Bipolar targeting small nucleic acid drug nas01 and application thereof

Abstract

The application discloses a two-pole targeted small nucleic acid drug NAS01 and application thereof, and relates to a siRNA composed of a sense strand and an antisense strand RNA, which is obtained by siRNA drug design on ACC1 and PIK3CB genes, and has a nucleotide sequence of SEQ ID NO: 1-10; and the modified siRNA is conjugated with GalNAc(L96) to form the two-pole targeted small nucleic acid drug NAS01. The NAS01 can target and internalize liver cells, and effectively silence ACC1 and PIK3CB at the mRNA level at the same time, thereby relieving the fatty degeneration of non-alcoholic fatty liver disease, and reducing the weight of obese mice, so that the NAS01 potentially becomes a drug for treating non-alcoholic fatty liver disease or non-alcoholic fatty hepatitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a bipolar targeted small nucleic acid drug NAS01 and its applications. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is a chronic, progressive liver disease caused by overnutrition and insulin resistance (IR) in genetically susceptible individuals. The disease spectrum includes non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), and related fibrosis and cirrhosis. NAFLD is characterized by hepatic steatosis and is often accompanied by symptoms such as inflammation, oxidative stress, mitochondrial dysfunction, and hepatocellular damage.

[0003] Acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme in de novo fatty acid synthesis, and the ACC1 isoform is a key driver of hepatic lipid synthesis. In NAFLD / NASH, the expression and activity of ACC1 are significantly upregulated, promoting excessive accumulation of hepatic lipids. Therefore, ACC1 is recognized as an important target for the treatment of NAFLD / NASH. For example, Chinese patent CN114206884 discloses thienopyrimidine derivatives as ACC inhibitors and their uses, involving small molecule inhibitors.

[0004] In metabolic-associated fatty liver disease (NAFLD), the PIK3CB gene plays a crucial role as a mediator of insulin signaling and an amplifier of metabolic disorders. Its encoded phosphoinositide 3-kinase catalytic subunit beta (PI3Kβ) is one of the important catalytic subunits of the PI3K / AKT signaling pathway, playing a key role in insulin signaling, glucose metabolism, lipid synthesis, cell survival, and inflammation regulation. When the body ingests excessive energy or develops insulin resistance, PIK3CB (as part of the PI3K / AKT pathway) is activated, promoting the maturation and nuclear translocation of SREBP-1c (sterol regulatory element-binding protein). Once in the nucleus, SREBP-1c directly binds to the promoter region of the ACC1 gene, leading to its high expression. ACC1 then catalyzes the conversion of acetyl-CoA to malonyl-CoA, promoting fatty acid synthesis while preventing fatty acid oxidation. PIK3CB dysfunction may lead to decreased hepatocyte responsiveness to insulin, increased fatty acid synthesis, gluconeogenesis imbalance, and exacerbated metabolic inflammation, thereby driving the occurrence and progression of NAFLD. High expression of ACC1 directly leads to the accumulation of malonyl-CoA, which in turn leads to the continuous synthesis of saturated fatty acids such as palmitic acid. These fatty acids not only accumulate into triglycerides, but their toxic metabolites also trigger inflammation and oxidative stress, accelerating the progression of the disease from simple fatty liver to hepatitis.

[0005] Small interfering RNA (siRNA) is a double-stranded RNA molecule of 21-23 nucleotides in length that provides a novel strategy for disease treatment by specifically silencing the expression of target genes at the posttranscriptional level. For example, Chinese patent CN119732910 discloses an siRNA nanoliposome and its application in the preparation of a drug for treating non-alcoholic fatty liver disease (NAFLD), using LNP delivery of siRNA for the preparation of this drug. Chinese patent CN101892226 discloses an siRNA that inhibits the expression of the ACC gene in human adipocytes, its expression vector, and its application, using the pSilencer 4.1-PPARγ2P-neo-D siRNA vector carrying an adipocyte-specific promoter and siRNA sequence for the preparation of a weight-loss drug. However, siRNA molecules themselves are easily degraded by serum nucleases, have low cellular uptake efficiency, and lack tissue targeting, which limits their clinical application. GalNAc-L96 is a tri-antennae N-acetylgalactosamine molecule that specifically binds to the desialization glycoprotein receptor (ASGPR), which is highly expressed on the surface of hepatocytes, mediating highly efficient liver-targeted delivery of siRNA. Currently, GalNAc-siRNA conjugation technology has become one of the most advanced delivery platforms in the field of liver disease. For example, Chinese patent CN116640762 discloses siRNA targeting diglyceride acyltransferase and its applications, designing siRNA targeting DGAT2 (diglyceride acyltransferase) to prepare a treatment for non-alcoholic fatty liver disease.

