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] This divisional application is a divisional application of an earlier application filed on August 12, 2025, with application number 2025111243293 and invention title "An siRNA that inhibits GPR75 gene expression and its application". Technical Field

[0002] This application belongs to the field of biomedical technology, specifically relating to an siRNA that inhibits GPR75 gene expression and its application. Background Technology

[0003] The G protein-coupled receptor 75 (GPR75) gene encodes a 540-amino acid membrane protein receptor with typical G protein-coupled receptor structural features, namely seven transmembrane domains, with the N-terminus located extracellularly and the C-terminus intracellularly. The GPR75 gene is expressed in multiple human tissues, including the brain, liver, kidneys, and heart, and it is primarily involved in physiological processes such as energy metabolism, inflammatory responses, and metabolic disorders.

[0004] Studies have shown that overexpression of the GPR75 gene directly promotes diseases such as obesity, diabetes, cardiovascular disease, cancer, and fatty liver by regulating appetite, insulin sensitivity, vascular tone, and cancer cell migration. Therefore, reducing the expression level of GPR75 can influence the progression of related diseases, and inhibiting GPR75 gene expression has become a potential therapeutic strategy.

[0005] Small interfering RNA (siRNA) is a double-stranded RNA molecule that specifically silences the expression of target genes through RNA interference (RNAi) mechanisms. siRNA pairs complementaryly with the target mRNA, is processed into small fragments by the enzyme Dicer, and then binds to the RNA-induced silencing complex (RISC). The RISC then cleaves the target mRNA, thereby inhibiting its translation. Developing effective siRNAs targeting the GPR75 gene could provide new insights for the treatment of related diseases. Summary of the Invention

[0006] The purpose of this application is to provide an siRNA that inhibits GPR75 gene expression and its application, so as to provide a new treatment strategy for diseases related to GPR75 gene overexpression.

[0007] To achieve the above objectives, the first aspect of this application provides an siRNA for inhibiting GPR75 gene expression, comprising a sense strand and an antisense strand, wherein the antisense strand has a nucleotide sequence that differs from the nucleotide sequence shown in any one of SEQ ID NO.109~216 and SEQ ID NO.226~231 by no more than 3 nucleotides; the sense strand and the antisense strand are at least partially complementary 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 to 108 or any one of SEQ ID NO. 220 to 225, and the antisense strand has a nucleotide sequence shown in one of SEQ ID NO. 109 to 216 or SEQ ID NO. 226 to 231 corresponding to the sense strand.

[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 includes one or more combinations of 2'-methoxy modification, 2'-fluoro modification, thiophosphate modification, vinyl phosphate modification and 2'-deoxynucleotide modification.

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

[0013] In one or more embodiments, at least the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 of the antisense strand are linked by thiophosphate groups in the direction from the 5' end to the 3' end.

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

[0015] In one or more embodiments, the nucleotide at least at position 1 of the antisense strand is a vinyl phosphate modified nucleotide, in the direction from the 5' end to the 3' end.

[0016] In one or more embodiments, the nucleotide at the 1st and / or 21st position of the positive strand is linked to a reverse debasing nucleotide in the direction from the 5' end to the 3' end, and the nucleotide at the 1st and / or 21st position of the positive strand is linked to the reverse debasing nucleotide by a thiophosphate group.

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

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

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

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

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

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

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

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

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

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

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

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

[0029] In one or more embodiments, the siRNA is coupled to one or more nucleotides on at least one strand or coupled to one or more target groups or functional molecules via a adapter or vector.

[0030] In one or more embodiments, the siRNA has a targeting group or functional molecule coupled internally or at the end of the sense and / or antisense strands.

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

[0032] To achieve the above objectives, a second aspect of this application provides the use of the siRNA described in any of the above embodiments in the preparation of a medicament for the prevention or treatment of diseases related to GPR75 gene expression.

[0033] To achieve the above objectives, a third aspect of this application provides a GPR75 inhibitor comprising the siRNA described in any of the above embodiments.

[0034] To achieve the above objectives, the fourth aspect of this application provides a medicament for the prevention or treatment of diseases related to GPR75 gene expression, comprising the siRNA described in any of the above embodiments; and a pharmaceutically acceptable vector.

