An antisense oligonucleotide conjugate targeting stk25 mRNA and uses thereof

Abstract

The application discloses an antisense oligonucleotide conjugate targeting Stk25 mRNA and application thereof, and belongs to the technical field of biological medicines. The antisense oligonucleotide conjugate comprises: 1) an antisense oligonucleotide with a length of 16 nucleotides, wherein the oligonucleotide sequence is GATCTTGATGGCCACC, as shown in SEQ ID NO:1; and 2) a trivalent GalNAc conjugation part covalently connected to the oligonucleotide. The application provides an antisense oligonucleotide conjugate targeting Stk25 mRNA, and experiments prove that the antisense oligonucleotide conjugate can realize liver-specific, high-efficiency and long-acting STK25 knockdown in vivo, and simultaneously improve pathological indexes such as lipid accumulation, abnormal blood glucose and obesity of body weight related to MASLD, thereby providing a novel gene therapy drug with high targeting, long-lasting curative effect and high safety for the treatment of metabolic related fatty liver disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antisense oligonucleotide conjugate targeting Stk25 mRNA and its applications. Background Technology

[0002] Metabolic dysfunction-associated steatotic liver disease (MASLD, formerly known as NAFLD) is a chronic liver disease closely related to metabolic disorders. Its main pathological features include excessive lipid accumulation (steatohepatitis), immune cell infiltration (inflammation), and fibrosis mediated by hepatic stellate cell activation. This disease is closely associated with metabolic diseases such as obesity, type II diabetes, and cardiovascular disease, seriously threatening patients' health.

[0003] The treatment of MASLD still faces challenges: although lifestyle interventions (such as diet control and exercise) are the basic treatment, clinical practice shows that patients have low long-term adherence. Therefore, it is still necessary to find targeted therapeutic drugs.

[0004] Serine / threonine kinase 25 (Stk25) is a key protein kinase regulating hepatic lipid homeostasis and the development of metastatic lipid disorder (MASLD). It directly encapsulates intracellular lipid droplets (LDs) and maintains hepatic lipid metabolism homeostasis by regulating downstream pathways. Previous studies have confirmed a close link between Stk25 and the occurrence and progression of MASLD and hepatocellular carcinoma (HCC). By constructing a high-fat diet-induced Stk25 gene knockout mouse model and performing quantitative hepatic proteomic analysis on knockout and normal mice, 131 proteins and 60 phosphorylated proteins were screened. Their expression and regulation are related to Stk25, including a large number of proteins responsible for important physiological functions such as peroxisome function, ubiquitination-mediated protein hydrolysis, and antioxidant activity. These results suggest that regulating peroxisome function and metabolic stress response may be an important molecular mechanism by which Stk25 controls the occurrence and progression of MASLD.

[0005] Antisense oligonucleotide (ASO) therapeutics typically consist of 15-30 chemically modified nucleotides linked primarily by phosphothioester bonds. Once inside the cell, ASO drugs bind to complementary target mRNAs according to the complementary pairing principle, and are then degraded by ribonuclease H1, thereby inhibiting the expression of the target gene.

[0006] The present invention aims to provide a liver-targeted antisense oligonucleotide conjugate targeting human Stk25. Summary of the Invention

[0007] The first objective of this invention is to provide an antisense oligonucleotide conjugate targeting Stk25 mRNA, and the second objective of this invention is to provide the application of the antisense oligonucleotide conjugate.

[0008] The first objective of this invention is achieved by providing an antisense oligonucleotide conjugate targeting Stk25 mRNA, comprising: 1) An antisense oligonucleotide of 16 nucleotides in length, the sequence of which is shown in SEQ ID NO:1; 2) Covalently linked to the trivalent N-acetylgalactosamine (GalNAc) conjugate of the oligonucleotide.

[0009] The second objective of this invention is achieved by using the antisense oligonucleotide conjugate in the preparation of a drug for treating MASLD.

