SiRNA for inhibiting GPR75 gene expression and application thereof

Abstract

The invention discloses siRNA for inhibiting GPR75 gene expression and application of the siRNA. The siRNA comprises a positive-sense strand and an antisense strand, and the antisense strand has a nucleotide sequence which is different from a nucleotide sequence shown in any one of SEQ ID NO.109-216 and SEQ ID NO.226-231 by not more than 3 nucleotides; the antisense strand and the positive-sense strand are at least partially complementary to form a double-strand region. The invention provides siRNA for inhibiting GPR75 gene expression, the siRNA is modified to ensure the stability and inhibitory activity of the siRNA, and experiments prove that multiple groups of siRNA have obvious inhibitory activity on GPR75 gene expression, and show application prospects in preparation of drugs for treating or preventing GPR75 expression related diseases in human bodies.

Description

[0001] The divisional application is a divisional application of the prior application with the application date of August 12, 2025, the application number of 2025111243293, and the invention name of "siRNA for inhibiting GPR75 gene expression and application thereof". TECHNICAL FIELD

[0002] The application belongs to the technical field of biological medicine, and particularly relates to siRNA for inhibiting GPR75 gene expression and application thereof. BACKGROUND

[0003] G-protein-coupled receptor 75 (GPR75) encodes a membrane protein receptor containing 540 amino acids, which has the structural characteristics of a typical G-protein-coupled receptor, that is, seven transmembrane domains, the N-terminal is located in the extracellular, and the C-terminal is located in the intracellular. GPR75 gene is expressed in multiple tissues of the human body, including the brain, liver, kidney, heart, etc., which mainly participates in physiological processes such as energy metabolism, inflammatory response and metabolic disorders.

[0004] Studies have shown that overexpression of GPR75 gene directly promotes diseases such as obesity, diabetes, cardiovascular disease, cancer and fatty liver by regulating appetite, insulin sensitivity, vascular tension and cancer cell migration. Therefore, by reducing the expression level of GPR75, the progress of related diseases can be affected, and inhibition of GPR75 gene expression becomes a potential treatment strategy.

[0005] Small interfering RNA (siRNA) is a double-stranded RNA molecule that can specifically silence the expression of target genes through the RNA interference (RNAi) mechanism. siRNA is complementary to the target mRNA and is processed into small fragments under the action of Dicer enzyme, and then binds to the RNA-induced silencing complex (RISC), guiding the RISC complex to cut the target mRNA, thereby inhibiting its translation. Developing effective siRNA targeting GPR75 gene can provide a new idea for the treatment of related diseases. SUMMARY

[0006] The purpose of the present application is to provide siRNA for inhibiting GPR75 gene expression and application thereof, so as to provide a new treatment strategy for diseases related to overexpression of GPR75 gene.

[0007] In order to achieve the above-mentioned object, the first aspect of the present application provides an siRNA for inhibiting the expression of a GPR75 gene, comprising a sense strand and an antisense strand, wherein the antisense strand has a nucleotide sequence which differs from the nucleotide sequence shown in any one of SEQ ID NO. 109-216 or SEQ ID NO. 226-231 by no more than 3 nucleotides; and the sense strand is at least partially complementary to the antisense strand to form a double-stranded region.

[0008] In one or more embodiments, the sense strand has a nucleotide sequence shown in any one of SEQ ID NO. 1-108 or a nucleotide sequence shown in any one of SEQ ID NO. 220-225, and the antisense strand has a nucleotide sequence shown in a corresponding one of SEQ ID NO. 109-216 or SEQ ID NO. 226-231.

[0009] In one or more embodiments, at least one nucleotide in the sense strand and / or the antisense strand is a modified nucleotide.

[0010] In one or more embodiments, the modification comprises one or more of a 2'-methoxy modification, a 2'-fluoro modification, a phosphorothioate group modification, a vinyl phosphonate modification, and a 2'-deoxynucleotide modification.

[0011] In one or more embodiments, at least the nucleotides located at positions 1-6, 8, 12-21 of the sense strand and / or at least the nucleotides located at positions 1, 3-5, 7-9, 11, 13, 15, 17-21 of the antisense strand are 2'-methoxy modified nucleotides, in the direction from the 5' end to the 3' end.

[0012] In one or more embodiments, at least the nucleotides located at positions 7, 9-11 of the sense strand and / or at least the nucleotides located at positions 2, 6, 10, 14, 16 of the antisense strand are 2'-fluoro modified nucleotides, in the direction from the 5' end to the 3' end.

[0013] In one or more embodiments, the nucleotides between the first and second positions, between the second and third positions, between the 19th and 20th positions, and between the 20th and 21st positions of the antisense strand are connected by a phosphorothioate group, in the direction from the 5' end to the 3' end.