[0006] Although ACC1 is widely recognized as an important therapeutic target for regulating lipid production, and PIK3CB, a key molecule involved in insulin signaling and glucose and lipid metabolism regulation, is increasingly showing its interventional value in metabolic-related fatty liver disease, and GalNAc delivery technology has made significant progress in the field of liver-targeted nucleic acid drugs, there are currently no publicly reported cases of specific siRNA sequences targeting both ACC1 and PIK3CB for the comprehensive treatment of NAFLD / NASH with significant efficacy. Therefore, developing a novel, highly efficient drug that simultaneously regulates lipid synthesis and metabolic signaling pathways by targeting both ACC1 and PIK3CB has significant clinical implications and broad market value. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a bipolar targeted small nucleic acid drug, NAS01, and its application. By designing siRNAs that dually target ACC1 and PIK3CB and conjugating them with GalNAc, the drug targets and internalizes hepatocytes while simultaneously silencing the ACC1 and PIK3CB genes. This alleviates the steatotic degeneration of non-alcoholic fatty liver disease and reduces the weight of obese mice, thereby enabling the preparation of drugs for treating non-alcoholic steatohepatitis (NASH) or non-alcoholic steatohepatitis.

[0008] To achieve the above objectives, the present invention provides a bipolar targeted small nucleic acid drug NAS01, wherein the core effector molecule in the bipolar targeted small nucleic acid drug NAS01 is siRNA, and the siRNA is selected from any one of the following five siRNAs composed of sense strand RNA and antisense strand RNA: (1) siRNA composed of the sense strand RNA shown in SEQ ID NO:1 and the antisense strand RNA shown in SEQ ID NO:2; (2) siRNA composed of the sense strand RNA shown in SEQ ID NO:3 and the antisense strand RNA shown in SEQ ID NO:4; (3) siRNA composed of the sense strand RNA shown in SEQ ID NO:5 and the antisense strand RNA shown in SEQ ID NO:6; (4) siRNA composed of the sense strand RNA shown in SEQ ID NO:7 and the antisense strand RNA shown in SEQ ID NO:8; (5) siRNA consisting of the sense strand RNA shown in SEQ ID NO:9 and the antisense strand RNA shown in SEQ ID NO:10.

[0009] Preferably, the bipolar targeted small nucleic acid drug NAS01 can simultaneously downregulate the expression of ACC1 and PIK3CB genes.

[0010] Preferably, the siRNA has been chemically modified.

[0011] More preferably, the chemical modification is phosphodiester bond modification and / or ribose 2' modification.

[0012] More preferably, the phosphodiester bond modification is that one or more of the phosphodiester bonds at positions 1 and 2 of the sense strand and positions 1, 2, and the last two phosphodiester bonds of the antisense strand are modified with thiophosphate; the ribose 2' modification is that one or more of the 2' positions of the nucleotides at positions 7, 9, 10, and 11 of the sense strand and positions 2, 6, 14, and 16 of the antisense strand are modified with fluorine and the 2' position of the remaining nucleotides is modified with methoxy.

[0013] Preferably, the siRNA is conjugated with a conjugate to form the bipolar targeted small nucleic acid drug NAS01.

[0014] More preferably, the conjugate is GalNAc or a GalNAc derivative (L96).

[0015] More preferably, the siRNA and the conjugate are connected by covalently linking the conjugate to the 3' end of the positive strand of the siRNA.

[0016] The present invention also provides the use of the bipolar targeted small nucleic acid drug NAS01 in the preparation of drugs for the treatment and / or prevention of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

[0017] Preferably, the drug comprises one or more pharmaceutically acceptable carriers.

[0018] The beneficial effects of this invention are as follows: This invention designs siRNAs that target the ACC1 and PIK3CB genes, effectively silencing the expression of ACC1 and PIK3CB mRNAs. Conjugating the siRNA to GalNAc(L96) allows for targeting and internalization of hepatocytes, alleviating steatosis in non-alcoholic fatty liver disease, and reducing the weight of obese mice. Therefore, it can be used to prepare drugs for treating non-alcoholic steatohepatitis (NAFLD) or non-alcoholic steatohepatitis. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the modification scheme of the siRNA-ACC1-PIK3CB-1-L96 conjugate in this invention.

[0020] Figure 2 This is a bar chart showing the expression level of ACC1 mRNA in HepG2 cells in Example 2; **** in the figure indicates P<0.0001.

[0021] Figure 3 This is a bar chart showing the expression level of PIK3CB mRNA in HepG2 cells in Example 2; **** in the figure indicates P<0.0001.

[0022] Figure 4 This is a bar chart showing the expression level of ACC1 mRNA in HepG2 cells in Example 3; **** in the figure indicates P<0.0001.