[0010] The beneficial effects of this invention are as follows: This invention provides an antisense oligonucleotide conjugate targeting Stk25 mRNA. Experimental verification shows that this antisense oligonucleotide conjugate can achieve liver-specific, efficient, and long-lasting STK25 knockdown in vivo, and simultaneously improve pathological indicators related to MASLD such as lipid accumulation, abnormal blood glucose, and obesity. This provides a novel gene therapy drug with strong targeting, long-lasting efficacy, and high safety for the treatment of metabolic-related fatty liver disease. Attached Figure Description

[0011] Figure 1 This is the mass spectrum of the GalNAc conjugate Gc337 of this invention; Figure 2 The image shows lipid accumulation in HepG2 cells after treatment with the antisense nucleotide c337 of this invention. The left image is a confocal microscope image of lipid droplet staining in different treatment groups, and the right image is a quantitative bar chart obtained by quantitative analysis of the fluorescence area of ​​lipid droplets in each group using ImageJ software. Figure 3 This is a dose-dependent bar chart showing the effect of Gc337 on STK25 mRNA expression in mouse liver.

[0012] Figure 4 This is a Western blot electrophoresis image showing the effect of Gc337 on STK25 protein expression in mouse liver.

[0013] Figure 5 This is a graph showing the trend of weight change in each group of mice during the treatment period.

[0014] Figure 6 The blood glucose-time curves for the intraperitoneal glucose tolerance test (IPGTT) in each group of mice are shown.

[0015] Figure 7 This is a statistical bar chart showing the area under the glucose curve (AUC Glucose) in the IPGTT experiment for each group of mice.

[0016] Figure 8 The blood glucose-time curves for the intraperitoneal insulin tolerance test (IPITT) in each group of mice are shown.

[0017] Figure 9 This is a statistical bar chart showing the area under the blood glucose curve (AUC) in the IPITT experiment for each group of mice.

[0018] Figure 10 A bar chart showing the effect of Gc337 treatment on STK25 mRNA expression in mouse livers at different time points.

[0019] Figure 11 Western blot electrophoresis image showing the effect of Gc337 treatment on STK25 protein expression in mouse liver at different time points. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0021] This invention provides an antisense oligonucleotide conjugate targeting Stk25 mRNA, comprising: 1) An antisense oligonucleotide of 16 nucleotides in length, wherein the oligonucleotide sequence is GATCTTGATGGCCACC, as shown in SEQ ID NO:1; 2) Covalently linked to the trivalent N-acetylgalactosamine (GalNAc) conjugate of the oligonucleotide.

[0022] The antisense oligonucleotide has a 3-10-3 spacer structure: the nick region contains 10 2'-deoxynucleotides, and the 5' and 3' ends are each connected to a wing composed of 3 nucleotides; wherein, each nucleotide in the 5' wing and each nucleotide in the 3' wing are modified with cEt sugar, each nucleotide in the nick region is modified with 2'-deoxy sugar, the internucleotide bonds in the entire spacer are phosphate thioester bonds (P=S), and all cytosine residues are 5-methylcytosine.

[0023] The trivalent GalNAc conjugate is GalNAc3o, with the structure shown in Equation I: I.

[0024] The 5′ end of the oligonucleotide is covalently linked to GalNAc3o via a phosphodiester bond (P=O).

[0025] The present invention also provides the application of the antisense oligonucleotide conjugate targeting Stk25 mRNA, one application being its use in inhibiting Stk25 mRNA expression.

[0026] Another application of the antisense oligonucleotide conjugate targeting Stk25 mRNA is in the preparation of drugs for the prevention and treatment of diseases related to Stk25 mRNA overexpression.

[0027] The diseases associated with Stk25 mRNA overexpression are hyperlipidemia or metabolic dysfunction-related steatohepatitis.

[0028] The metabolic dysfunction-related steatohepatitis mentioned above is non-alcoholic steatohepatitis.

[0029] The present invention further provides a pharmaceutical composition comprising the aforementioned antisense oligonucleotide conjugate and a pharmaceutically acceptable carrier.

[0030] The definitions of terms used in the following embodiments are as follows: "Antisense oligonucleotide" or "ASO" refers to an oligonucleotide having a nucleobase sequence complementary to that of a target nucleic acid or a region or segment thereof. Antisense oligonucleotides can specifically hybridize with a target nucleic acid segment, and this hybridization leads to RNase H-mediated cleavage of the target nucleic acid.