[0014] In one or more embodiments, at least the nucleotide located at the 12th position of the antisense strand is a 2'-deoxynucleotide, in the direction from the 5' end to the 3' end.

[0015] In one or more embodiments, the nucleotide at the 1stposition of the antisense strand is a phosphorothioate modified nucleotide, in the 5' to 3' direction of the antisense strand.

[0016] In one or more embodiments, the nucleotide at the 1stposition and / or the 21stposition of the sense strand is linked with an inverted abasic nucleotide, and the nucleotide at the 1stposition and / or the 21stposition of the sense strand is linked with the inverted abasic nucleotide through a phosphorothioate group, in the 5' to 3' direction of the sense strand.

[0017] In one or more embodiments, the sense strand has a nucleotide sequence of SEQ ID NO. 55, and the antisense strand has a nucleotide sequence of SEQ ID NO. 163.

[0018] In one or more embodiments, the sense strand has a nucleotide sequence of SEQ ID NO. 56, and the antisense strand has a nucleotide sequence of SEQ ID NO. 164.

[0019] In one or more embodiments, the sense strand has a nucleotide sequence of SEQ ID NO. 57, and the antisense strand has a nucleotide sequence of SEQ ID NO. 165.

[0020] In one or more embodiments, the sense strand has a nucleotide sequence of SEQ ID NO. 84, and the antisense strand has a nucleotide sequence of SEQ ID NO. 192.

[0021] In one or more embodiments, the sense strand has a nucleotide sequence of SEQ ID NO. 86, and the antisense strand has a nucleotide sequence of SEQ ID NO. 194.

[0022] In one or more embodiments, the sense strand has a nucleotide sequence of SEQ ID NO. 99, and the antisense strand has a nucleotide sequence of SEQ ID NO. 207.

[0023] In one or more embodiments, the sense strand has a nucleotide sequence of SEQ ID NO. 220, and the antisense strand has a nucleotide sequence of SEQ ID NO. 226.

[0024] In one or more embodiments, the sense strand has a nucleotide sequence of SEQ ID NO. 221, and the antisense strand has a nucleotide sequence of SEQ ID NO. 227.

[0025] In one or more embodiments, the sense strand has a nucleotide sequence of SEQ ID NO. 222, and the antisense strand has a nucleotide sequence of SEQ ID NO. 228.

[0026] In one or more embodiments, the sense strand has a nucleotide sequence of SEQ ID NO. 223, and the antisense strand has a nucleotide sequence of SEQ ID NO. 229.

[0027] In one or more embodiments, the sense strand has a nucleotide sequence of SEQ ID NO. 224, and the antisense strand has a nucleotide sequence of SEQ ID NO. 230.

[0028] In one or more embodiments, the sense strand has a nucleotide sequence of SEQ ID NO. 225, and the antisense strand has a nucleotide sequence of SEQ ID NO. 231.

[0029] In one or more embodiments, the siRNA is coupled with one or more nucleotides on at least one strand or one or more targeting groups or functional molecules via a linker or a carrier.

[0030] In one or more embodiments, the siRNA has a targeting group or a functional molecule coupled internally or terminally to the sense strand and / or the antisense strand.

[0031] In one or more embodiments, the targeting group is a small molecule, a polypeptide or an antibody targeting transferrin receptor (TfR), and the functional molecule is 2'-O-hexadecyl.

[0032] To achieve the above object, the second aspect of the present application provides an application of the siRNA of any of the above embodiments in the preparation of a drug for preventing or treating a disease related to the expression of GPR75 gene.

[0033] To achieve the above object, the third aspect of the present application provides a GPR75 inhibitor comprising the siRNA of any of the above embodiments.

[0034] To achieve the above object, the fourth aspect of the present application provides a drug for preventing or treating a disease related to the expression of GPR75 gene, comprising the siRNA of any of the above embodiments; and a pharmaceutically acceptable carrier.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] The application provides siRNA for inhibiting GPR75 gene expression, and the siRNA is modified to ensure its stability and inhibitory activity. Experiments prove that the plurality of siRNA provided by the application has obvious inhibitory activity on GPR75 gene expression in cells, wherein the inhibition rates of hGPR75_443AM13, hGPR75_1347AM13, hGPR75_1348AM13, hGPR75_1349AM13 and hGPR75_1764AM13 on GPR75 gene expression in cells at a concentration of 0.1 nM all reach more than 90%, and the application prospect of the siRNA in the preparation of drugs for treating or preventing diseases related to GPR75 expression in human body is shown.

[0037] In the siRNA provided by the application, the half-inhibitory concentration IC50 of hGPR75_1348AM13, hGPR75_1347AM13 and hGPR75_1764AM13 on GPR75 gene in cells is less than 0.02 nM, and the siRNA has significant inhibitory activity.

