity on the GPR75 gene.

[0085] Example 4 Evaluation of in vivo activity

[0086] To evaluate the effect of duplex targeting GPR75 to reduce GPR75 mRNA level in vivo, a single 150 μg dose of the RNAi agent in Table 2 was administered to humanized GPR75 (hGPR75) mice by intracerebroventricular injection at day 0. At day 14 or 21 post administration, animals were sacrificed, cerebral cortex samples were collected, and the level of GPR75 mRNA was quantified by qPCR. The specific qPCR experimental procedure was as follows:

[0087] The cerebral cortex tissue was taken for RNA extraction using MagicPure 96 Total RNA Kit (full style-EC521-96-11), and then the extracted tissue RNA was reverse transcribed into cDNA using PrimeScript TM RT Master Mix (Takara-RR036B), and finally TB Premix Ex Taq II TM (Takara-RR820B) was used for qPCR experiment on the obtained cDNA.

[0088] The expression level of target gene mRNA of each sample was calculated by ΔΔCT relative quantification method. The relative expression of the target gene was represented by 2-ΔΔCT. Specifically, the CT value of the target gene in each sample was subtracted from the CT value of the internal reference gene (Gapdh) to obtain the ΔCT value, then the ΔCT value of the administration group was subtracted from the ΔCT value of the control group to obtain the ΔΔCT value, and finally the ΔΔCT was converted by 2-ΔΔCT to obtain the relative expression value of GPR75 mRNA (value of sample).

[0089] The inhibition rate of GPR75 gene mediated by the RNAi agent was calculated according to the following formula, and the calculation results are shown in Table 4.

[0090] GPR75 gene inhibition rate % = (1-value of sample / Average value of Control)*100.

[0091] Table 4

[0092] No. Average inhibition(%) No. Average inhibition(%) X1M B X3M B X3M1 B X3M2 B X5M B X6M B X7M B X8M B X9M B X10M B X12M B X14M B X15M B

[0093] Wherein, B≥40%.

[0094] As can be seen from Table 4, the RNAi agent of the present application has good inhibitory activity on GPR75 gene.

[0095] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. An RNAi agent for inhibiting GPR75 gene expression, or a pharmaceutically acceptable salt thereof, characterized in that, The RNAi agent comprises a sense strand and an antisense strand, the sense strand and the antisense strand being at least partially complementary, the sense strand and the antisense strand being selected from the sequences shown in Table 1, wherein at least one nucleotide on the sense strand and / or the antisense strand is modified, the modification being selected from fatty acid chains.

2. The RNAi agent according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The fatty acid chain is selected from: hexadecanesulfonylaminophosphate nucleoside interlinking (C16muP).

3. The RNAi agent according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, characterized in that, At least one nucleotide at positions 3-10, starting from the 5' end of the positive strand, is modified by a fatty acid chain, preferably the nucleotide at position 5, 6, or 7.

4. The RNAi agent according to claim 3, or a pharmaceutically acceptable salt thereof, characterized in that, The 5th nucleotide starting from the 5' end of the positive chain is modified with 2'-O-hexadecyl (C16).

5. The RNAi agent according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, characterized in that, The first nucleotide starting from the 3' end of the positive chain is modified by a fatty acid chain.

6. The RNAi agent according to claim 5, or a pharmaceutically acceptable salt thereof, characterized in that, The first nucleotide starting from the 3' end of the positive strand is modified with DTx-01-08.

7. The RNAi agent according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, characterized in that, The nucleotides on the sense and / or antisense strands may also be modified by other means, wherein the modified nucleotides are selected from: Alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoronucleotides, deoxynucleotides, 5'-methylphosphonucleotides, 5'-C-methylphosphonucleotides, 2′-deoxy-2′-fluoronucleotides, vinylphosphonate nucleotides (VP), thiophosphonucleotides, dithiophosphonucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), and glycol nucleic acids (GNA).

8. The RNAi agent according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The modified RNAi agent is selected from the sequences shown in Table 2.

9. A pharmaceutical composition for inhibiting GPR75 gene expression, characterized in that, The pharmaceutical composition comprises the RNA agent as described in any one of claims 1-8, or a pharmaceutically acceptable salt thereof.

10. The use of the RNAi agent according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 9, in the preparation for the treatment of GPR75 gene-related diseases, conditions, or symptoms, wherein the disease is selected from obesity, etc.

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