[0035] The advantages of this application, which differ from existing technologies, are:

[0036] This application provides siRNA for inhibiting GPR75 gene expression, and modifies the siRNA to ensure its stability and inhibitory activity. Experiments have shown that multiple groups of siRNAs in this application have significant inhibitory activity against GPR75 gene expression in cells. Among them, hGPR75_443AM13, hGPR75_1347AM13, hGPR75_1348AM13, hGPR75_1349AM13 and hGPR75_1764AM13 all achieved an inhibition rate of over 90% against GPR75 gene expression in cells at a concentration of 0.1 nM, demonstrating their application prospects in the preparation of drugs for the treatment or prevention of diseases related to GPR75 expression in humans.

[0037] Among the siRNAs provided in this application, hGPR75_1348AM13, hGPR75_1347AM13, and hGPR75_1764AM13 exhibit significant inhibitory activity against the GPR75 gene in cells, with an IC50 of less than 0.02 nM.

[0038] The siRNA provided in this application can be used to perform dual-target silencing by mixing two siRNAs, and the inhibitory activity is better than that of a single siRNA, showing a synergistic enhancement effect. Among them, hGPR75_1348AM13+hGPR75_1764AM13 has an IC50 of less than 0.01 nM against the GPR75 gene in cells, and has very outstanding inhibitory activity. Detailed Implementation

[0039] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application are clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application.

[0040] It should be noted that, in this invention, "modified nucleotide" is selected from at least one of the following groups: 2'-methoxy-modified nucleotides, 2'-fluoro-modified nucleotides, phosphate thioester-modified nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, locked nucleotides, unlocked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, reverse nucleotides, reverse base-free nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides. - Modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl- modified nucleotides, morpholino nucleotides, aminophosphates, nucleotides containing non-natural bases, tetrahydropyran modified nucleotides, 1,5-dehydrohexyl modified nucleotides, cyclohexenyl modified nucleotides, glycol nucleic acid nucleotides (GNA), open-ring nucleotides (UNA), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimics, or terminal nucleotides linked to cholesterol derivatives or dodecanoic acid bisdecamide groups.

[0041] In one embodiment of the present invention, "2'-fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with fluorine, which has the structure shown in the following formula (1);

[0042] In one embodiment of the present invention, "2'-methoxy modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group, which has the structure shown in the following formula (2);

[0043] In one embodiment of the present invention, "2'-deoxynucleotide modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a hydrogen atom, which has the structure shown in the following formula (3); wherein B refers to the modified or unmodified nucleotide base A, U, G, C, T or other nucleotide bases.

[0044]

[0045] In one embodiment of the present invention, the thiophosphate group in the "thiophosphate-modified nucleotide" has a structure as shown in formula (4). In one embodiment of the present invention, the nucleotide linked to the thiophosphate group is shown in formula (5):

[0046]

[0047] In one embodiment of the present invention, the VP-modified nucleotide is a vinyl phosphate ester modified nucleotide. In one embodiment of the present invention, the VP-modified and methoxy-modified nucleotide, i.e., the nucleotide modified with 5'-(E)-vinyl-2'-methoxy-modified phosphonate group (5'-(E)-VP-2'-OMe), is as shown in formula (6); in one embodiment of the present invention, the VP-modified, methoxy-modified, and thiophosphate-modified nucleotide, i.e., the 5'-PS-modified nucleotide (i.e., the 5'-(E)-vinyl-2'-methoxy-modified thiophosphonate ester-modified nucleotide), is as shown in formula (7).

[0048]

[0049] In one embodiment of the present invention, the structural formula of the "reverse debasing nucleotide" is shown in formula (8).

[0050]

[0051] In one embodiment of the present invention, the siRNA is coupled to a functional molecule, the coupled functional molecule including N-acetylgalactosamine (GalNAc), lipophilic molecules, peptides, small molecule drugs, antibodies, etc.

[0052] In one embodiment of the present invention, siRNA may be coupled to one or more coupling groups containing functional molecules;

[0053] In one embodiment of the present invention, the functional molecule is a small molecule, polypeptide, or antibody that targets the transferrin receptor (TfR);

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

[0055] In one embodiment of the invention, the functional molecule is a lipophilic molecule (or a lipophilic or lipophilic moiety). The siRNA comprises one or more lipophilic moieties coupled or conjugated to one or more nucleotides on at least one chain via a linker or vector. 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 varying chain lengths.