[0038] The siRNA provided by the application can be mixed with two siRNAs to perform double-target point silencing, and the inhibitory activity is better than that of single siRNA, and the siRNA has a synergistic enhancement effect, wherein the half-inhibitory concentration IC50 of hGPR75_1348AM13+hGPR75_1764AM13 on GPR75 gene in cells is less than 0.01 nM, and the siRNA has very outstanding inhibitory activity. DETAILED DESCRIPTION

[0039] In order to enable personnel in the technical field to better understand the technical solutions in the application, the technical solutions in the embodiments of the application are clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the application, not all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor should belong to the protection scope of the application.

[0040] It is to be noted that the "modified nucleotide" in the present application is selected from at least one of the following: 2'-methoxy modified nucleotide, 2'-fluoro modified nucleotide, phosphorothioate modified nucleotide, 2'-deoxy-nucleotide, 3'-terminal deoxy-thymine (dT) nucleotide, locked nucleotide, non-locked nucleotide, configuration restricted nucleotide, restricted ethyl nucleotide, abasic nucleotide, inverted nucleotide, inverted abasic nucleotide, 2'-amino-modified nucleotide, 2'-O-allyl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2'-hydroxyl-modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl-modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing non-natural base, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, glycol nucleic acid nucleotide (GNA), unlinked nucleotide (UNA), nucleotide containing methylphosphonate group, nucleotide containing 5'-phosphate, and nucleotide containing 5'-phosphate mimic, or terminal nucleotide linked with cholesteryl derivative or dodecanoic acid dipalmitoyl amide group.

[0041] In an embodiment of the present application, the "2'-fluoro modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group is replaced with fluorine, and has the following structure shown in formula (1);

[0042] In an embodiment of the present application, the "2'-methoxy modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is replaced with methoxy, and has the following structure shown in formula (2);

[0043] In an embodiment of the present application, the "2'-deoxy-nucleotide modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is replaced with hydrogen atom, and has the following structure shown in formula (3); wherein B refers to a modified or unmodified nucleotide base A, U, G, C, T or other nucleotide base.

[0044]

[0045] In an embodiment of the present application, the "phosphorothioate group modified nucleotide" has a phosphorothioate group with the structure shown in formula (4). In an embodiment of the present application, the phosphorothioate group linked nucleotide has the following structure shown in formula (5):

[0046]

[0047] In an embodiment of the present application, the VP-modified nucleotide is a vinylphosphonate modification. In an embodiment of the present application, the VP-modified and methoxy-modified nucleotide, i.e., 5’-(E)-vinyl-2’-methoxy modified phosphonate group (5’-(E)-VP-2’-OMe) modified nucleotide, is shown in formula (6). In an embodiment of the present application, the VP-modified, methoxy-modified, and phosphorothioate group-modified nucleotide, i.e., 5’-PS modified nucleotide (i.e., 5’-(E)-vinyl-2’-methoxy modified phosphorothioate group modified nucleotide), is shown in formula (7).

[0048]

[0049] In an embodiment of the present application, the structure of the “inverted abasic nucleotide” is shown in formula (8).

[0050]

[0051] In an embodiment of the present application, the siRNA is conjugated to a functional molecule, and the conjugated functional molecule includes N-acetylgalactosamine (GalNAc), a lipophilic molecule, a polypeptide, a small molecule drug, an antibody, etc.

[0052] In an embodiment of the present application, the siRNA can be conjugated to one or more functional molecule-containing conjugation groups.

[0053] In an embodiment of the present application, the functional molecule is a small molecule, a polypeptide, or an antibody targeting transferrin receptor (TfR).

[0054] The types of linkers, targeting groups, and the manner of attachment to the siRNA are described in more detail in WO2019113393A1, WO2023056388A1, US20230256112A1 (the entire contents of which are incorporated herein by reference).

[0055] In an embodiment of the present application, the functional molecule is a lipophilic molecule (or lipophilic or lipophilic moiety). The siRNA comprises one or more lipophilic moieties conjugated or conjugated to one or more nucleotides on at least one strand via a linker or carrier. The lipophilic moieties include, but are not limited to, one or more of saturated alkanes, unsaturated alkanes, saturated fatty acids, unsaturated fatty acids, and cholesterol of different chain lengths.

[0056] In an embodiment of the present application, the lipophilic moiety is C16, C16 represents 2'-O-hexadecyl, which is a short lipid chain attached to siRNA, has lipophilicity, and can interact with cell membranes or membrane proteins. The structure of 2'-O-hexadecyl modified nucleotide is shown in formula (9), and B represents a modified or unmodified nucleotide base A, U, G, C, T or other nucleotide base.

[0057]

[0058] In an embodiment of the present application, the C16 can be connected to any nucleotide of the sense strand.

[0059] More examples of the lipophilic moiety are described in detail in WO2019217459A1 and WO2024216155A1 (the entire contents of which are incorporated herein by reference).

