Small interfering RNA of GDF15 gene as well as preparation method and application of small interfering RNA

Abstract

The invention relates to a small interfering RNA (Ribonucleic Acid) of a GDF15 gene, and a preparation method and application of the small interfering RNA. The small interfering RNA comprises a main body part and a modification part, the main body part contains a positive-sense strand and an antisense strand, the positive-sense strand is at least one nucleic acid sequence in SEQ ID NO.1-9, and the antisense strand is a corresponding accounting sequence of at least one of SEQ ID NO.1-9 '; the small interfering RNA of the GDF15 gene can cut and degrade mRNA of the GDF15 to mediate gene silencing, reverse a tumor immunosuppression microenvironment and recover the anti-tumor effect of immune cells, and a potential clinical scheme is provided for tumor treatment.

Description

Technical Field

[0001] This disclosure relates to the field of biomedical technology, specifically to a small interfering RNA of the GDF15 gene, its preparation method, and its application. Background Technology

[0002] Liver cancer treatment options are limited. Early-stage treatment primarily involves surgery, but due to its insidious nature, most patients are diagnosed at an intermediate or advanced stage, resulting in a high recurrence rate 5 years post-surgery. Chemotherapy and targeted therapy are the main treatments for intermediate and advanced stages, but their efficacy is unsatisfactory. In recent years, immunotherapy has shown promise, but the hepatic suppressive tumor microenvironment (TME) and complex mechanisms present challenges. While its objective response rate in advanced liver cancer is approximately 20%, and overall survival is not ideal, it still holds positive significance. The applicant's previous work (WO2021197171A1) demonstrated that growth differentiation factor 15 (GDF15) is a novel target for liver cancer immunotherapy. Monoclonal antibody blocking of GDF15 can reverse the TME suppression state in liver cancer, inhibit tumor growth, and has no autoimmune side effects; however, it requires long-term use, lacks tumor targeting, and may induce adverse reactions such as anti-drug antibodies (ADA). Gene editing to knock out GDF15, while significantly inhibiting tumor growth, raises clinical safety concerns. Therefore, a highly effective and safe targeted complex for liver cancer is urgently needed. Summary of the Invention

[0003] This disclosure aims to address the problems existing in the prior art by providing a small interfering RNA for the GDF15 gene, its preparation method, and its application.

[0004] According to a first aspect of this disclosure, a small interfering RNA for the GDF15 gene is provided, comprising: The main body comprises a positive strand and an antisense strand; the positive strand comprises at least one nucleic acid sequence from SEQ ID NO.1 to SEQ ID NO.9, and the antisense strand comprises at least one nucleic acid sequence from SEQ ID NO.1' to SEQ ID NO.9'; the positive strand and the antisense strand correspond one-to-one. The modification portion is connected to the 3' end of the justice chain.

[0005] In one embodiment of this disclosure, the modified portion is N-acetylgalactosamine.

[0006] According to a second aspect of this disclosure, a method for preparing small interfering RNA of the GDF15 gene is also provided, comprising the following steps: S1: Using phosphoramide solid-phase synthesis technology, each base is added one by one to the 5' hydroxyl group of the cyclic primer chain to synthesize the main body of the small interfering RNA.

[0007] S2: N-acetylgalactosamine is attached to the 3' end of the positive strand of the small interfering RNA via a synthetic reaction involving phosphoramidite of thymine nucleotide modified with N-acetylgalactosamine.

[0008] In one embodiment of this disclosure, the primer chain includes an upstream primer and a downstream primer, the sequence of the upstream primer being 5'-AGATCAAGACGAGCCTGCAC-3', and the sequence of the downstream primer being 5'-ATAGGTCTGGAGCGACACCC-3'.

[0009] In one embodiment of this disclosure, the synthetic reaction includes: The deprotection reaction uses a dichloromethane solution of 3% trichloroacetic acid. A condensation reaction in which the phosphorusamide-protected nucleotide monomer is mixed with 5-ethylthio-1-H-tetrazole at a volume ratio of 1:1.5; A blocking reaction, wherein blocking solution A and blocking solution B are used to block the 5'-terminal hydroxyl group; An oxidation reaction, wherein the oxidation reaction uses a 0.02–0.05 mol / L iodine solution, wherein the iodine solution is a mixture of tetrahydrofuran, pyridine, and water; and Product post-processing and purification steps to obtain the target product.

[0010] In one embodiment of this disclosure, the synthetic reaction further includes 2'-oxymethyl, 2'-fluorine modified adenosine, guanosine, cytidine, uridine nucleoside phosphoramidide monomers, and a CPG carrier preloaded with a specific nucleoside.

[0011] In one embodiment of this disclosure, the molar ratio of the N-acetylgalactosamine modification to the GDF15 small interfering RNA is 1:1.

[0012] According to a third aspect of this disclosure, the use of a small interfering RNA of the GDF15 gene in the preparation of a GDF15 gene-related product for the treatment of liver cancer is also provided.

[0013] In one embodiment of this disclosure, the relevant product is a drug or a pharmaceutical composition; the drug or pharmaceutical composition further includes diluents, excipients and / or carriers that can be used in the pharmaceutical field.

[0014] According to the fourth aspect of this disclosure, the use of a small interfering RNA of the GDF15 gene in the preparation of GDF15 gene-related products for the treatment of tumors is also provided.

[0015] One beneficial effect of this disclosure is that the GDF15 small interfering RNA targeting liver cancer, through its linkage with N-acetylgalactosamine, enhances the liver cancer targeting specificity of the GDF15 small interfering RNA. N-acetylgalactosamine can specifically bind to desialyl glycoprotein expressed on the surface of hepatocytes, allowing the small interfering RNA to accumulate in the liver and be internalized by liver cancer cells. This leads to the cleavage and degradation of GDF15 mRNA, thereby mediating GDF15 gene silencing. This persistently reverses the immunosuppressive microenvironment within tumor cells, restoring the anti-tumor function of immune cells and achieving the goal of treating liver cancer. This approach avoids the limitations of repeated doses in traditional therapies and the safety issues associated with gene editing methods, reducing treatment costs and improving efficacy. Therefore, delivering GDF15 small interfering RNA to the liver cancer region to mediate GDF15 gene silencing, inhibit GDF15 function, and reverse the suppressive immune microenvironment of liver cancer represents a potential clinical treatment option.

[0016] Other features and advantages of this disclosure will become clear from the following detailed description of exemplary embodiments with reference to the accompanying drawings. Attached Figure Description

[0017] The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments of the present disclosure and, together with their description, serve to explain the principles of the present disclosure.