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

[0057]

[0058] In one embodiment of the present invention, the C16 may be attached to any nucleotide of the positive strand.

[0059] The lipophilic portion is described in more 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 tone, and cancer cell migration. Therefore, reducing GPR75 expression levels can influence the progression of related diseases, making GPR75 gene inhibition a potential therapeutic strategy.

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

[0062] Example 1: Sequence Design

[0063] First, the applicant designed multiple siRNA sequences based on the mRNA sequence encoding the GPR75 gene from the NCBI database. Please refer to Table 1 below for the specific siRNA sequences.

[0064] Table 1. Sequences and IDs of siRNAs that inhibit GPR75

[0065]

[0066]

[0067]

[0068]

[0069] Example 2: siRNA Modification

[0070] To improve the stability of siRNA and effectively suppress 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 above-mentioned siRNA is as follows:

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

[0073] Following the direction from the 5' end to the 3' end, the nucleotides located at positions 7 and 9-11 of the sense strand are 2'-fluorinated, and the nucleotides located at positions 2, 6, 10, 14, and 16 of the antisense strand are 2'-fluorinated.

[0074] In the direction from the 5' end to the 3' end, the nucleotides located at the 1st and 2nd positions, the nucleotides at the 2nd and 3rd positions, the nucleotides at the 19th and 20th positions, and the nucleotides at the 20th and 21st positions of the antisense strand are linked by thiophosphate groups.

[0075] The nucleotide at position 12 of the antisense strand is modified with 2'-deoxynucleotide in the direction from 5' end to 3' end. If the nucleotide at position 12 is uracil nucleotide, it is replaced with thymine deoxyribonucleotide.

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

[0077] Following the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 21 of the positive strand are linked to the reverse debasing nucleotide (InvAb), and the nucleotides at positions 1 and 21 of the positive strand are linked to the reverse debasing nucleotide (InvAb) via thiophosphate groups.

[0078] Based on the above modification scheme, the modified siRNA was obtained, as detailed in Table 2 below.

[0079] In Table 2 below, m represents a nucleotide whose left adjacent nucleotide is 2'-methoxy modified; f represents a nucleotide whose left adjacent nucleotide is 2'-fluoro modified; s represents a nucleotide whose left and right adjacent nucleotides are modified with a thiophosphate group; (d) represents a nucleotide whose left adjacent nucleotide is 2'-deoxynucleotide modified; (InvAb) represents a reverse debasing nucleotide; and VP represents a nucleotide whose right adjacent nucleotide is vinyl phosphate modified.

[0080] Table 2 Modified siRNA sequences

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

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

[0089] To verify the inhibitory activity of the aforementioned siRNA on GPR75 gene expression, the applicant provided an on-target activity assay for the modified siRNAs listed in Table 2. The assay used a plasmid vector constructed with the psiCHECK2 vector, a type of plasmid vector that monitors changes in the expression of a target gene fused with a reporter gene. This vector uses Renali luciferase as the primary reporter gene. The target fragment is cloned into a multiple cloning site downstream of the translation stop codon of Renali luciferase. The RNAi process, initiated by the synthesized siRNA, targets the target gene, leading to the cleavage and subsequent degradation of the fused mRNA. By detecting changes in Renali luciferase activity, it can be determined whether a targeting relationship exists between the siRNA and the target gene fragment.

[0090] The experimental procedure is as follows:

[0091] Step 1: Plasmid Construction Detection

[0092] A single copy of the insert sequence having the nucleotide sequence shown in SEQ ID NO. 217 was cloned into the Xho I / Not I site of the psiCHECKTM-2 plasmid (Suzhou Gemma Gene Co., Ltd., catalog number C09005) to obtain the detection plasmid. The insert sequence was a fragment extracted from the full-length human GPR75 mRNA sequence, and included target sequences that were completely complementary to the antisense strands of all siRNAs shown in Table 1.

[0093] Step 2: Cell Culture and Transfection

[0094] The siRNA to be tested was diluted with DEPC water to obtain the siRNA dilution solution. Then, the siRNA dilution solution was added to the 96-well plate at a rate of 5 μL / well.

[0095] Add 12.5 μL of opti-MEM (Gibco, catalog number 31985-070) containing 20 ng of detection plasmid to a 96-well plate, add 32.5 μL of opti-MEM to a 96-well plate, add 0.3 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, catalog number 11668-019) to a 96-well plate, and let stand at room temperature (25 ℃) for 15 min to obtain the mixed system.