[0060] Overexpression of the GPR75 gene directly promotes diseases such as obesity, diabetes, cardiovascular disease, cancer and fatty liver by regulating appetite, insulin sensitivity, vascular tension and cancer cell migration. Therefore, by reducing the expression level of GPR75, the progress of related diseases can be affected, and inhibiting the expression of the GPR75 gene becomes a potential treatment strategy.

[0061] To this end, the applicant has developed an siRNA that can knock down the expression level of the GPR75 gene based on RNA interference technology, which is described in detail below.

[0062] Example 1: Sequence design

[0063] First, the applicant designed multiple groups of siRNA sequences based on the mRNA sequence encoding the GPR75 gene in the NCBI database, and the specific siRNA sequences are shown in Table 1 below.

[0064] Table 1: siRNA sequences and numbers for inhibiting GPR75

[0065]

[0066]

[0067]

[0068]

[0069] Example 2: Modification of siRNA

[0070] In order to improve the stability of siRNA and effectively inhibit the expression of the target gene, the applicant further modified the siRNA sequences in Table 1 above.

[0071] Specifically, in one embodiment, the modification scheme of the siRNA is as follows:

[0072] According to the direction from the 5' end to the 3' end, the nucleotides at positions 1-6, 8, 12-21 of the sense strand are 2'-methoxy modified, and the nucleotides at positions 1, 3-5, 7-9, 11, 13, 15, 17-21 of the antisense strand are 2'-methoxy modified;

[0073] According to the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9-11 of the sense strand are 2'-fluoro modified, and the nucleotides at positions 2, 6, 10, 14, 16 of the antisense strand are 2'-fluoro modified;

[0074] According to the direction from the 5' end to the 3' end, the nucleotides between positions 1 and 2, between positions 2 and 3, between positions 19 and 20, and between positions 20 and 21 of the antisense strand are connected by phosphorothioate groups;

[0075] According to the direction from the 5' end to the 3' end, the nucleotide at position 12 of the antisense strand is 2'-deoxynucleotide modified, and if the nucleotide at position 12 is a uracil nucleotide, it is replaced by a thymine deoxyribonucleotide;

[0076] According to the direction from the 5' end to the 3' end, the nucleotide at position 1 of the antisense strand is vinyl phosphate modified;

[0077] According to the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 21 of the sense strand are connected to inverted abasic nucleotides (InvAb), and the nucleotides at positions 1 and 21 of the sense strand and the inverted abasic nucleotides (InvAb) are connected by phosphorothioate groups.

[0078] Based on the above modification scheme, the modified siRNA is obtained, which can be specifically seen in Table 2 below.

[0079] In Table 2 below, m represents that the nucleotide adjacent to the left side is a 2'-methoxy modified nucleotide; f represents that the nucleotide adjacent to the left side is a 2'-fluoro modified nucleotide; s represents that the two nucleotides adjacent to the left and right sides are phosphorothioate modified, (d) represents that the nucleotide adjacent to the left side is a 2'-deoxynucleotide modified nucleotide, (InvAb) represents an inverted abasic nucleotide, and VP represents that the nucleotide adjacent to the right side is a vinyl phosphate modified nucleotide.

[0080] Table 2 Modified siRNA sequence

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Example 3: Detection of on-target activity of modified siRNA for inhibiting GPR75 expression

[0089] In order to verify the inhibitory activity of the above siRNA on the expression of GPR75 gene, the applicant provides an on-target activity detection experiment for the modified siRNA in the above Table 2, in which a psiCHECK2 vector is used to construct a plasmid vector for detection. The psiCHECK2 vector is a plasmid vector that can monitor the change in expression of a target gene fused with a reporter gene. The vector uses a Renilla luciferase as the main reporter gene, and the target fragment is cloned into the multiple cloning site downstream of the Renilla luciferase translation termination codon. The RNAi process against the target gene triggered by the synthesized siRNA leads to the cleavage and subsequent degradation of the fusion mRNA. By detecting the change in Renilla luciferase activity, it can be determined whether there is a targeting relationship between the siRNA and the target gene fragment.

[0090] The experimental process is as follows:

[0091] Step one: detection of plasmid construction

[0092] An insert sequence with the nucleotide sequence shown in SEQ ID NO. 217 is single-copied into the Xho I / Not I site of the psiCHECKTM-2 plasmid (Suzhou Jimake Gene Co., Ltd., item number C09005) to obtain a detection plasmid. The insert sequence is taken from a fragment of the full-length sequence of human GPR75 mRNA, which includes a target sequence completely complementary to the antisense strands of all siRNAs shown in Table 1.