[0018] Figure 1 A simplified structural diagram of the GDF15 small interfering RNA provided in this disclosure; Figure 2 A schematic diagram of the synthesis steps of GalNAc-siGDF15 provided in this disclosure; Figure 3 The image shows the Western blot results of GDF15 protein expression level detection in cell samples transfected with 9 types of GalNAc-siGDF15 in this disclosure; Figure 4 To Figure 3 A statistical graph of GDF15 protein expression levels obtained from grayscale analysis; Figure 5 The image shows the qRT-PCR results of GDF15 mRNA expression levels in cell samples transfected with GalNAc-siGDF15 provided in this disclosure. Figure 6 The images show in vivo images of the five groups of mice in this disclosure, along with corresponding fluorescence value statistics. Figure 7 This is a diagram showing the size of liver tumors in each group of mice on day 22 of the experiment in this embodiment of the present disclosure; Figure 8 This is a statistical graph showing the changes in mouse body weight and liver weight ratio during the experiment disclosed in this publication; Figure 9 This is a statistical chart showing the survival time of five groups of mice in the embodiments of this disclosure; Figure 10 This is a graph showing the relative expression level of GDF15 mRNA in mouse liver tumor cells detected by qPCR in this embodiment of the present disclosure; Figure 11 This is a statistical graph showing the GDF15 content in the serum of 5 groups of mice after treatment, detected by ELISA in this embodiment of the present disclosure. Figure 12 The images show immunohistochemical staining of liver tumor sections and statistical graphs of GDF15 expression levels in the tissues of five groups of mice after treatment in this embodiment of the present disclosure. Figure 13 This is a statistical graph showing the proportion of infiltrating NK cells in mouse liver cancer tumor tissue detected by flow cytometry in this embodiment of the present disclosure. Figure 14 This is a statistical graph showing the proportion of CD8+ T cells infiltrating mouse liver cancer tumor tissue detected by flow cytometry in this embodiment of the present disclosure. Detailed Implementation

[0019] To make the inventive objectives, technical solutions, and beneficial technical effects of this application clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the embodiments described in this specification are merely illustrative and not intended to limit the scope of this application.

[0020] For simplicity, this paper only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an undefined range; and any lower limit can be combined with other lower limits to form an undefined range, just as any upper limit can be combined with any other upper limit to form an undefined range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can serve as its own lower or upper limit and be combined with any other point or individual value, or with other lower or upper limits, to form an undefined range.

[0021] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.

[0022] The foregoing description of this invention is not intended to describe every disclosed embodiment or implementation. Instead, the following description provides more specific examples of exemplary embodiments. Throughout the application, guidance is provided through a series of embodiments that can be used in various combinations. The examples listed are merely representative and should not be construed as exhaustive.

[0023] Immunotherapy plays a crucial role in cancer treatment and has been widely proven to be an effective method for treating various types of solid tumors, such as hepatocellular carcinoma, gastric cancer, lung cancer, and colon cancer. Unlike chemotherapy drugs that directly kill cancer cells, immunotherapy primarily works by enhancing the body's anti-tumor immune response.

[0024] The hepatocellular carcinoma (HCC) suppressive tumor microenvironment (TME) refers to the immunosuppressive tumor microenvironment present in HCC tissues. Specifically, tumor cells secrete cytokines and other substances within the tumor microenvironment, recruiting and maintaining immunosuppressive cells, while simultaneously inducing phenotypic and functional changes in effector immune cells (such as cytotoxic T cells). This creates an environment around the liver tumor that severely suppresses immune cell function, preventing the immune system from effectively recognizing and attacking cancer cells, thus leading to tumor immune escape and progression. In recent years, several monoclonal antibodies targeting GDF15 have entered clinical trials; however, they require long-term use, lack precise tumor targeting, and may produce adverse reactions. The small interfering RNA (SRNA) of the GDF15 gene disclosed in this paper exhibits high targeting and high safety in addressing the HCC suppressive TME and its complex mechanisms.

[0025] N-acetylgalactosamine is a derivative obtained by replacing the hydroxyl group at the C-2 position of galactose with an amino group and then acetylifying it. N-acetylgalactosamine is a high-affinity targeting ligand for the desialyl glycoprotein receptor. Desialyl glycoprotein is an endocytic receptor that is highly specifically expressed on the membrane surface of hepatocytes. Its endocytosis, along with clathrin-mediated endocytosis, can effectively transport N-acetylgalactosamine from the cell surface to the cytoplasm to form endosomes, thereby bringing a sufficient amount of siRNA into the cell.

[0026] To overcome the shortcomings of existing liver cancer treatment technologies, such as poor targeting, low gene silencing efficiency, and safety risks, this disclosure provides a small interfering RNA (SRNA) for the GDF15 gene. By constructing the GDF15 SRNA and covalently modifying the 3' end of the positive strand with N-acetylgalactosamine (hereinafter referred to as "GalNAc-siGDF15"), the SRNA can be internalized into cells through the specific binding of N-acetylgalactosamine to desialized glycoprotein, thereby silencing the GDF15 gene in liver cancer cells and reversing the therapeutic effect of the tumor immunosuppressive microenvironment. The specific embodiments of this disclosure are described below with reference to the accompanying drawings.

[0027] This disclosure provides a small interfering RNA for the GDF15 gene, comprising a main body and a modified part. The main body includes a sense strand and an antisense strand; the sense strand includes at least one nucleic acid sequence from SEQ ID NO.1 to SEQ ID NO.9, and the antisense strand includes at least one nucleic acid sequence from SEQ ID NO.1' to SEQ ID NO.9'; the sense strand and the antisense strand correspond one-to-one.

[0028] As shown in Table 1, the nucleotide sequences of the small interfering RNA for the GDF15 gene used in this invention are as follows: the sense strand sequences are shown in SEQ ID NO.1 to NO.9, and the corresponding antisense strand sequences are shown in SEQ ID NO.1' to NO.9'. Table 1

[0029] Furthermore, it also includes a chemically modified small interfering RNA nucleotide sequence of the GDF15 gene. The modified portion is attached to the 3' end of the positive strand.

[0030] Furthermore, the aforementioned chemical modification methods may include phosphate backbone modification, ribose modification, base modification, end-cap structure modification, Poly(A) tail modification, pseudouracil modification, RNA editing, chemical synthesis modification, peptide nucleotide modification, locked nucleotide modification, and methoxygenation. Or ethoxy modification and other modifications, etc.

[0031] Small interfering RNA (siRNA) is a double-stranded RNA molecule that can guide the silencing of specific genes, typically 20^64 molecules in length. 25 nucleotides. siRNA binds to its corresponding mRNA, triggering an RNA-induced silencing complex (RISC), which leads to the degradation of the target mRNA and prevents its translation into a protein. This mechanism is used in gene function research and therapeutic gene silencing.

[0032] To alter or improve the performance of siRNA, such as stability and reducing its immunogenicity, various chemical modifications can be applied to small interfering RNA. These modifications include ribosome modification, phosphodiester backbone modification, and terminal modification. These chemical modifications may alter the pharmacokinetic properties of siRNA, including but not limited to its stability, cellular uptake efficiency, and in vivo distribution.

[0033] In this disclosure, the small interfering RNA of the GDF15 gene is modified with N-acetylgalactosamine and is attached to the 3' end of the aforementioned positive strand. For example... Figure 1 As shown, the liver cancer-targeting GDF15 small interfering RNA modified with N-acetylgalactosamine possesses both a main body and a modified portion. From Figure 1The double-stranded structure and modification sites of the small interfering RNA are clearly visible in the figure. The gene sequence shown is the GalNAc-siGDF15-7 gene sequence, where the GalNAc modification is located at the 3' end of the siRNA's sense strand. The sense and antisense strands of the siRNA form hydrogen bonds with corresponding bases according to the complementary base pairing principle, resulting in the siRNA's double-stranded structure.