[0031] "Stk25 nucleic acid" refers to any nucleic acid that encodes Stk25. In some embodiments, Stk25 nucleic acid includes a DNA sequence encoding Stk25 and an RNA sequence transcribed therefrom (pre-mRNA sequence, including introns and exons), as well as an mRNA sequence encoding Stk25.

[0032] "cEt" or "restricted ethyl" refers to a bicyclic nucleotide having a bicyclic sugar moiety comprising a bridge connecting a 4'-carbon and a 2'-carbon, wherein the bridge has the formula: 4'-CH(CH3)-O-2'. "cEt-modified nucleotide" refers to a bicyclic nucleotide having a bicyclic sugar moiety comprising a bridge connecting a 4'-carbon and a 2'-carbon, wherein the bridge has the formula: 4'-CH(CH3)-O-2'. Its structural formula is as follows:

[0033] Where Bx represents any nucleobase. Sugars with restricted ethyl modification are modified sugars.

[0034] "5-Methylcytosine" refers to cytosine with a methyl group attached to the 5-position. 5-Methylcytosine is a modified nucleobase.

[0035] A "spacer" or "gapmer" refers to an antisense oligonucleotide containing an inner region of multiple nucleotides that support RNase H cleavage between an outer region containing one or more nucleotides, wherein the nucleotides containing the inner region are chemically distinct from the one or more nucleotides containing the outer region. The inner region may be referred to as a "gap" or "segment," and the outer region may be referred to as a "wing." In some embodiments, the antisense oligonucleotide is a spacer.

[0036] N-acetylgalactosamine (GalNAc) is a ligand for the desialyl glycoprotein receptor (ASGPR) on the surface of liver cells. It is linked with antisense oligonucleotides to form antisense oligonucleotide conjugates with liver targeting. In Example 1, the targeting portion comprises three GalNAc moieties arranged in a triantennary pattern (triantennary GalNAc).

[0037] Unless otherwise stated, all reagents and solutions used in the synthesis of the oligonucleotide compounds were commercially available. Standard phosphoramidite structural units and solid supports were used to incorporate nucleoside residues, including, for example, T, A, G, and mC residues. A phosphodiester-linked GalNAc3o ligand was introduced at the 5' end of the oligonucleotide chain using a GalNAc phosphoramidite compound (0.1 M, soluble in anhydrous acetonitrile). The GalNAc phosphoramidite compound was a monomer having the formula (GalNAc3o)-phosphamidite, which could be prepared according to methods known in the art (e.g., phosphoramidation derivatization based on a trivalent GalNAc ligand structure as described in patent CN 117903225A).

[0038] Example 1: Preparation of the antisense oligonucleotide conjugate Gc337 The antisense oligonucleotide conjugate Gc337 described in this embodiment is formed by conjugating the antisense oligonucleotide c337 with a gapmer structure and the tri-antenna type N-acetylgalactosamine (GalNAc3o) via a phosphodiester bond. The sugar ring and nucleobase modification of ASO and the liver-targeting design of GalNAc3o are all in accordance with the aforementioned definitions of this invention. The specific preparation method is as follows: 1. Materials and Reagents Monomer solutions: β-D-2'-deoxyribonucleoside phosphoramidide solution concentration was 0.06M (solvent was anhydrous acetonitrile); cEt-modified phosphoramidide solution concentration was 0.1M (solvent was anhydrous acetonitrile); 5-methylcytosine phosphoramidide (containing the modified nucleobase "5-methylcytosine", used to synthesize all cytosine residues in ASO).

[0039] 2. Preparation method 2.1 Solid-phase synthesis of oligonucleotide chains The oligonucleotide chain c337 (nucleotide sequence shown in SEQ ID No: 1) was synthesized using a phosphoramide coupling method on a Universal CPG solid support synthesis column (Dinacinco, 2 μmol loading) packed in an LK-48E synthesizer. This oligonucleotide chain was designed as a 3-10-3 spacer: the notch contained 10 2'-deoxynucleosides, and the 5' and 3' ends each had a wing consisting of 3 nucleosides; all nucleosides in the 5' and 3' wings were modified with cEt sugars, and all nucleosides in the notch were modified with 2'-deoxynucleosides. All internucleotide bonds in the spacer were phosphate thioester (P=S) bonds, and all cytosine residues in the spacer were 5-methylcytosine.