[0093] Step two: cell culture and transfection

[0094] The siRNA to be tested is diluted with DEPC water to obtain an siRNA diluent, and then the siRNA diluent is added to the 96-well plate at an addition amount of 5 μL / well;

[0095] opti-MEM (purchased from Gibco, item number 31985-070) containing 20 ng of detection plasmid was added to the 96-well plate at an addition amount of 12.5 μL / well, and opti-MEM was added to the 96-well plate at an addition amount of 32.5 μL / well, and Lipofectamine RNAiMAX transfection reagent (purchased from Thermo, item number 11668-019) was added to the 96-well plate at an addition amount of 0.3 μL / well, and the mixed system was allowed to stand at room temperature (25 ℃) for 15 min.

[0096] After the standing was completed, DMEM complete medium (purchased from Transgen Biotech, item number FI101-01) containing 2×10 5 cells / mL of 293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences) was added to the 96-well plate at an addition amount of 50 μL / well, and the 96-well plate was cultured in a 5% (v / v) CO2, 37 ℃ cell incubator for 24 h, and was used for subsequent dual luciferase detection experiments.

[0097] In the culture system, the final concentration of siRNA was 1 nM, and in the experiment, a blank control group MOCK and a negative control group NC were set. The blank control group MOCK was a group to which only the interference reagent was added without any sequence, and the negative control group NC was added with a final concentration of 1 nM of NC in the well. The NC sequence is shown in Table 3.

[0098] Table 3 Sequence of the negative control group

[0099]

[0100] Step three: dual luciferase detection experiment

[0101] After the culture was completed, the cells in the 96-well plate were subjected to dual luciferase detection, and 3 replicate wells were set for each siRNA to be detected in the experiment. The specific detection method is as follows:

[0102] A dual luciferase detection kit (purchased from Promega, item number E2940) was taken, and the 5× lysis solution in the kit was diluted with water to 1× lysis solution, and substrate 1 and substrate 2 were configured according to the instructions of the kit;

[0103] A 96-well plate was taken, and the supernatant was discarded. After dilution and washing twice with PBS buffer (purchased from Hyclone, item number SH30256.01) per well, 1× lysis solution was added to the 96-well plate at an addition amount of 50 μL / well, and the cells in the 96-well plate were lysed by incubation at room temperature (25 ℃) for 20 min, to obtain the lysis solution;

[0104] The lysis solution was sucked out from the original 96-well plate, and then substrate 1 and substrate 2 were added into the new 96-well plate in an addition amount of 30 μL / well, after which the 96-well plate was detected by a multifunctional enzyme label meter after each addition of the substrate, and the numerical results of Firefly luciferase and Renilla luciferase were obtained respectively;

[0105] The luminescence ratio of each well of the 96-well plate was calculated based on the following formula:

[0106] The luminescence ratio of each well of the 96-well plate was calculated based on the following formula:

[0107] The luminescence ratio of each test group (i.e., the group added with the siRNA to be tested), the blank control group and the negative control group was the average of the luminescence ratios of the three replicate wells;

[0108] The luminescence ratios of each test group and the negative control group were normalized based on the luminescence ratio of the blank control group, and the ratio R of luminescence ratio (test) / luminescence ratio (control) was obtained, which represented the expression level of the Renilla luciferase reporter gene (i.e., the relative residual activity);

[0109] After obtaining the relative residual activity of the Renilla luciferase reporter gene, the inhibition rate of siRNA on GPR75 gene was calculated according to the formula: inhibition rate = (1-R) x 100%, and the following Table 4 data was obtained.

[0110] Table 4: Dual luciferase detection experiment results of Example 3

[0111]

[0112]

[0113] As shown in the above table data, all siRNAs except hGPR75_1269AM13 showed inhibition effect on the expression of GPR75 gene, among which, hGPR75_443AM13, hGPR75_1347AM13, hGPR75_1348AM13, hGPR75_1349AM13 and hGPR75_1764AM13, which were equivalent to the blank control group, had an inhibition rate of more than 90% on the expression of GPR75 gene in 293T cells at a concentration of 0.1 nM, showing significant inhibitory activity.

[0114] Example 4: Inhibitory activity detection of different concentrations of siRNA on GPR75 gene expression

[0115] To further study the effect of siRNA concentration in the culture system on the inhibitory activity, the applicants selected siRNAs with an inhibition rate greater than 75% in Example 3 for further experiments, i.e., hGPR75_435AM13, hGPR75_437AM13, hGPR75_438AM13, hGPR75_443AM13, hGPR75_568AM13, hGPR75_937AM13, hGPR75_967AM13, hGPR75_1345AM13, hGPR75_1346AM13, hGPR75_1347AM13, hGPR75_1348AM13, hGPR75_1349AM13, hGPR75_1504AM13, hGPR75_1582AM13, hGPR75_1681AM13, hGPR75_1687AM13, hGPR75_1688AM13, hGPR75_1727AM13, hGPR75_1736AM13, hGPR75_1737AM13, hGPR75_1738AM13, hGPR75_1743AM13, hGPR75_1750AM13, hGPR75_1764AM13, hGPR75_1770AM13, hGPR75_1789AM13, hGPR75_1973AM13.