[0034] Furthermore, the motifs of the small interfering RNAs of the above 9 GDF genes are abbreviated. Taking M23886-siGdf15-7 as an example, after N-acetylgalactosamine modification, the detailed motifs of the sense and antisense strands of GalNAc-siGDF15-7 should be as follows: GalNAc-siGDF15-7's Chain of Justice: (mG)(mC)(mA)(mG)(mG)(mC)(2FA)(mA)(2FC)(2FU)(2FC)(mU)(mU)(mG)(mA)(mA)(mG)(mA)(mC)(mU)(mU)(dT)(dT)-galnac The ansaurus of GalNAc-siGDF15-7: (mA)(2FA)(mG)(mU)(mC)(2FU)(mU)(mC)(mA)(mA)(mG)(mA)(mG)(2FU)(mU)(2FG)(mC)(mC)(mU)(mG)(mC)(dT)(dT) Wherein, (mN) represents 2'-methoxy modification, (2FN) represents fluorinated modification, and galnac represents N-acetylgalactosamine modification.

[0035] Furthermore, the in vivo mechanism of action of the liver cancer-targeting GDF15 small interfering RNA modified with N-acetylgalactosamine is as follows: From recognition and entry into cells to exerting its interfering effect, GalNAc-modified siRNA drugs can bind to the ASGPR receptor on the surface of hepatocytes, enabling GalNAc-modified siRNA to target the liver. Simultaneously, through endocytosis, the siRNA enters the hepatocytes and is released into the cytoplasm, where it is assembled into RISC, mediating the cleavage and degradation of GDF15 mRNA, thereby reducing the expression of GDF15 protein.

[0036] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods used in the art, and the reagents used are all analytical grade reagents that can be obtained from legitimate suppliers.

[0037] Example 1: This disclosure also provides a method for preparing the small interfering RNA of the GDF15 gene mentioned above, comprising the following steps: S1: The main body of the small interfering RNA was synthesized by adding each base one by one to the 5' hydroxyl group of the cyclic primer strand using phosphoramide solid-phase synthesis technology. S2: N-acetylgalactosamine is attached to the 3' end of the positive strand of the small interfering RNA via a synthetic reaction involving phosphoramidite of thymine nucleotide modified with N-acetylgalactosamine.

[0038] In the preparation method disclosed herein, the molar ratio of N-acetylgalactosamine modification to GDF15 small interfering RNA is 1:1.

[0039] The primer chains used in the preparation method of small interfering RNA of GDF15 gene include upstream and downstream primers, and the sequences of upstream and downstream primers are shown in Table 2: Table 2

[0040] refer to Figure 2 The synthesis of N-acetylgalactosamine-modified liver cancer-targeting GDF15 small interfering RNA (hereinafter referred to as GalNAc-siGDF15) includes: deprotection reaction, condensation reaction, blocking reaction, oxidation reaction, and product post-processing and purification steps. The synthesis reaction also requires the addition of 2'-oxymethyl, 2'-fluorine modified adenosine, guanosine, cytidine, uridine nucleoside phosphoramidite monomers, and a CPG vector pre-loaded with specific nucleosides. In the embodiments of this disclosure, the vector used is a CPG vector. Two types of CPG vectors for GalNAc-siGDF15 were purchased from Wuhu Huaren Technology Co., Ltd., product catalog number: HR-01006003; their pore size is 500 Å; the construction method is loading via amino linkage of L96 and CPG; the appearance and loading capacity of the CPG vector need to be monitored after linking to ensure they meet expectations.

[0041] The GalNAc-CPG structure used for the Justice Chain is shown in the following figure:

[0042] The general 3'-CPG structure used for antisense chains is shown in the figure below:

[0043] Detailed information about GalNAc-L96-CPG is shown in Table 3 below: Table 3

[0044] In this embodiment of the invention, the method of ammonolysis of the CPG carrier is as follows: the synthesized DNA fragment is linked to the solid-phase carrier CPG, so the fragment needs to be cut off; ammonia can cleave the ester bond between the CPG linking compound and the initial nucleoside; specifically, the CPG with the synthesized sequence is soaked in ammonia for more than 1 hour.

[0045] The specific operational steps of the synthesis reaction in this embodiment are as follows: (1) Deprotection reaction: 3% trichloroacetic acid (TCA) in dichloromethane solution is introduced into the synthesis column (single-strand nucleic acid synthesizer-12P) and reacted for 1~2 min to remove the DMT (dimethoxytriphenylmethyl) protecting group of the nucleotide linked to the CPG (Controlled Pore Glass) to obtain the free 5'-hydroxy terminus for the next condensation reaction; (2) Condensation reaction: The phosphoramidite-protected nucleotide monomer (referring to the phosphoramidite group attached to the hydroxyl group of the C3 of the pentose nucleotide to protect the C3 hydroxyl group) is mixed with 5-ethylthio-1-H-tetrazole (concentration 0.25~0.5 mol / L) at a volume ratio of 1:1.5 and passed into the synthesis column. The reaction is carried out for 3~5 min to form the phosphoramidite-tetrazole active intermediate. The phosphoramidite-tetrazole active intermediate undergoes a condensation reaction with the deprotected 5'-hydroxyl group on the CPG support to form a 3',5'-phosphite bond, thereby extending the oligonucleotide chain forward by one base. The reaction time is 9~15 min. (3) Capping: After the condensation reaction, in order to prevent the unreacted 5'-hydroxyl group from being extended in the subsequent cycle reaction, the terminal hydroxyl group is often capped by acetylation; the capping solution A (phenoxyacetic anhydride acylation agent uses phenoxyacetic anhydride / THF / pyridine, the carbonyl carbon atom in its anhydride structure -CO-O-CO- is electron-deficient and is easily attacked by nucleophiles (such as hydroxyl -OH); THF, as an aprotic polar solvent, ensures that the reactants are in full contact; pyridine is a base to neutralize acidic substances and promote the reaction) and the capping solution B (10% N-methylimidazole / THF, 10% N-methylimidazole is a strong acylation reaction catalyst; THF, as an aprotic polar solvent, ensures that the reactants are in full contact with the catalyst) are introduced in sequence, and the reaction is carried out for 1~2 min; (4) Oxidation: Pass in 0.02~0.05 mol / L iodine solution (the solvent is a mixture of tetrahydrofuran, pyridine and water. THF provides excellent solubility and ensures that all reactants are homogeneous; pyridine acts as a base to neutralize harmful acid byproducts, protect the RNA chain, and stabilize the iodine solution; water is an essential reactant that combines with iodine to form a real active oxidant), react for 1~2 min, and oxidize the phosphite bond (P(III)) to the phosphotriester bond (P(V)) to enhance chain stability; (5) Post-processing: Wash three times with anhydrous acetonitrile for 30 seconds each time to remove residual oxidant; (6) Purification: The synthesized RNA was purified by HPLC to remove impurities and unreacted raw materials. A gradient elution was performed using acetonitrile / triethylamine acetate (TEAA) buffer as the mobile phase, with the target RNA and impurities eluted stepwise according to their retention time differences (e.g., acetonitrile concentration gradually increasing from 5% to 30%). The elution peaks were monitored using a UV detector (260 nm wavelength), and the fraction corresponding to the target product peak was collected to obtain high-purity RNA. The purity was calculated by the percentage of the main peak area or height to the total peak area or height to ensure that the purity met experimental requirements.