[0096] After settling, it will contain 2×10 5 293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were added to 96-well plates at a rate of 50 μL / well using DMEM complete medium (purchased from Transgen Biotech, catalog number FI101-01) and cultured in a 5% (v / v) CO2, 37 °C cell culture incubator for 24 h for subsequent dual-luciferase assay.

[0097] The final concentration of siRNA in the culture system was 1 nM. In the experiment, a blank control group MOCK and a negative control group NC were set up. The blank control group MOCK was the group with only interference reagent added and no sequence added. The negative control group NC was the group with a final concentration of 1 nM of NC added to the wells. The NC sequence is shown in Table 3.

[0098] Table 3 Negative control group sequences

[0099] +

[0100] Step 3: Dual-luciferase assay

[0101] After culture, the cells in the 96-well plate were subjected to dual-luciferase assay. Three replicates were used for each siRNA assay. The specific assay method is as follows:

[0102] Take the dual-luciferase assay kit (purchased from Promega, catalog number E2940), dilute the 5× lysis buffer in the kit with water to make 1× lysis buffer, and prepare substrate 1 and substrate 2 according to the kit instructions.

[0103] Take a 96-well plate, discard the supernatant, dilute each well with PBS buffer (purchased from Hyclone, catalog number SH30256.01) and wash twice, then add 1× lysis buffer at a rate of 50 μL / well to the 96-well plate and incubate at room temperature (25 ℃) for 20 min to lyse the cells in the 96-well plate to obtain lysis buffer;

[0104] The lysis buffer was aspirated from the original 96-well plate and added to a new opaque 96-well plate at a rate of 30 μL / well. Substrate 1 and Substrate 2 were then added to the 96-well plate in portions of 30 μL / well / time. After each addition of substrate, the 96-well plate was analyzed using a multi-mode microplate reader to obtain the numerical results of firefly luciferase and Renilla luciferase, respectively.

[0105] The emission ratio of each well in a 96-well plate is calculated using the following formula:

[0106] The luminescence ratio is calculated as follows: Renilla luciferase value / Firefly luciferase value. The luminescence ratio of each well in the 96-well plate is calculated.

[0107] The luminescence ratio of each test group (i.e., the group with added siRNA), blank control group, and negative control group is the average of the luminescence ratios of the three replicates;

[0108] Using the luminescence ratio of the blank control group as a benchmark, the luminescence ratios of each test group and negative control group were normalized to obtain the ratio R of luminescence ratio (test) / luminescence ratio (control), which represents the expression level (i.e., relative residual activity) of Renaissance luciferase reporter gene.

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

[0110] Table 4. Results of dual-luciferase assay in Example 3

[0111]

[0112]

[0113] As shown in the table above, all siRNAs except hGPR75_1269AM13 exhibited inhibitory effects on GPR75 gene expression. Among them, equivalent to the blank control group, hGPR75_443AM13, hGPR75_1347AM13, hGPR75_1348AM13, hGPR75_1349AM13, and hGPR75_1764AM13 all showed an inhibition rate of over 90% on GPR75 gene expression in 293T cells at a concentration of 0.1 nM, demonstrating significant inhibitory activity.

[0114] Example 4: Detection of the inhibitory activity of different concentrations of siRNA on GPR75 gene expression

[0115] To further investigate the effect of siRNA concentration on inhibitory activity in the culture system, the applicant selected siRNAs with inhibition rates greater than 75% from Example 3 for further experiments, namely hGPR75_435AM13, hGPR75_437AM13, hGPR75_438AM13, hGPR75_443AM13, hGPR75_568AM13, hGPR75_937AM13, hGPR75_967AM13, hGPR75_1345AM13, hGPR75_1346AM13, hGPR75_1347AM13, hGPR75_1348AM13, and hGPR75_1349A. M13, 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, in Example 4, the same method as in Example 3 was used for the experiment. The final concentrations of the siRNA to be tested in the culture system were set to 0.1 nM and 0.01 nM, respectively, and the data in Table 5 below were obtained.