[0116] Specifically, Example 4 used the same method as Example 3 to perform experiments, and the final concentration of each group of siRNAs in the culture system was set to 0.1 nM and 0.01 nM, respectively, and the following Table 5 data was obtained.

[0117] Table 5 Results of dual luciferase detection experiments of Example 4

[0118]

[0119] As can be seen from the data in Table 5, the inhibitory activity of each siRNA on GPR75 gene expression at a final concentration of 0.01 nM was lower than that at a final concentration of 0.1 nM, but still showed an inhibitory effect on GPR75 gene expression.

[0120] Among them, at a final concentration of 0.01 nM, equivalent to the blank control group, hGPR75_1347AM13 and hGPR75_1348AM13 had an inhibition rate of GPR75 gene expression in 293T cells of more than 64%, showing significant inhibitory activity.

[0121] Example 5: Half-inhibitory concentration IC50 detection

[0122] Applicant further selected siRNAs showing excellent inhibitory activity at two final concentrations in Example 4 for experiments, namely: hGPR75_1348AM13, hGPR75_1347AM13, hGPR75_1764AM13, hGPR75_443AM13, hGPR75_1687AM13, hGPR75_1727AM13, hGPR75_1349AM13.

[0123] In addition, two siRNAs were mixed by Applicant and introduced into a mixed test group, specifically: hGPR75_1348AM13 + hGPR75_1764AM13, hGPR75_443AM13 + hGPR75_1727AM13, hGPR75_1687AM13 + hGPR75_1349AM13, hGPR75_1347AM13 + hGPR75_1764AM13, and the concentrations of the two siRNAs in the mixed test group were the same.

[0124] Specifically, Example 5 used the same method as Example 3 for experiments, and the final concentrations of the siRNAs to be tested in each group in the culture system were set to be 10 nM, 1 nM, 0.3 nM, 0.1 nM, 0.01 nM, 0.01 nM and 0.003 nM, respectively. After calculating the inhibition rates of GPR75 gene expression at different final concentrations, the half-inhibitory concentration IC50 of each group of siRNAs was calculated by applying GraphPad Prism software, and the following Table 6 data was obtained.

[0125] Table 6 Calculation results of IC50 of Example 5

[0126]

[0127] As shown in Table 6 data, the IC50 of the test group of single siRNA to GPR75 gene in 293T cells was less than 0.1 nM, among which the IC50 of hGPR75_1348AM13, hGPR75_1347AM13 and hGPR75_1764AM13 to GPR75 gene in 293T cells was less than 0.02 nM, showing significant inhibitory activity.

[0128] In addition, the IC50 of each mixed test group was lower than the IC50 of the single siRNA included therein, showing a synergistic enhancement effect; among them, the IC50 of hGPR75_1348AM13 + hGPR75_1764AM13 to GPR75 gene in 293T cells was less than 0.01 nM, having very outstanding inhibitory activity.

[0129] Example 6: Detection of the on-target activity of unmodified siRNA for inhibiting GPR75 expression

[0130] To verify the inhibitory activity of unmodified siRNA sequences on GPR75 gene expression, the applicant selected some siRNAs with better inhibitory activity in Example 3, and tested the inhibitory activity of the corresponding unmodified siRNAs on GPR75 gene expression, including hGPR75_1347, hGPR75_1348, hGPR75_1349, hGPR75_1687, hGPR75_1727, hGPR75_1764, and hGPR75_443.

[0131] Meanwhile, to verify the inhibitory activity of the allosteric sequences of the above siRNAs on GPR75 gene expression, the applicant further allosterically modified hGPR75_1347, hGPR75_1349, hGPR75_1687, hGPR75_1764, and hGPR75_443, including replacing the nucleotides at the 5' end and / or 3' end of the sense strand, and / or replacing the nucleotides at the 5' end and / or 3' end of the antisense strand, to obtain the allosteric siRNA sequences: hGPR75_1347G, hGPR75_1349G, hGPR75_1687G, hGPR75_1764G, hGPR75_443A, and hGPR75_443AG. The specific siRNA sequences before and after allosteric modification can be seen in Table 7 below.

[0132] Table 7 Allosteric sequences of siRNAs

[0133]

[0134] Specifically, Example 6 used the same method as Example 3 to test the unmodified siRNAs shown in Table 7, and the final concentration of each group of siRNAs in the culture system was set to 1 nM, and the following data in Table 8 was obtained.