[0040] In the coupling step, DNA phosphoramidite monomers were delivered in an amount more than four times the loading on the solid carrier and phosphoramidite condensation was carried out for 10 min; cEt phosphoramidite monomers were delivered in an amount more than four times the loading on the solid carrier and phosphoramidite condensation was carried out for 20 min. The activator was a 0.35 M BTT anhydrous acetonitrile solution containing 0.5% NMI. The dimethoxytriphenylmethyl (DMT) group on the 5'-hydroxyl group of the nucleotide was removed using a 3% trichloroacetic acid solution in dichloromethane. Phosphophosphate bonds were introduced by sulfurization in a pyridine / acetonitrile (1:1) solution of 0.2 M PADS for 3 min; phosphite bonds were introduced by oxidation in a THF / pyridine solution of 0.05 M I2 for 2 min.

[0041] 2.2 Linkage of GalNAc3o ligands After the oligonucleotide chain was synthesized, the GalNAc3o ligand was directly ligated on a solid-phase synthesizer: GalNAc phosphorous amide compound (0.1 M) was delivered in an amount exceeding four times the loading on the solid support, the coupling time lasted for 20 minutes, and the activator was a 0.35 M BTT anhydrous acetonitrile solution containing 0.5% NMI. After the ligation reaction was completed, a stable phosphodiester bond was formed using the same oxidation step as in the chain synthesis (oxidation in 0.05 M I2 THF / pyridine solution for 2 minutes), thus obtaining the crude product of the complete antisense oligonucleotide conjugate Gc337 with GalNAc3o conjugated at the 5' end. The chemical structure of the GalNAc3o- group is shown in Formula I. I.

[0042] 2.3 Post-processing and purification After synthesis, the solid support was suspended in ammonia (25-30 wt%) and heated at 55 °C for 4 h. The solid support was then filtered off, and the ammonia was removed under reduced pressure. The residue was purified by high-performance liquid chromatography (HPLC) with the following column parameters: Waters XBridge C18 column (2.1 × 100 mm, particle size 3.5 μm); mobile phase: Phase A: water / acetonitrile = 95 / 5, 0.1 M hexafluoroisopropanol and 0.02 M n-hexylamine; Phase B: water / acetonitrile = 80 / 20; gradient: increasing the proportion of Phase B from 5% to 100% over 15 minutes; flow rate: 0.8 ml / min. The eluent of the obtained product was concentrated, and the pH was adjusted to above 11 with 0.5 M sodium hydroxide aqueous solution. The mixture was stirred at room temperature for 3 h, desalted using a C18 desalting column, and lyophilized to prepare the antisense oligonucleotide conjugate Gc337. Its molecular weight was verified by mass spectrometry. The results are as follows: Figure 1 As shown in Table 1.

[0043] Table 1. Molecular weight of the antisense oligonucleotide conjugate Gc337

[0044] Example 2: Detection of the inhibitory effect of antisense oligonucleotide C337 on oleic acid-induced lipid accumulation in HepG2 cells. Cell spreaders were placed into 12-well cell culture plates, and HepG2 cells were then seeded into the 12-well plates at a seeding density of 2 × 10⁶ cells / well. 4 Approximately 1 mL of fresh DMEM medium (containing 10% serum) was added, followed by oleic acid (final concentration: 0.25 mM) to induce the formation of a MASLD cell model. After 24 hours of culture, control ASO (202, sequence: GGCCAATACGCCGTCA) and the antisense oligonucleotide c337 of this invention were transfected using a calcium phosphate transfection kit (Beyotime, Cat#C0508), with three biological replicates for each group. The ASO solution was prepared using RNase-free water to obtain a 20 μM ASO solution. The final concentration of ASO used during transfection was 100 nM, and the transfection procedure was strictly performed according to the kit instructions. After transfection, the cells were cultured for another 48 hours. Then, lipid droplet staining was performed. First, the culture medium was discarded, and 1 mL of PBS was added to wash the cell surface. Then, BODIPY and Hoechst staining solutions diluted 1:20000 were added to stain the cells in the dark for 10 minutes. After that, the supernatant dye was discarded, and PBS was added to wash the cells three times, 5 minutes each time. After fixation with 4% paraformaldehyde for 10 minutes, the supernatant was discarded, and PBS was added for washing three times, 5 minutes each time. Finally, the slide was mounted onto a glass slide and photographed using a confocal microscope (scale bar: 10 μm). The fluorescence area of ​​lipid droplets in HepG2 cells was quantitatively analyzed using ImageJ software.