[0117] Table 5. Results of dual-luciferase assay in Example 4

[0118]

[0119] As shown in Table 5, the inhibitory activity of each siRNA on GPR75 gene expression was reduced at a final concentration of 0.01 nM compared to a final concentration of 0.1 nM, but it still showed an inhibitory effect on GPR75 gene expression.

[0120] At a final concentration of 0.01 nM, equivalent to the blank control group, hGPR75_1347AM13 and hGPR75_1348AM13 inhibited GPR75 gene expression in 293T cells by more than 64%, demonstrating significant inhibitory activity.

[0121] Example 5: IC50 detection of half-maximal inhibitory concentration

[0122] The applicant further selected the siRNAs that exhibited excellent inhibitory activity at both final concentrations in Example 4 for experiments, namely: hGPR75_1348AM13, hGPR75_1347AM13, hGPR75_1764AM13, hGPR75_443AM13, hGPR75_1687AM13, hGPR75_1727AM13, and hGPR75_1349AM13.

[0123] In addition, the applicant also introduced two siRNAs into the mixed test group, specifically hGPR75_1348AM13+hGPR75_1764AM13, hGPR75_443AM13+hGPR75_1727AM13, hGPR75_1687AM13+hGPR75_1349AM13, and hGPR75_1347AM13+hGPR75_1764AM13, with the concentrations of the two siRNAs being the same in the mixed test group.

[0124] Specifically, in Example 5, the same method as in Example 3 was used for the experiment. The final concentrations of the siRNAs to be tested in the culture system were set to 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 rate of GPR75 gene expression at different final concentrations, the half-maximal inhibitory concentration (IC50) of each siRNA group was calculated using GraphPad Prism software, and the data in Table 6 below were obtained.

[0125] Table 6 IC50 calculation results for Example 5

[0126]

[0127] As shown in Table 6, the IC50 of the single siRNA test group against the GPR75 gene in 293T cells was all below 0.1 nM. Among them, hGPR75_1348AM13, hGPR75_1347AM13 and hGPR75_1764AM13 showed significant inhibitory activity against the GPR75 gene in 293T cells with an IC50 below 0.02 nM.

[0128] In addition, the IC50 of each mixed test group was lower than that of the single siRNA it included, showing a synergistic enhancement effect; among them, hGPR75_1348AM13+hGPR75_1764AM13 had an IC50 of less than 0.01 nM against the GPR75 gene in 293T cells, showing 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 modified siRNAs with superior inhibitory activity from Example 3 and tested the inhibitory activity of their corresponding unmodified siRNAs on GPR75 gene expression. Specifically, these included hGPR75_1347, hGPR75_1348, hGPR75_1349, hGPR75_1687, hGPR75_1727, hGPR75_1764, and hGPR75_443.

[0131] To verify the inhibitory activity of the modified siRNA sequences on GPR75 gene expression, the applicant further modified hGPR75_1347, hGPR75_1349, hGPR75_1687, hGPR75_1764, and hGPR75_443. The modification methods included replacing nucleotides at the 5' and / or 3' ends of the sense strand and / or replacing nucleotides at the 5' and / or 3' ends of the antisense strand, resulting in the modified siRNA sequences: hGPR75_1347G, hGPR75_1349G, hGPR75_1687G, hGPR75_1764G, hGPR75_443A, and hGPR75_443AG. The specific pre- and post-modification siRNA sequences are shown in Table 7 below.

[0132] Table 7. Allosteric sequences of siRNA

[0133]

[0134] Specifically, in Example 6, the unmodified siRNAs shown in Table 7 were tested using the same method as in Example 3. The final concentration of each group of siRNAs to be tested in the culture system was set to 1 nM, and the data in Table 8 below were obtained.

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

[0136]

[0137] As shown in Table 8, the inhibition rate of unmodified siRNA on GPR75 gene expression in 293T cells was slightly lower than that of modified siRNA, but it still showed significant inhibitory activity. At a final concentration of 1 nM, equivalent to the blank control group, hGPR75_1764 inhibited GPR75 gene expression in 293T cells by 91.92%, showing significant inhibitory activity.

[0138] Furthermore, as shown in Table 8, the allosteric sequence of siRNA also showed a significant inhibitory effect on GPR75 gene expression, with an inhibition rate similar to that before the allosteric sequence. Specifically, at a final concentration of 1 nM, equivalent to the blank control group, hGPR75_1764G inhibited GPR75 gene expression in 293T cells by 91.46%, demonstrating significant inhibitory activity.