[0135] Table 8 Results of dual luciferase detection experiments in Example 6

[0136]

[0137] As shown in Table 8, the unmodified siRNAs had slightly lower inhibitory rates on GPR75 gene in 293T cells than the modified siRNAs, but still showed significant inhibitory activity. Among them, at a final concentration of 1 nM, corresponding to the blank control group, the inhibitory rate of hGPR75_1764 on GPR75 gene expression in 293T cells reached 91.92%, showing significant inhibitory activity.

[0138] Further, as shown in Table 8, the allosteric sequences of siRNA also showed significant inhibition effect on the expression of GPR75 gene, and the inhibition rate was similar to that before allostery. Among them, at a final concentration of 1 nM, equivalent to the blank control group, the inhibition rate of hGPR75_1764G on the expression of GPR75 gene in 293T cells reached 91.46%, showing significant inhibitory activity.

[0139] Example 7

[0140] The applicant further compares the inhibitory activity of the siRNA sequences of the present application with the siRNA sequences targeting GPR75 gene in the prior art.

[0141] Specifically, the applicant found the closest prior art US20230392156A1 through the patent database of PatSnap. The comparison of the siRNA sequences in Table 7 of the present application with the closest sequences in US20230392156A1 is shown in Table 9 below:

[0142] Table 9 Sequence alignment table of the present application and US20230392156A1

[0143]

[0144] Referring to the comparison group 1 of Table 9, the closest prior art to the sequences hGPR75_1347, hGPR75_1348, hGPR75_1349, hGPR75_1347G, hGPR75_1349G of the present application is AD-1424540, see Table 4 on page 137 of the specification of US20230392156A1, the remaining amount of RLuc / FLuc of AD-1424540 under the condition of a final concentration of 10 nM is 17.507%, and the inhibition rate is 82.493%. Referring to Example 6 of the present application, the inhibition rates of hGPR75_1347, hGPR75_1348, hGPR75_1349, hGPR75_1347G, hGPR75_1349G sequences under the condition of 1 nM are 87.38%, 84.83%, 82.58%, 84.84% and 83.44% respectively, which are still improved by 4.89%, 2.34%, 0.09%, 2.35% and 0.95% respectively compared with AD-1424540 at a lower concentration; and referring to Example 3 of the present application, the inhibition rates of hGPR75_1347AM13, hGPR75_1348AM13, hGPR75_1349AM13 under the condition of 1 nM can reach 90.41%, 92.68% and 91.08% respectively, which are significantly improved compared with the prior art.

[0145] Referring to Table 9, the closest prior art to hGPR75_443, hGPR75_443A, hGPR75_443AG sequence is AD-1423767, see US20230392156A1 patent specification page 138 Table 4, AD-1423767 has a RLuc / FLuc residual amount of 73.371% and an inhibition rate of 26.62% at a final concentration of 10 nM. Referring to Example 6 of the present application, hGPR75_443, hGPR75_443A, hGPR75_443AG have inhibition rates of 83.89%, 83.65%, and 72.17% respectively at 1 nM, all of which are significantly higher than AD-1423767, and among them, hGPR75_443 achieves an inhibition effect that is 3.15 times that of AD-1423767 at a lower concentration. In addition, referring to Example 4 of the present application, hGPR75_443AM13 can still achieve an inhibition effect of more than 80% at 0.1 nM, which is unpredictable by those skilled in the art.

[0146] Referring to Table 9, the closest prior art to hGPR75_1764 and hGPR75_1764G sequence is AD-1424920, see US20230392156A1 patent specification page 137 Table 4, AD-1424920 has a RLuc / FLuc residual amount of 12.141% and an inhibition rate of 87.859% at a final concentration of 10 nM. Referring to Example 6 of the present application, hGPR75_1764 and hGPR75_1764G sequence have inhibition rates of 91.92% and 91.46% respectively at 1 nM, and referring to Example 3 of the present application, hGPR75_1764AM13 has an inhibition rate of 93.63% at 1 nM, which is significantly improved compared with the prior art.

[0147] Referring to Table 9, the closest prior art to hGPR75_1687 and hGPR75_1687G sequence is AD-1424841, see US20230392156A1 patent specification page 137 Table 4, AD-1424841 has a RLuc / FLuc residual amount of 14.574% and an inhibition rate of 85.426% at a final concentration of 10 nM. Referring to Example 6 of the present application, hGPR75_1687 and hGPR75_1687G sequence have inhibition rates of 84.96% and 74.39% respectively at 1 nM, and achieve similar inhibition effects as AD-1424841 at a lower concentration.

[0148] Referring to Table 9, the closest prior art to the sequence of hGPR75_1727 is AD-1424880, see Table 4 on page 137 of the specification of US20230392156A1 patent, AD-1424880 has a RLuc / FLuc residual amount of 36.367% at a final concentration of 10 nM, and an inhibition rate of 63.633%. Referring to Example 6 of the present application, the hGPR75_1727 of the present application has an inhibition rate of 74.79% at a concentration of 1 nM, and the inhibition effect achieved at a lower concentration is still 11.16% higher than that of AD-1424880. Referring to Example 3 of the present application, the hGPR75_1727 AM13 can still achieve an inhibition effect of more than 80% at a concentration of 1 nM, which is unpredictable by those skilled in the art.