[0045] Results: As shown in Table 2 and Figure 2 As shown, compared with the control antisense oligonucleotide (ASO), the proportion of lipid droplet fluorescence area in HepG2 cells treated with the antisense oligonucleotide c337 of this invention decreased by 70.44%, which was significantly lower than that in the negative control group, indicating that c337 can significantly reduce the lipid droplet content in the lipid MASLD model.

[0046] Table 2. Inhibition efficiency of c337 on HepG2 lipid droplet synthesis

[0047] Example 3: Detection of STK25 mRNA knockdown efficiency of the antisense oligonucleotide conjugate Gc337 in mice. 1. qPCR detection of Stk25 mRNA levels Twenty-one 6-week-old wild-type male C57BL / 6 mice were randomly divided into 7 groups (n=3 per group) and injected subcutaneously with different doses of Gc337 (0 mg / kg, 1.25 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, and 30 mg / kg). Gc337 was prepared as a 1 mg / mL solution using 0.9% saline. Three days after injection, the mice were sacrificed, and liver tissue was collected for analysis. Approximately 50 mg of liver tissue was used to extract total RNA using the Trizol method. RNA concentration and purity were determined using a micro-spectrophotometer; the A260 / A280 ratio was consistently between 1.8 and 2.1. 500 ng of total RNA was used to perform genomic DNA removal and reverse transcription using the HiScript III RT SuperMix for qPCR kit (Vazyme) according to the manufacturer's instructions to obtain cDNA. Using cDNA diluted to 200 μL with ddH2O as a template, amplification was performed on a QuantStudio® 5 real-time quantitative PCR instrument (384-well module) using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) kit. The reaction system and cycling procedure were performed according to the kit instructions.

[0048] Result: As Figure 3 As shown in Table 3, the inhibitory effect of Gc337 on STK25 mRNA expression in liver tissue exhibited a significant dose-dependent effect. Compared with the 0 mg / kg control group: at a dose of 1.25 mg / kg, STK25 expression showed no significant change; at doses ≥2.5 mg / kg, its mRNA level showed a significant decreasing trend with increasing dose, and all high-dose groups (2.5 mg / kg and above) showed statistical significance.

[0049] Table 3. Knockdown efficiency of Stk25 mRNA levels by different dose groups of Gc337

[0050] 2. Western Blot detection of STK25 protein levels In each group of mouse liver tissue (approximately 50 mg per tissue sample), 500 μL of protein extraction buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF, 0.1% SDS) was added and homogenized using a tissue homogenizer. The samples were centrifuged at 20,000 × g for 10 min at 4°C, and the supernatant was collected into new EP tubes. The total protein concentration was determined using a BCA protein assay kit (Invitrogen, Singapore).

[0051] Take 50 μg of protein sample for protein electrophoresis. The steps are as follows: (1) Prepare SDS-PAGE gel. First, prepare the separating gel: mix acrylamide solution, SDS, Tris-HCl (pH 8.8), APS (catalyst) and TEMED (coagulant), pour into the glass plate to 70% height, cover with isopropanol and flatten. Then prepare the stacking gel: after the separating gel solidifies (about 15 minutes), pour off the isopropanol, pour in the stacking gel (acrylamide / Tris-HCl pH 6.8), and insert the comb. (2) Sample loading: after removing the comb, fill the well with electrophoresis buffer (Tris-glycine-SDS), add the sample to the well, and leave one well for pre-stained protein marker. Electrophoresis conditions: constant voltage 80V (stacking gel stage, about 20 minutes). Switch to 120V (separating gel stage, about 1 hour, stop when bromophenol blue indicator reaches the bottom of the gel).