[0139] Example 7

[0140] The applicant further compared the inhibitory activity of the siRNA sequence of this application with that of siRNA sequences targeting the GPR75 gene in the prior art.

[0141] Specifically, the applicant found the closest prior art to be US20230392156A1 through a search of the PatSnap patent database. The comparison between the siRNA sequence in Table 7 of this application and the closest sequence in US20230392156A1 is shown in Table 9 below:

[0142] Table 9 Sequence Alignment Table between this Application and US20230392156A1

[0143]

[0144] Referring to comparison group 1 in Table 9, the prior art closest to the hGPR75_1347, hGPR75_1348, hGPR75_1349, hGPR75_1347G, and hGPR75_1349G sequences of this application is AD-1424540. See Table 4 on page 137 of the specification of US20230392156A1. AD-1424540 has an RLuc / FLuc residual of 17.507% and an inhibition rate of 82.493% at a final concentration of 10 nM. Referring to Example 6 of this application, the inhibition rates of hGPR75_1347, hGPR75_1348, hGPR75_1349, hGPR75_1347G, and hGPR75_1349G sequences at 1 nM were 87.38%, 84.83%, 82.58%, 84.84%, and 83.44%, respectively. Compared with AD-1424540, the inhibition effect was still improved by 4.89%, 2.34%, 0.09%, 2.35%, and 0.95% at lower concentrations, respectively. Furthermore, referring to Example 3 of this application, the inhibition rates of hGPR75_1347AM13, hGPR75_1348AM13, and hGPR75_1349AM13 at 1 nM could reach 90.41%, 92.68%, and 91.08%, respectively, which are significantly improved compared with the prior art.

[0145] Referring to comparison group 2 in Table 9, the prior art closest to the hGPR75_443, hGPR75_443A, and hGPR75_443AG sequences is AD-1423767. See Table 4 on page 138 of the specification of US20230392156A1. AD-1423767 has an RLuc / FLuc residual of 73.371% and an inhibition rate of 26.62% at a final concentration of 10 nM. Referring to Example 6 of this application, the inhibition rates of hGPR75_443, hGPR75_443A, and hGPR75_443AG at 1 nM were 83.89%, 83.65%, and 72.17%, respectively, all significantly higher than AD-1423767. Notably, hGPR75_443 achieved an inhibition effect 3.15 times that of AD-1423767 at a lower concentration. Furthermore, referring to Example 4 of this application, hGPR75_443AM13 still achieved an inhibition effect of over 80% at 0.1 nM, which was unexpected by those skilled in the art.

[0146] Referring to comparison group 3 in Table 9, AD-1424920 is the closest to the hGPR75_1764 and hGPR75_1764G sequences. See Table 4 on page 137 of the specification of US20230392156A1. At a final concentration of 10 nM, AD-1424920 has an RLuc / FLuc residual of 12.141% and an inhibition rate of 87.859%. Referring to Example 6 of this application, the hGPR75_1764 and hGPR75_1764G sequences have inhibition rates of 91.92% and 91.46% respectively at 1 nM. Furthermore, referring to Example 3 of this application, hGPR75_1764AM13 achieves an inhibition rate of 93.63% at 1 nM, representing a significant improvement compared to existing technologies.

[0147] Referring to comparison group 4 in Table 9, the prior art closest to the hGPR75_1687 and hGPR75_1687G sequences is AD-1424841. See Table 4 on page 137 of the specification of US20230392156A1. At a final concentration of 10 nM, AD-1424841 showed an RLuc / FLuc residual of 14.574% and an inhibition rate of 85.426%. In contrast, according to Example 6 of this application, the hGPR75_1687 and hGPR75_1687G sequences showed inhibition rates of 84.96% and 74.39% respectively at 1 nM, achieving similar inhibitory effects to AD-1424841 at lower concentrations.

[0148] Referring to comparison group 5 in Table 9, the prior art closest to the hGPR75_1727 sequence is AD-1424880. See Table 4 on page 137 of the specification of US20230392156A1. AD-1424880, at a final concentration of 10 nM, has an RLuc / FLuc residual of 36.367% and an inhibition rate of 63.633%. However, referring to Example 6 of this application, hGPR75_1727 in this application achieves an inhibition rate of 74.79% at 1 nM, and the inhibition effect at a lower concentration is still 11.16% higher than that of AD-1424880. Furthermore, referring to Example 3 of this application, hGPR75_1727AM13 can still achieve an inhibition effect of over 80% at 1 nM, which is unexpected by those skilled in the art.