[0149] In order to further compare the inhibition activity of the modified siRNA, the applicant also synthesized the modified sequences of the above-mentioned sequences shown in the US20230392156A1 patent, see Table 10 below for the specific sequences, and used the same method as in Example 3 to conduct experiments, and the final concentration of each group of siRNA in the culture system was set to 0.1 nM, and the following Table 11 data was obtained.

[0150] Table 10 Synthesized prior sequences

[0151]

[0152] Table 11 Dual luciferase assay results of prior sequences

[0153]

[0154] Referring to the experimental results of Example 4 and the data of Table 11, the following Table 12 is obtained.

[0155] Table 12 Comparison of inhibition rates of siRNA sequences of the present application and prior sequences

[0156]

[0157] As shown in Table 12 above, the inhibition rate of the siRNA sequence of the present application on GPR75 gene expression is significantly higher than that of the prior sequence, which has an unexpected technical effect.

[0158] It is apparent that a person skilled in the art can, without departing from the scope of the application, implement the application in other specific forms, and thus the embodiments described above are to be considered in all respects only as illustrative and non-restrictive, the scope of the application being indicated by the claims hereafter rather than the above description, and all changes falling within the meaning and range of equivalency of the claims are therefore to be embraced therein.

[0159] Furthermore, it should be understood that although the description is made according to the embodiments, not every embodiment contains only one independent technical solution, and the description is made in this way only for the sake of clarity, and a person skilled in the art should consider the description as a whole, and the technical solutions in each embodiment can also be properly combined to form other embodiments that can be understood by a person skilled in the art.

Claims

1. A siRNA that inhibits GPR75 gene expression, characterized in that, It includes a justice chain and an antisense chain, wherein the justice chain and the antisense chain are at least partially complementary to form a dual-chain region; Wherein, the nucleotide sequence of the sense strand is shown in SEQ ID NO. 99, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 207; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO.223, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

229.

2. The siRNA according to claim 1, characterized in that, At least one nucleotide in the sense strand and / or the antisense strand is a modified nucleotide.

3. The siRNA according to claim 2, characterized in that, The modifications include one or more combinations of 2'-methoxy modification, 2'-fluoro modification, thiophosphate modification, vinyl phosphate modification and 2'-deoxynucleotide modification.

4. The siRNA according to claim 3, characterized in that, In the direction from the 5' end to the 3' end, at least the nucleotides at positions 1-6, 8, and 12-21 of the sense strand are 2'-methoxy modified nucleotides, and at least the nucleotides at positions 1, 3-5, 7-9, 11, 13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides; and / or, In the direction from the 5' end to the 3' end, at least the nucleotides at positions 7 and 9-11 of the sense strand are 2'-fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are 2'-fluorinated nucleotides; and / or, In a direction from the 5' end to the 3' end, at least the nucleotides located at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 of the antisense strand are linked by phosphate thioester groups; and / or, Following the direction from the 5' end to the 3' end, at least the nucleotide located at position 12 of the antisense strand is a 2'-deoxynucleotide; and / or, In the direction from the 5' end to the 3' end, the nucleotide at position 1 and / or position 21 of the positive strand is linked to a reverse debase nucleotide, and the nucleotide at position 1 and / or position 21 of the positive strand is linked to the reverse debase nucleotide by a phosphate thioester group; and / or, In the direction from the 5' end to the 3' end, at least the nucleotide at the first position of the antisense strand is a vinyl phosphate modified nucleotide.

5. The siRNA according to any one of claims 1 to 4, characterized in that, The siRNA is coupled to one or more nucleotides on at least one strand or to one or more target groups or functional molecules via a linker or vector.

6. The siRNA as described in claim 5, characterized in that, The siRNA has a targeting group or functional molecule coupled to its internal or terminal strands of the sense and / or antisense strands.

7. The siRNA as described in claim 6, characterized in that, The targeting group is a small molecule, polypeptide, or antibody that targets the transferrin receptor (TfR); the functional molecule is 2'-O-hexadecyl.

8. The use of the siRNA according to any one of claims 1 to 7 in the preparation of a medicament for the prevention or treatment of diseases related to GPR75 gene expression.

9. A GPR75 inhibitor, characterized in that, Includes the siRNA described in any one of claims 1 to 7.

10. A medicament for the prevention or treatment of diseases associated with GPR75 gene expression, characterized in that, Includes the siRNA as described in any one of claims 1 to 7; and pharmaceutically acceptable vectors.

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