[0052] Then, the transfer and antibody incubation were performed as follows: (1) Cut the gel to the target molecular weight range and place it in transfer buffer (Tris-glycine + 20% methanol) for equilibration for 10 minutes. (2) Assemble the "transfer sandwich" (from negative to positive electrode): sponge pad → filter paper → gel → PVDF membrane (pre-activated with methanol for 15 seconds) → filter paper → sponge pad. Ensure no air bubbles and that the membrane and gel adhere tightly. Transfer conditions: (3) After the transfer is complete, the PVDF membrane is washed 3 times with TBST (TBS + 0.1% Tween 20) for 5 minutes each time. Add 5% skim milk and block at room temperature with shaking for 1 hour. (4) Dilute the primary antibody with TBST and add it to the membrane. Incubation conditions: 4℃ overnight. After incubation, wash 3 times with TBST for 5 minutes each time. (5) Dilute the HRP-labeled secondary antibody with TBST and incubate at room temperature with shaking for 1 hour. Wash 3 times with TBST for 5 minutes each time to remove the non-binding secondary antibody. (6) Mix ECL luminescent solution A / B at a 1:1 ratio, cover the membrane surface, and react in the dark for 1 minute. Capture the signal with an imaging system and generate an image. Use ImageJ software to count the gray values ​​of the bands and calculate the knockdown efficiency of STK25 protein.

[0053] Results: Western blot results showed that when the Gc337 dose reached 20 mg / kg, the protein expression level of STK25 was significantly reduced compared with the blank control group (Gc337 dose 0 mg / kg) (reduction of 73.71%, P<0.05). Figure 4 As shown in Table 4.

[0054] Table 4. Knockdown efficiency of Stk25 protein levels by different dose groups of Gc337

[0055] Example 4: Effects of Gc337 and Resmetiro on Body Weight in a High-Fat Diet-Induced MASLD Mouse Model To study the therapeutic effect of Gc337 in vivo, this embodiment uses a high-fat diet (HFD)-induced metabolic-associated fatty liver disease (MASLD) model and uses resimeltiro as a benchmark to evaluate the therapeutic effect of Gc337.

[0056] Experimental Methods: Six-week-old wild-type male mice (C57BL / 6) were fed a high-fat diet (HFD, 60 kcal% fat, Synergy Bio, Cat#XTHF60) for 13 weeks to establish a MASLD model. They were then randomly divided into three treatment groups (n=6-8 per group): a high-fat diet control group (HFD-NC), a high-fat diet + Gc337 group (HFD-Gc337), and a high-fat diet + resmetidine group (HFD-Resme). Age-matched mice fed a standard diet (SD, 15 kcal% fat, Synergy Bio, Cat# XTI01CR-010) served as the negative control group (SD-NC). During the 8-week treatment period, mice in the HFD-NC and SD-NC groups received weekly subcutaneous injections of 20 ml / kg of saline and daily gavage of 3 ml / kg of a solvent (0.6% methylcellulose + 0.5% Tween 80 + 98.9% water); mice in the HFD-Gc337 group received weekly subcutaneous injections of 20 mg / kg of Gc337 (prepared with saline) and daily gavage of 3 ml / kg of a solvent (0.6% methylcellulose + 0.5% Tween 80 + 98.9% water); mice in the HFD-Resme group received daily gavage of 3 mg / kg of resimetilol and weekly subcutaneous injections of 20 ml / kg of saline. Throughout the 8-week treatment period, the mice's physiological status and body weight changes were monitored weekly.

[0057] Results: During the 8-week treatment period, the body weight of mice in both the HFD-Gc337 group and the HFD-Resme group gradually decreased significantly, such as... Figure 5 As shown in Table 5.

[0058] Table 5. Changes in body weight of mice in each group.