[0046] Example 2: This disclosure uses the N-acetylgalactosamine-modified liver cancer-targeting GDF15 small interfering RNA (hereinafter referred to as GalNAc-siGDF15) from Example 1 to verify and screen the efficiency of silencing the GDF15 gene in Hepa1-6 cells in vitro, and selects drugs for subsequent in vivo experiments. The experiment includes cell transfection, qRT-PCR detection of the relative expression level of GDF15 mRNA and Western blotting (WB) detection of GDF15 protein expression level. The specific operation steps are as follows: 2.1 Cell transfection One day before transfection, 1 × 10⁵ Hepa1-6 cells were seeded in 24-well plates and cultured in antibiotic-free DMEM high-glucose complete medium overnight in a cell culture incubator. The RNA obtained in Example 1 was then dissolved and diluted to 20 μM using RNase H₂O.

[0047] (1) Preparation of the transfection system: ① Dilute 20 nM siRNA with 50 µL Opti-MEM and mix by pipetting 3-5 times; ② Dilute 0.5µL of transfection reagent with 50µL Opti-MEM (using Lipofectamine 2000 transfection reagent from Thermo Fisher Scientific (Shanghai)) and let stand at room temperature for 5 min; ③ Mix siRNA and transfection reagent, and let stand at room temperature for 20 min to form a transfection complex; (2) Transfection procedure: Add 100 µL of the transfection complex to each well of a 24-well plate and mix well; place the cell plate in an incubator with sterile conditions, 37°C, 95%~100% humidity, darkness, 5% CO2 and 95% air for 18~48 h; after 4~6 h of transfection, the culture medium can be replaced with fresh medium (use DMEM medium containing 10% fetal bovine serum, all from Gibco (USA)). (3) Cell collection: Discard the culture medium, add 1 ml of trypsin to digest until the cells detach, add 2 ml of PBS buffer to stop digestion, centrifuge in 15 ml centrifuge tube (1200 rpm, 4 ℃, 5 min) to collect the cell pellet, resuspend the cell pellet in 2 ml of PBS buffer, centrifuge (1200 rpm, 4 ℃, 5 min) and collect the cell pellet. Repeat the above operation 3 times to obtain Hepa1-6 cell pellet.

[0048] 2.2 qRT-PCR detection of relative expression level of GDF15 mRNA (1) RNA extraction: ① After weighing the above cell pellet samples, place them in EP tubes. Add 1 μl of Trizol lysis buffer for every 100 μg of tissue. Use a Tissue Prep cell disruptor to disrupt the cells at low temperature (using 1-2 mm diameter zirconia grinding beads, frequency 28 Hz, run for 30 seconds, pause for 30 seconds, repeat 3 times). After that, let it stand at 4°C. Vortex once every 10 min (each vortex for 30 seconds), repeat 3 times. ② Centrifuge (1200 rpm, 4℃, 10 min), take the supernatant, add Trizol lysis buffer to make up the system to 1 ml, add 200 μl chloroform, mix by pipetting and shaking, and let stand at 4℃ for 3 min; ③ Centrifuge the above liquid (1200 rpm, 4℃, 10 min), take the uppermost colorless aqueous phase liquid, add an equal volume of isopropanol and mix by pipetting, let stand at -20℃ for 4 h and then centrifuge (1200 rpm, 4℃, 10 min), and discard the supernatant. ④ Add 75% ethanol at 4°C to the above precipitate, mix by pipetting, centrifuge (1200 rpm, 4°C, 10 min), discard the supernatant, repeat this step twice to obtain RNA.

[0049] (2) Reverse transcription (RT) ①Removal of genomic DNA: Add 5 μl of 4×gDNA wiper Mix, RNA template, and RNase-free ddH2O to an RNase-free PCR tube to bring the total volume to 16 μl. Mix well by pipetting and incubate at 42°C for 3 min. ② Prepare the reverse transcription system: Add 4 μl of 5×HiScriptⅡqRT SuperMixⅡ to the PCR tube above and mix well; set the reverse transcription conditions to 50℃ for 15 min; 85℃ for 5 s. After the reaction is complete, obtain the sample cDNA and use it immediately for qPCR reaction or store it at -20℃. (3) Real-time quantitative PCR (qPCR) ① Dilute the cDNA sample obtained above 5 times with RNase-free ddH2O to obtain cDNA dilution solution; ② Reaction system (20 μL): Prepare the reaction system by mixing 10 μL of 2×ChamQ SYBR qPCR Master Mix, 0.6 μL each of upstream and downstream primers, 0.6 μL of 50×ROX Reference Dye II, 2 μL of cDNA dilution buffer, and 7 μL of RNase-free ddH2O, and then add it to an eight-tube strip and centrifuge briefly (RCF2000×g, 30s, 4℃). ③ Place the above eight tubes into a 7500Fast real-time quantitative PCR system and set the reaction conditions as follows: 95℃, 15 s; 95℃, 10 s; 60℃, 45 s; repeat for 40 cycles. ④ Melting curve analysis: The instrument collects data under the default program preset reaction conditions and analyzes it with ABI Prism7500 SDS software; the software performs mathematical processing on the original fluorescence value (RFU) vs. temperature (T) data (usually an S-shaped curve), calculates its negative derivative (-d(RFU) / dT), and plots the negative derivative against the temperature to obtain the melting peak diagram.

[0050] 2.3 Western Blotting (WB) Detection of GDF15 Protein Expression The Western blot (WB) assay disclosed herein was performed using Future PAGE from Boyi Biotechnology (Changzhou). TM Protein pregels were prepared, and the protein samples were pretreated using the following method. The specific pretreatment steps are as follows: (1) Extraction of tissue protein ① Prepare protein lysis buffer: Add 1 Protease Inhibitor Cocktail Tablet to 10 mL of RIPA lysis buffer (strong), mix thoroughly, and place on ice for later use; ② Weigh the tissue sample from liquid nitrogen or freshly collected and place it in an EP tube. Add 500µL of protein lysis buffer to every 100mg of tissue sample and place on ice. The amount of tissue in each tube should be controlled between 50-100mg. ③ Add pre-cooled tissue homogenate beads and homogenize using a TissuePrep rapid tissue cell disruptor; ④ Transfer the homogenized liquid to a new EP tube and let it stand on ice for 30 minutes, vortexing it once every 10 minutes during this period; ⑤ Centrifuge the EP tube from the previous step (13000g, 4℃, 15min), and carefully aspirate the supernatant into a new EP tube to obtain the tissue protein lysis buffer.

[0051] (2) Determination of protein concentration by BCA method Protein concentration was measured using the BCA Protein Assay Kit (Enhanced Version), supplied by Beyotime (Shanghai).