[0149] To further compare the inhibitory activity of the modified siRNA, the applicant also synthesized a modified sequence of the above sequence shown in US20230392156A1 patent. The specific sequence is shown in Table 10 below. The experiment was conducted using the same method as in Example 3. The final concentration of each group of siRNA to be tested in the culture system was set to 0.1 nM, and the data in Table 11 below were obtained.

[0150] Table 10. Existing sequences synthesized

[0151]

[0152] Table 11. Experimental results of dual-luciferase detection of existing sequences.

[0153]

[0154] The experimental results of Example 4 were compared with the data in Table 11, and the following Table 12 was obtained.

[0155] Table 12 Comparison of inhibition rates of the siRNA sequence of this application with existing sequences

[0156]

[0157] As shown in Table 12 above, the siRNA sequence of this application has a significantly higher inhibition rate on GPR75 gene expression than existing sequences, and has unexpected technical effects.

[0158] It will be apparent to those skilled in the art that this application is not limited to the details of the exemplary embodiments described above, and that this application can be implemented in other specific forms without departing from the spirit or essential characteristics of this application. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of this application is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within this application.

[0159] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An siRNA that inhibits the expression of a GPR75 gene, characterized in that, complementary to the antisense strand to form a double-stranded region; wherein the nucleotide sequence of the sense strand is as set forth in SEQ ID NO. 10, and the nucleotide sequence of the antisense strand is as set forth in SEQ ID NO. 118; or, the nucleotide sequence of the sense strand is as set forth in SEQ ID NO. 224, and the nucleotide sequence of the antisense strand is as set forth in SEQ ID NO. 230; or, the nucleotide sequence of the sense strand is as set forth in SEQ ID NO. 225, and the nucleotide sequence of the antisense strand is as set forth in SEQ ID NO.

231.

2. The siRNA of claim 1, wherein At least one of the nucleotides in the sense strand and / or the antisense strand is a modified nucleotide.

3. The siRNA of claim 2, wherein The modification comprises one or more of 2'-methoxy modification, 2'-fluoro modification, phosphorothioate modification, vinylphosphonate modification and 2'-deoxynucleotide modification in combination.

4. The siRNA of claim 3, wherein According to the direction from 5' end to 3' end, at least the nucleotides at positions 1-6, 8, 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, 17-21 of the antisense strand are 2'-methoxy modified nucleotides; and / or, According to the direction from 5' end to 3' end, at least the nucleotides at positions 7, 9-11 of the sense strand are 2'-fluoro modified nucleotides, and at least the nucleotides at positions 2, 6, 10, 14, 16 of the antisense strand are 2'-fluoro modified nucleotides; and / or, According to the direction from 5' end to 3' end, at least 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 group; and / or, According to the direction from 5' end to 3' end, at least the nucleotide at position 12 of the antisense strand is a 2'-deoxynucleotide; and / or, According to the direction from 5' end to 3' end, the nucleotide at position 1 and / or 21 of the sense strand is connected with an inverted abasic nucleotide, and the nucleotide at position 1 and / or 21 of the sense strand and the inverted abasic nucleotide are connected by phosphorothioate group; and / or, According to the direction from 5' end to 3' end, at least the nucleotide at position 1 of the antisense strand is a vinylphosphonate modified nucleotide.

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

6. The siRNA as claimed in claim 5, wherein, The siRNA has a targeting group or a functional molecule coupled internally or terminally to the sense strand and / or the antisense strand.

7. The siRNA as claimed in claim 6, wherein, The targeting group is a small molecule, a polypeptide or an antibody targeting transferrin receptor (TfR); and the functional molecule is 2'-O-hexadecyl.

8. Use of the siRNA according to any one of claims 1 to 7 in the preparation of a medicament for preventing or treating a disease associated with the expression of GPR75 gene.

9. A GPR75 inhibitor, characterized in that, The siRNA according to any one of claims 1 to 7.

10. A medicament for preventing or treating a disease associated with GPR75 gene expression, characterized by, The siRNA comprising any one of claims 1 to 7; and a pharmaceutically acceptable carrier.

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