[0059] Example 5: Effects of Gc337 and Resmetiro on Glucose Regulation and Insulin Resistance in a High-Fat Diet-Induced MASLD Mouse Model Insulin resistance is a major driver of metabolic-associated fatty liver disease (MASLD) and a key challenge in its management. In addition to its role in hepatic lipid metabolism, STK25 is also involved in systemic glucose homeostasis. In this example, after the 8-week treatment period described in Example 4, SPF-grade wild-type male C57BL / 6J mice from each group in Example 4 underwent intraperitoneal glucose tolerance tests (IPGTT) and intraperitoneal insulin tolerance tests (IPITT) to evaluate the effect of Gc337 on improving glucose regulation and insulin resistance.

[0060] I. Intraperitoneal Glucose Tolerance Test (IPGTT): Assessing glucose regulation capacity 1. Experimental Methods Mice to be tested were fasted for 12-16 hours one day in advance. The next day, all mice were weighed, and their fasting blood glucose levels (0 minutes) were immediately measured and recorded. Subsequently, a 20% glucose solution was injected intraperitoneally at a dose of 0.75 g / kg. Blood glucose levels were measured again at 30, 60, 90, and 120 minutes after injection.

[0061] 2. Experimental Results like Figure 6 As shown in Table 6, compared with the HFD-NC group, the fasting blood glucose levels of mice in the HFD-Gc337 group and the HFD-Resme group were significantly reduced. After glucose injection, blood glucose levels in all groups peaked at 30 minutes; however, the peak level in the HFD-Gc337 treatment group was lower and returned to baseline levels within 90 minutes, indicating that Gc337 can effectively improve glucose tolerance in MASLD model mice.

[0062] Table 6. Changes in blood glucose levels in mice of different groups during the IPGTT experiment.

[0063] 3. Further quantitative analysis of the area under the blood glucose-time curve (AUC) further confirmed the above conclusions. Figure 7 (See Table 7): Compared with the HFD-NC group, the AUC of both the HFD-Gc337 group and the HFD-Resme group was significantly reduced, with the HFD-Gc337 group showing a reduction of 26.5% (P<0.001).

[0064] Table 7. Improvement rate of glucose tolerance in each group of mice

[0065] II. Intraperitoneal insulin tolerance test (IPITT): Assessing the effect of insulin resistance improvement 1. Experimental Methods Mice were fasted for 6 hours prior to injection. Mice were weighed, and their fasting blood glucose levels at 0 minutes were measured and recorded. Subsequently, insulin solution (prepared with 0.9% saline, injection volume calculated based on the weight of each mouse) was injected intraperitoneally at a dose of 0.75 U / kg. Blood glucose levels in the tail vein of each group of mice were measured at 30, 60, 90, and 120 minutes after injection.

[0066] 2. Experimental Results like Figure 8As shown in Table 8, compared with the HFD-NC control group, the fasting blood glucose levels in the HFD-Gc337 and HFD-Resme groups were significantly lower. After insulin injection, blood glucose levels in all groups reached their lowest point within 15-30 minutes. Specifically, the HFD-NC control group mice showed a smaller decrease in blood glucose and a rapid rebound at subsequent time points. Conversely, the HFD-Gc337 and HFD-Resme groups mice showed a greater decrease in blood glucose and maintained lower blood glucose levels throughout the experiment, with a significantly slower recovery to baseline compared to the control group. This indicates that Gc337 can effectively improve insulin resistance induced by a high-fat diet.

[0067] Table 8. Changes in blood glucose levels in mice of each group during the IPITT experiment.

[0068] The quantitative results of the area under the blood glucose-time curve (AUC) further confirm the above conclusions. Figure 9 (See Table 9). With the HFD-NC group as the control, the area under the curve in the treatment groups was significantly reduced, and the insulin resistance in the mice in the treatment groups was significantly alleviated (HFD-Resme group: 34.5% reduction, P<0.001; HFD-Gc337 group: 31.7% reduction, P<0.001).