[0052] ①Prepare protein standards: Add 1.2 mL of protein standard preparation solution to 30 mg BSA and dissolve thoroughly to obtain a 25 mg / mL protein standard solution; ②Preparation of BCA working solution: Mix reagents A and B thoroughly at a volume ratio of 50:1, let stand at room temperature, and prepare fresh before use; ③ Protein concentration detection: Dilute the protein standard with standard diluent to prepare standard protein solutions of different concentration gradients of 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL and 0.5 mg / mL. Add 20 µL of each solution to a 96-well plate, and set up 3 replicates for each concentration. ④ Dilute the protein sample to be tested 10 times and add it to a 96-well plate at a volume of 20µL per well, with 3 replicates for each concentration; ⑤ Add 200 μl of BCA working solution to each well and incubate at 37°C for 30 min; ⑥ The absorbance of each well at a wavelength of 562 nm was measured using an Epoch microplate spectrophotometer; ⑦ Plot a standard curve based on the absorbance of standard protein solutions of varying concentrations, and calculate the protein concentration of the sample based on the curve; ⑧ Add 1 / 4 volume of protein loading buffer (5×) to the protein sample, heat in a boiling water bath for 5 minutes to fully denature the protein; ⑨ Centrifuge at 12000g for 10 min at 4℃, then perform Western blotting or store at -20℃.

[0053] After the above pretreatment of the protein samples, this disclosure uses Western blotting (WB) to detect the expression level of GDF15 protein, including SDS-PAGE protein gel electrophoresis, wet transfer membrane formation, blocking and antibody incubation, and chemiluminescence imaging. The specific operation steps are as follows: (1) SDS-PAGE protein gel electrophoresis: The protein sample supernatant and protein marker were loaded. An appropriate volume of protein loading buffer (1×) was added to the wells at both ends of the sample, and the bands were made flush. Electrophoresis was performed at a constant voltage of 280V (the electrophoresis was terminated at the bromophenol blue position). The protein loading buffer (1×) used was obtained by adding 4 volumes of deionized water to SDS-PAGE protein loading buffer (5×) from Beyotime (Shanghai). (2) Protein wet transfer membrane: Cut out the desired gel region and immerse it in 1× transfer buffer (1× transfer buffer is made by adding 7.2g of tris(hydroxymethyl)aminomethane and 34.6g of glycine to ultrapure water to a final volume of 1920ml, stirring with a magnetic stirrer until completely dissolved, then adding 480ml of methanol and mixing well) for 10 min at 4℃; After the PVDF membrane is activated with methanol, assemble it according to the "sponge-3 layers of filter paper-gel-PVDF membrane-3 layers of filter paper-sponge" configuration, transfer at a constant current of 300mA at 4℃ for 50 min, then remove the PVDF membrane and immerse it in 1×TBST buffer for later use; (3) Blocking and antibody incubation: Block the PVDF membrane with 5% skim milk at room temperature for 2 hours → Dilute the primary antibody with antibody diluent and incubate overnight at 4°C → Wash the PVDF membrane 3 times with 1×TBST buffer for 10 minutes each time → Dilute the secondary antibody with antibody diluent and incubate overnight at 4°C → Wash the above PVDF membrane 3 times with 1×TBST buffer for 10 minutes each time. (4) Chemiluminescence imaging: Equal volumes of solutions A and B from the ECL kit (supplier: BeyoECL Plus Ultrasensitive ECL Chemiluminescence Kit, model: P0018S, 100ml) were mixed (protected from light) and uniformly added to the PVDF membrane. Images were acquired using a fully automated chemiluminescence imaging system, and the images were recorded after appropriate processing. Reference Figure 3 , Figure 3This image shows the Western blotting (WB) results of GDF15 protein expression levels in cell samples transfected with nine different GalNAc-siGDF15 transfection methods. The Hepa1-6 cells were divided into 11 groups, from left to right: "PC" represents cells treated with PBS buffer; "NC" represents cells transfected with GalNAc-siNC; and bands 1-9 represent cells transfected with GalNAc-siGDF15-1 to GalNAc-siGDF15-9. After culturing these 11 groups of cells under the same conditions for a period of time, cell samples were collected for WB analysis to detect GDF15 protein expression levels. The lighter the band, the lower the expression level. Among the nine GalNAc-siGDF15 bands, GalNAc-siGDF15-2, GalNAc-siGDF15-7, and GalNAc-siGDF15-9 showed lighter band colors than the others, with GalNAc-siGDF15-7 showing the lightest color, indicating that it had the best effect in interfering with GDF15 protein expression.

[0054] like Figure 4 As shown, this figure is for... Figure 3 The GDF15 protein expression level was statistically analyzed using grayscale analysis of the Western blot chemiluminescence (WB) images, and analyzed using ImageJ 1.54k software. Three data points were included for each sample because three points were selected from different locations within the same band of the same chemiluminescence image to reduce error. The graph clearly shows that the GDF15 protein expression level was lowest in cells transfected with GalNAc-siGDF15-7, indicating the best interference effect.

[0055] In this embodiment, all figures are based on the two-tailed Mann-Whitney U test; the bar chart data are expressed as mean ± standard deviation (mean ± SD). ns: no significant difference, "*" indicates P < 0.05, "**" indicates P < 0.01, "***" indicates P < 0.001.

[0056] refer to Figure 5Figure A in the figure shows the qRT-PCR results of GDF15 mRNA expression levels in cell samples transfected with nine different GalNAc-siGDF15 strains. It can be seen that the relative expression level of GDF15 mRNA in cells transfected with GalNAc-siGDF15-7 is the lowest, indicating that GalNAc-siGDF15-7 has the best effect in interfering with GDF15 mRNA expression. Cells transfected with GalNAc-siGDF15-2, GalNAc-siGDF15-3, and GalNAc-siGDF15-9 also have low relative expression levels of GDF15 mRNA, indicating that GalNAc-siGDF15-2, GalNAc-siGDF15-3, and GalNAc-siGDF15-9 have relatively good effects in interfering with GDF15 mRNA expression.

[0057] To further screen for subsequent in vivo experimental drugs, Hepa1-6 cells were transfected again with GalNAc-siGDF15-2, GalNAc-siGDF15-3, GalNAc-siGDF15-7, and GalNAc-siGDF15-9. After a period of culture, the relative expression level of GDF15 mRNA in Hepa1-6 cells was detected by qRT-PCR. Figure 5 As shown in Figure B, the cells transfected with GalNAc-siGDF15-7 exhibited the lowest relative expression level of GDF15 mRNA, indicating that GalNAc-siGDF15-7 was the most effective at interfering with GDF15 mRNA expression. Therefore, GalNAc-siGDF15-7 was preliminarily identified as the investigational drug for subsequent in vivo experiments. Next, GalNAc-siGDF15-7 will be used as the target GDF15 small interfering RNA drug, and its in vivo tumor-suppressive effect will be validated and evaluated.

[0058] Example 3: The tumor-suppressing effect of GalNAc-siGDF15 provided in this disclosure was validated in the Hepa1-6 orthotopic hepatocellular carcinoma mouse model, including the construction of the mouse orthotopic hepatocellular carcinoma model, model evaluation and in vivo imaging, as well as drug administration and detection. The specific experimental steps are as follows: 3.1 Construction of a mouse orthotopic hepatocellular carcinoma model (1) Take 80 mice, completely shave the hair on the chest and abdomen below the neck, weigh them, and then administer 0.6% sodium pentobarbital solution to each mouse for anesthesia by intraperitoneal injection at 1g / 10µl, and closely observe their activity status. (2) After fixing the mice in a supine position, disinfect them with 75% ethanol. Make an opening of about 1 cm about 1 cm below the xiphoid process of the mouse chest to expose the left lobe of the mouse liver. (3) Mix physiological saline and Matrigel gel in a 1:1 ratio to resuspend the cells. 1.5 × 10⁶ Hepa1-6-Luc cells are resuspended in 10 µl of the above mixture. 20 µl of the cell resuspension solution is injected into the left lobe of the liver of each mouse. After hemostasis by pressing, the liver is returned to its original position and sutured. (4) After the mice were revived in a 37°C environment, they were fed normally.

[0059] 3.2 Model Evaluation and Live Imaging (1) Model evaluation After feeding the mice for a period of time, they were sacrificed on day 22. The livers of the mice were dissected and the fur was cleaned with PBS buffer. After removing unnecessary organs, the mice were placed on paper to absorb moisture and weighed in groups. The mice were sorted by weight and photographed. The tumor formation of the livers in each group was observed and compared. The occurrence and development of liver cancer in the mice were assessed by the liver photographs and the ratio of liver to body weight.

[0060] (2) In vivo imaging (IVISlumina II) ① Starting from the 7th day after inoculation with Hepa1-6-Luc cell suspension, small animal in vivo imaging was performed every 3 days. Before each imaging, the hair on the chest and abdomen of the mouse was examined. If there was obvious hair, it was removed. ② After ensuring that there is enough oxygen and isoflurane remaining, open the oxygen valve (to a partial pressure of about 0.1 MPa) and the isoflurane valve (to a scale of about 2.5), start the small animal live imaging instrument and imaging software, and perform instrument and software initialization and imaging preparation work (determine the storage path, etc.). ③ Take out the required amount of 1×D-Luciferin Solution in advance, melt it in the dark, and briefly vortex it. Inject 0.1 mL of 1×D-Luciferin Solution into each mouse intraperitoneally. ④ Five minutes after anesthetizing the mice, the mice were placed in a supine position for imaging, with the tumor inoculation site centered. The data were recorded and saved.

[0061] 3.3 Drug administration and detection (1) Group administration If the above 80 mice are divided into 5 groups (A, B, C, D, and E), and administered the drug once every 3 days starting from day 7, and small animal imaging is performed once, for a total of 5 administrations; the specific treatment for each group of mice is as follows: Group A mice were injected subcutaneously with 200 μl of physiological saline; Group B mice were subcutaneously injected with 200 μl of 1 μg / μl siNC solution (dissolved in physiological saline); Group C mice were subcutaneously injected with 200 μl of 1 μg / μl GalNAc-siGDF15-7 solution (dissolved in physiological saline); Group D mice were subcutaneously injected with 200 μl of 2 μg / μl GalNAc-siGDF15-7 solution (dissolved in physiological saline); Mice in group E were intraperitoneally injected with 200 μl of 1 μg / μl GDF15 antibody G15A solution (dissolved in physiological saline).

[0062] The grouping of the above 5 groups of mice was based on the following criteria: Group A and Group B served as negative control groups. Group A was used to exclude the influence of the injection procedure itself, the solvent, and the animal's own disease progression on the results; Group B was used to exclude the immune response or off-target effects caused by the non-specific introduction of the siRNA molecule itself (such as its chemical structure, charge, etc.). Groups C and D were used as treatment experimental groups. Group C was a low-concentration treatment group, used to explore whether GalNAc-siGDF15-7 had a therapeutic effect on orthotopic liver cancer in mice; Group D was a high-concentration treatment group, used to explore whether the therapeutic concentration of GalNAc-siGDF15-7 had a significant effect on its therapeutic effect. Group E served as the positive control group. The GDF15 antibody G15A had a clear anti-tumor effect and was used as a positive control for the treatment effect of orthotopic liver cancer in mice, and to explore which treatment strategy (gene silencing vs. antibody neutralization) was more advantageous (e.g., stronger efficacy, fewer side effects).

[0063] (2) Sample collection and testing ① Blood was collected from the eyeballs on day 22. After standing and centrifuging, the supernatant was collected and the serum GDF15 protein was detected by ELISA. ② After euthanizing the mice, dissect the liver tumor tissue, weigh it, and photograph it; divide the tumor tissue from each mouse into 3 equal parts: a. Extract RNA and detect GDF15 mRNA by qRT-PCR; b. Immunohistochemical staining to detect GDF15 expression; c. Flow cytometry was used to detect the proportion of tumor-infiltrating NK cells and CD8+ T cells; ③At the same time, another batch of mice was treated in the same way, and their survival time was statistically analyzed. For details, please refer to [link to relevant documentation]. Figure 11 .

[0064] In this embodiment, all figures are based on the two-tailed Mann-Whitney U test; the bar chart data are expressed as mean ± standard deviation (mean ± SD). ns: no significant difference, "*" indicates P < 0.05, "**" indicatesP < 0.01, "***" indicates P < 0.001.

[0065] In a specific embodiment of this disclosure, such as Figure 6 Figure A shows in vivo imaging of mice in each group on days 7 and 22. Before imaging, each mouse was injected intraperitoneally with 100 μL of fluorescein potassium salt. Figure 6 Figure B shows the statistical analysis of the imaging fluorescence values ​​of each group of mice. The software used was Caliper Life Sciences Living Image 4.2. It can be seen that compared with the saline group and the siNC group, the tumor fluorescence value of mice in the low-dose siGDF15 group was significantly reduced. This indicates that GalNAc-siGDF15 has a good therapeutic effect on mouse orthotopic liver cancer. There was no statistically significant difference in tumor fluorescence values ​​between the G15A group and the high-dose siGDF15 group. Moreover, the tumor fluorescence value of mice in the high-dose siGDF15 group was significantly lower than that of mice in the low-dose siGDF15 group. This indicates that the dose affects the therapeutic effect of siGDF15 on mouse orthotopic liver cancer, and the higher the dose, the better the therapeutic effect.

[0066] Combination Figure 7 It can be seen that after the mice were sacrificed on day 22 of the experiment, the tumors were dissected and photographed according to size and group. It was obvious that the tumors in the treatment groups (including the low-dose siGDF15 group, the high-dose siGDF15 group, and the G15A group) of the five groups were significantly reduced.