[0069] Table 9. Improvement rate of insulin resistance in mice of each group

[0070] Example 6: Long-term knockdown of STK25 mRNA and protein by Gc337 in mice. To assess the persistence of Gc337-mediated gene silencing, this study involved a single subcutaneous injection of 20 mg / kg Gc337 (prepared with 0.9% saline) into wild-type male C57BL / 6 mice, with saline-treated mice serving as controls. Liver tissues were collected on days 3, 7, 14, 21, 28, and 35 post-injection. Total RNA and total protein were extracted from the liver tissues using the method described in Example 3. The expression level of STK25 mRNA was detected by RT-qPCR, and the expression level of STK25 protein was detected by Western blot, monitoring the knockdown effect and duration of Gc337 on STK25.

[0071] Experimental results: 1. RT-qPCR analysis results showed that ( Figure 10(See Table 10). Significant inhibition of STK25 messenger ribonucleic acid (mRNA) was observed on day 3 (reduction of 46.1%, P<0.001). The knockdown efficiency peaked on days 14–21 (day 7: reduction of 50.7%, P<0.001; day 14: reduction of 62.4%, P<0.001; day 21: reduction of 59.7%, P<0.001; day 28: reduction of 54.8%, P<0.01), and a sustained silencing effect was observed on day 35 (reduction of 42.1%, P<0.05).

[0072] Table 10 Changes in Stk25 mRNA expression levels at different time points after Gc337 treatment.

[0073] 2. Western blot analysis results showed that ( Figure 11 (Tables 11 and 12) Compared with the high-fat diet control group (HFD-NC), the expression level of STK25 protein in the liver tissue of mice treated with Gc337 was also significantly reduced, indicating that Gc337 has the potential to become a long-acting therapeutic drug for metabolic-associated fatty liver disease (MASLD).

[0074] Table 11 Changes in Stk25 protein expression levels at different time points after Gc337 treatment.

[0075] Table 12 Changes in Stk25 protein expression levels at different time points after Gc337 treatment.

Claims

1. An antisense oligonucleotide conjugate targeting Stk25 mRNA, characterized in that, The antisense oligonucleotide conjugate comprises: 1) An antisense oligonucleotide with a length of 16 nucleotides, the nucleotide sequence of which is: GATCTTGATGGCCACC, as shown in SEQ ID NO:1; 2) Covalently linked to the trivalent GalNAc conjugate portion of the oligonucleotide.

2. The antisense oligonucleotide conjugate targeting Stk25 mRNA according to claim 1, characterized in that, The antisense oligonucleotide has a 3-10-3 spacer structure: the nick region contains 10 2'-deoxynucleotides, and the 5' and 3' ends are each connected to a wing composed of 3 nucleotides; wherein, each nucleotide in the 5' wing and each nucleotide in the 3' wing are modified with cEt sugar, each nucleotide in the nick region is modified with 2'-deoxy sugar, the internucleotide bonds in the entire spacer are phosphate thioester bonds, and all cytosine residues are 5-methylcytosine.

3. The antisense oligonucleotide conjugate targeting Stk25 mRNA according to claim 1, characterized in that, The trivalent GalNAc conjugate is GalNAc3o, with the structure shown in Equation I: I。 4. The antisense oligonucleotide conjugate targeting Stk25 mRNA according to claim 3, characterized in that, The 5′ end of the oligonucleotide is covalently linked to GalNAc3o via a phosphodiester bond.

5. The use of the antisense oligonucleotide conjugate targeting Stk25 mRNA as described in claim 1 in inhibiting Stk25 mRNA expression.

6. The use of the antisense oligonucleotide conjugate targeting Stk25 mRNA as described in claim 1 in the preparation of drugs for the prevention and treatment of diseases related to Stk25 mRNA overexpression.

7. The application according to claim 6, characterized in that, The diseases associated with Stk25 mRNA overexpression are hyperlipidemia or metabolic dysfunction-related steatohepatitis.

8. The application according to claim 7, characterized in that, The metabolic dysfunction-related steatohepatitis mentioned above is non-alcoholic steatohepatitis.

9. A pharmaceutical composition, characterized in that, Includes the antisense oligonucleotide conjugate of claim 1 and a pharmaceutically acceptable carrier.

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