[0067] refer to Figure 8 From day 7 to day 22, the weight of each group of mice was measured every 3 days to obtain the changes in mouse weight. Figure 8 Figure A shows no statistically significant difference in body weight among the groups of mice on day 22. However, after the mice were sacrificed on day 22, the dissected tumors were weighed, as shown in Figure A. Figure 8 As shown in Figure B, after calculating the liver tumor / body weight ratio, it was found that the liver weight ratio of mice in the low-dose siGDF15 group was significantly lower than that in the saline group and the siNC group. This indicates that GalNAc-siGDF15 has a good therapeutic effect on orthotopic liver cancer in mice. There was no statistically significant difference in liver weight ratio between the G15A group and the high-dose siGDF15 group, and the liver weight ratio of mice in the high-dose siGDF15 group was significantly lower than that in the low-dose siGDF15 group. This suggests that dosage affects the therapeutic effect of siGDF15 on orthotopic liver cancer in mice, and higher doses result in better therapeutic effects. (Reference) Figure 9Another batch of mice were subjected to the same procedure, and their survival time was statistically analyzed. It was found that compared with the saline group and the siNC group, the survival time of mice in the low-dose siGDF15 group was significantly prolonged. This indicates that GalNAc-siGDF15 has a good therapeutic effect on orthotopic hepatocellular carcinoma in mice. There was no statistically significant difference in survival time between the G15A group and the high-dose siGDF15 group. Moreover, the survival time of mice in the high-dose siGDF15 group was significantly longer than that of mice in the low-dose siGDF15 group. This indicates that the dose affects the effect of siGDF15 on prolonging the survival time of mice with orthotopic hepatocellular carcinoma, and the higher the dose, the longer the survival time and the better the treatment effect.

[0068] Depend on Figure 10 Data showed that the relative expression levels of GDF15 mRNA in tumor tissues of mice in different treatment groups were different: compared with the G15A group, saline group, and siNC group, the expression level of GDF15 mRNA in mice in the low-dose siGDF15 group was significantly reduced, indicating that GalNAc-siGDF15 can significantly reduce GDF15 mRNA in mouse orthotopic hepatocellular carcinoma tumor cells in vivo and has a good therapeutic effect on mouse orthotopic hepatocellular carcinoma; and the relative expression level of GDF15 mRNA in mice in the high-dose siGDF15 group was significantly lower than that in mice in the low-dose siGDF15 group, indicating that the dose affects the interference effect of siGDF15 on GDF15 mRNA expression in mouse orthotopic hepatocellular carcinoma tumor cells, and the higher the dose, the better the interference effect and the better the therapeutic effect. N-acetylgalactosamine-modified liver cancer-targeting GDF15 small interfering RNA can play a role in mice and successfully reduce the level of GDF15 gene mRNA in tumor cells of liver cancer tissue.

[0069] according to Figure 11 ELISA analysis of GDF15 protein expression levels in the serum of mice in different groups showed that the GDF15 content in the serum of mice in the low-dose siGDF15 group was significantly lower than that in the saline group and the siNC group, indicating that GalNAc-siGDF15 has a good therapeutic effect on orthotopic hepatocellular carcinoma in mice. There was no statistically significant difference in GDF15 content in the serum of mice in the G15A group and the high-dose siGDF15 group. Moreover, the GDF15 content in mice in the high-dose siGDF15 group was significantly lower than that in mice in the low-dose siGDF15 group, indicating that the dose affects the effect of siGDF15 on reducing GDF15 content in the serum of mice with orthotopic hepatocellular carcinoma, and the higher the dose, the better the reduction effect and the better the therapeutic effect.

[0070] according to Figure 12As shown in Figure A, after mice were sacrificed on day 22 and tumors were dissected, paraffin sections were prepared and immunohistochemical staining was performed. It can be seen that compared with the saline group and the siNC group, the brown staining (representing GDF15 expression level) of the tumor tissue of mice in the low-dose and high-dose siGDF15 groups and the G15A group was significantly reduced. This indicates that GalNAc-siGDF15 has a good therapeutic effect on orthotopic liver cancer in mice. The coloring map was further processed automatically using ImageJ to obtain a statistical graph. Figure 12 As shown in Figure B, compared to the saline and siNC groups, the relative expression level of GDF15 protein in the tumor tissue of mice in the low-dose siGDF15 group was significantly reduced. This indicates that GalNAc-siGDF15 can significantly reduce the expression level of GDF15 protein in mouse hepatocellular carcinoma tumor tissue and has a good therapeutic effect on mouse orthotopic hepatocellular carcinoma. There was no statistically significant difference in the relative expression level of GDF15 protein in the tumor tissue of mice in the G15A group and the high-dose siGDF15 group. Moreover, the survival time of mice in the high-dose siGDF15 group was significantly shorter than that of mice in the low-dose siGDF15 group, indicating that the dose affects the effect of siGDF15 in reducing the relative expression level of GDF15 protein in mouse liver tumor tissue, and the higher the dose, the better the reduction effect and the better the therapeutic effect. Therefore, N-acetylgalactosamine-modified hepatocellular carcinoma-targeting GDF15 small interfering RNA can reduce the level of GDF15 protein in the serum of mice with orthotopic hepatocellular carcinoma.

[0071] like Figure 13 As shown, N-acetylgalactosamine-modified GDF15 small interfering RNA can increase the proportion of infiltrating NK cells in mouse tumor tissue with orthotopic hepatocellular carcinoma. Figure 13 Figure A in the figure is a flow cytometry plot obtained after the tumor tissue was dissected and appropriately processed after the mice were sacrificed on day 22. Figure 13 Figure B shows the statistical analysis of NK cell proportions. It can be seen that compared with the saline group and the siNC group, the proportion of NK cells infiltrating the tumor tissue of mice in the low-dose siGDF15 group was significantly increased. This indicates that GalNAc-siGDF15 has a good therapeutic effect on orthotopic liver cancer in mice. Moreover, the proportion of NK cells infiltrating the tumor tissue of mice in the high-dose siGDF15 group was significantly higher than that of mice in the low-dose siGDF15 group. This indicates that the dose affects the effect of siGDF15 on increasing the proportion of NK cells in the tumor tissue of mice with orthotopic liver cancer, and the higher the dose, the better the effect and the better the therapeutic effect.

[0072] like Figure 14As shown in the figure, Figure A is the flow cytometry graph obtained by dissecting the tumor and processing the tumor tissue after the mice were sacrificed on day 22; Figure B is the statistical analysis graph of the proportion of CD8+ T cells. The flow cytometry analysis of the proportion of CD8+ T cells in tumor tissues of different groups of mice and the statistical graph show that N-acetylgalactosamine-modified GDF15 small interfering RNA can increase the proportion of CD8+ T cells in tumor tissues of mice with orthotopic hepatocellular carcinoma. Compared with the saline group and the siNC group, the proportion of CD8+ T cells in tumor tissues of mice in the low-dose siGDF15 group was significantly increased, indicating that GalNAc-siGDF15 has a good therapeutic effect on orthotopic hepatocellular carcinoma in mice. Furthermore, the proportion of CD8+ T cells in tumor tissues of mice in the high-dose siGDF15 group was significantly higher than that of mice in the low-dose siGDF15 group, indicating that the dose affects the effect of siGDF15 on increasing the proportion of CD8+ T cells in tumor tissues of mice with orthotopic hepatocellular carcinoma, and the higher the dose, the better the effect and the better the therapeutic effect.

[0073] This indicates that N-acetylgalactosamine-modified GDF15 small interfering RNA can increase the infiltration of CD8+ T cells and NK cells in tumor tissues of mice with orthotopic hepatocellular carcinoma, reverse the immunosuppressive tumor microenvironment, and enhance the killing effect of the immune system on tumor cells. The N-acetylgalactosamine-modified hepatocellular carcinoma-targeting GDF15 small interfering RNA disclosed in this disclosure can significantly inhibit the occurrence and development of Hepa1-6 orthotopic hepatocellular carcinoma in mice by silencing the GDF15 gene in hepatocellular carcinoma tumor cells, thus demonstrating a therapeutic effect on orthotopic hepatocellular carcinoma.

[0074] The N-acetylgalactosamine-modified hepatocellular carcinoma-targeting GDF15 small interfering RNA therapy disclosed herein significantly prolonged the survival of mice. Tumor tissue showed decreased GDF15 mRNA and protein expression, and serum GDF15 protein levels were reduced. The proportions of tumor-infiltrating NK cells and CD8+ T cells increased, indicating that GalNAc-siGDF15 can silence the GDF15 gene, reverse the immunosuppressive microenvironment, and inhibit hepatocellular carcinoma growth.

[0075] In summary, the N-acetylgalactosamine-modified hepatocellular carcinoma-targeting GDF15 small interfering RNA (SRNA) provided in this disclosure can specifically bind to desialyl glycoprotein expressed on the surface of hepatocytes, allowing the SRNA to accumulate in the liver and be internalized by hepatocellular carcinoma cells. GalNAc-siGDF15 induces the cleavage and degradation of GDF15 mRNA, thereby mediating GDF15 gene silencing, persistently reversing the immunosuppressive microenvironment within tumor cells, restoring the anti-tumor function of immune cells, and achieving the goal of treating hepatocellular carcinoma. This disclosure avoids the limitations of repeated doses in traditional therapies and the safety issues associated with gene editing methods, reduces treatment costs, and effectively improves treatment efficacy. Therefore, delivering GDF15 SRNA to the hepatocellular carcinoma region to mediate GDF15 gene silencing, inhibit GDF15 function, and reverse the suppressive immune microenvironment of hepatocellular carcinoma becomes a potential clinical treatment option.

[0076] In the above-mentioned trials disclosed herein, there were no cases of combined drug use; only N-acetylgalactosamine-modified small interfering RNA drugs were used in the trials; and there were no cases of simultaneous use of any two or more of GalNAc-siGDF15-1 to GalNAc-siGDF15-9. All trials in the above-mentioned trials used only one of GalNAc-siGDF15.

[0077] In one embodiment of this disclosure, the small interfering RNA of the GDF15 gene is used in the preparation of GDF15 gene-related products for the treatment of tumors. In orthotopic liver cancer solid tumors, the small interfering RNA of the GDF15 gene can directly inhibit the malignant phenotype of tumor cells, reverse the immunosuppressive microenvironment in the tumor, and restore the anti-tumor immune response.

[0078] The applications of small interfering RNA (SRNA) products related to the GDF15 gene in cancer treatment encompass multiple dimensions, including directly inhibiting the malignant phenotype of tumor cells, reshaping the immune microenvironment, overcoming radiotherapy and chemotherapy resistance, and combination therapy strategies. Their efficacy has been validated in preclinical models of orthotopic liver cancer. Future research requires further validation in various solid tumors, optimization of delivery systems, and advancement of clinical trials to achieve translation from laboratory to clinical practice.

[0079] In one embodiment of this disclosure, small interfering RNA of the GDF15 gene is used in the preparation of GDF15 gene-related products for the treatment of liver cancer. The related products are drugs or pharmaceutical compositions. The drugs or pharmaceutical compositions also include diluents, excipients and / or carriers that can be used in the pharmaceutical field, and the drug administration method is selected from oral, intravenous or intramuscular injection, etc.

[0080] In a preferred embodiment of this disclosure, the small interfering RNA of the GDF15 gene is first prepared as a lyophilized powder during pharmaceutical manufacturing, and then dissolved in physiological saline to form a solution for use via subcutaneous injection.

[0081] In one specific embodiment of this disclosure, N-acetylgalactosamine-modified small interfering RNA (SRNA) drugs can reduce the expression level or activity of GDF15 in liver cancer cells, effectively improving the tumor immune microenvironment and inhibiting liver cancer progression. In practical therapeutic applications, the GDF15 gene-related SRNA products provided in this disclosure can also be used in combination with other drugs for treating liver cancer and other tumor diseases. Before combining these drugs, in vivo mouse experiments and extensive clinical trials are required to ensure the safety of the combined treatment.

[0082] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, and are not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein. The scope of the invention is defined by the appended claims.

Claims

1. A small interfering RNA for the GDF15 gene, characterized in that, include: The main body comprises a positive strand and an antisense strand; the positive strand comprises at least one nucleic acid sequence from SEQ ID NO.1 to SEQ ID NO.9, and the antisense strand comprises at least one nucleic acid sequence from SEQ ID NO.1' to SEQ ID NO.9'; the positive strand and the antisense strand correspond one-to-one. The modification portion is connected to the 3' end of the justice chain.

2. The small interfering RNA of the GDF15 gene according to claim 1, characterized in that, The modified part is N-acetylgalactosamine.

3. A method for preparing small interfering RNA of the GDF15 gene as described in claim 1 or 2, characterized in that, Includes the following steps: S1: Using phosphoramide solid-phase synthesis technology, each base was added one by one to the 5' hydroxyl group of the cyclic primer chain to synthesize the main body of the small interfering RNA; S2: N-acetylgalactosamine is attached to the 3' end of the positive strand of the small interfering RNA via a synthetic reaction involving phosphoramidite of thymine nucleotide modified with N-acetylgalactosamine.

4. The preparation method according to claim 3, characterized in that, The primer chain includes an upstream primer and a downstream primer, the sequence of which is 5'-AGATCAAGACGAGCCTGCAC-3' and the sequence of which is 5'-ATAGGTCTGGAGCGACACCC-3'.

5. The preparation method according to claim 3, characterized in that, The synthetic reaction includes: The deprotection reaction uses a dichloromethane solution of 3% trichloroacetic acid. A condensation reaction in which the phosphorusamide-protected nucleotide monomer is mixed with 5-ethylthio-1-H-tetrazole at a volume ratio of 1:1.5; A blocking reaction, wherein blocking solution A and blocking solution B are used to block the 5'-terminal hydroxyl group; An oxidation reaction, wherein the oxidation reaction uses a 0.02–0.05 mol / L iodine solution, wherein the iodine solution is a mixture of tetrahydrofuran, pyridine, and water; and Product post-processing and purification steps to obtain the target product.

6. The preparation method according to claim 5, characterized in that, The synthetic reaction also includes 2'-oxymethyl, 2'-fluorine modified adenosine, guanosine, cytidine, uridine nucleoside phosphoramide monomers, and CPG carriers preloaded with specific nucleosides.

7. The preparation method according to claim 3, characterized in that, The molar ratio of the N-acetylgalactosamine modification to the GDF15 small interfering RNA is 1:

1.

8. The use of a small interfering RNA of the GDF15 gene as described in claim 1 or 2 in the preparation of GDF15 gene-related products for the treatment of liver cancer.

9. The application according to claim 8, characterized in that, The relevant products are pharmaceuticals or pharmaceutical compositions; the pharmaceuticals or pharmaceutical compositions also include diluents, excipients and / or carriers that can be used in the pharmaceutical field.

10. The use of a small interfering RNA of the GDF15 gene as described in claim 1 in the preparation of GDF15 gene-related products for the treatment of tumors.

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