[0023] Figure 5 This is a graph showing the weight changes of C57BL / 6 mice in Example 4.

[0024] Figure 6 The image shows the gross morphology of the liver tissue from C57BL / 6 mice in Example 4.

[0025] Figure 7 HE staining image of liver tissue from C57BL / 6 mice in Example 4 (200×).

[0026] Figure 8 Oil Red O staining image of liver tissue from C57BL / 6 mice in Example 4 (200×).

[0027] Figure 9shows the mRNA expression level of ACC1 in liver tissue of C57BL / 6 mice in Example 5, and **** in the figure indicates P<0.0001.

[0028] Figure 10 shows the mRNA expression level of PIK3CB in liver tissue of C57BL / 6 mice in Example 5, and **** in the figure indicates P<0.0001. Detailed Description of the Embodiments

[0029] The technical solution of the present invention will be further explained below with reference to the accompanying drawings and specific embodiments. It should be noted that the following examples are only preferred embodiments of the present invention, and should not be construed as limiting the present invention. The protection scope of the present invention shall be based on the contents recorded in the claims. Modifications and substitutions made to the technical solution of the present invention by those skilled in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] Main Materials and Reagents: C57BL / 6 mice: all are SPF grade, purchased from the Experimental Animal Center of Three Gorges University, production license number: SCXK (Hubei) 2022-0012; Human hepatoma cell line HepG2: provided by the research group of Professor Wu Jiangfeng of Three Gorges University; High-fat diet: purchased from Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd., XT310; RNA extraction kit: purchased from Nanjing Vazyme Biotech Co., Ltd., RC112-01; Reverse transcription reagent: purchased from Nanjing Vazyme Biotech Co., Ltd., R312-02; Oleic acid (OA): purchased from Sigma-Aldrich, USA, batch number: O1383; DMEM medium: purchased from Gibco, C11995500CP; Fetal bovine serum: purchased from Nanjing Vazyme Biotech Co., Ltd., F101-01; Penicillin-streptomycin solution: purchased from Wuhan Servicebio Technology Co., Ltd., GA2408008; Trypsin cell digestion solution: purchased from Wuhan Servicebio Technology Co., Ltd., G4010; Modified Oil Red O Staining Kit: purchased from Beijing Solarbio Science & Technology Co., Ltd., G1261; 2×Q3 SYBR qPCR Master mix: purchased from Shanghai Tolo Biotech Co., Ltd., 22204-01.

[0031] Oleic acid (OA) working solution: Dissolve 63 μL of oleic acid (OA) in 937 μL of 0.1 mM NaOH solution and dissolve in a water bath at 70 °C; dissolve bovine serum albumin (BSA) powder in the culture medium to prepare a 10% (w / v) BSA solution and dissolve in a water bath at 55 °C; mix the above two solutions at a ratio of 1:19 (v / v), aid dissolution at 55 °C, prepare a 10 mM OA working solution, filter to sterilize, and use for later use; High-fat feed: Customized purified feed for radiation-sterilized laboratory animals, Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., Formula No.: XT310.

[0032] Example 1 (1) Based on the human ACC1 gene sequence (NCBI Gene ID: 31) and PIK3CB gene sequence (NCBI Gene ID: 31) (GeneID: 5291) Five pairs of different siRNAs with positive and negative strand sequences were designed. The sequences of the five siRNAs and the L96 sequence were input into a solid-phase synthesizer, and siRNAs conjugated to L96 were synthesized using solid-phase synthesis. During the synthesis process, fluorine (2'-Fluoro, 2'-F) modification was performed on the 2' positions of nucleotides 7, 9, 10, and 11 of the positive strand and nucleotides 2, 6, 14, and 16 of the negative strand. In addition, methoxy (2'-O-Methyl, 2'-OMe) modification was performed on the 2' positions of all other nucleotides in the siRNA double strand. After synthesis, phosphate thioate (PS) modification was performed on the phosphodiester bonds at positions 1 and 2 of the positive strand and the phosphodiester bonds at positions 1, 2, and the last two positions of the negative strand, for a total of 6 modifications. The siRNA was connected to L96 by covalent coupling of the 3' end of the positive strand of the siRNA to L96. (2) The modified siRNA-L96 sense strand (3' end covalently coupled with L96) and antisense strand were purified by high performance liquid chromatography (HPLC) to obtain purified siRNA sense strand (3' end covalently coupled with L96) and antisense strand; (3) Take the purified siRNA sense strand (3' end covalently coupled with L96) and antisense strand, pair them, and place them in ddH2O in a molar ratio of 1:1. After boiling, anneal to room temperature to form 5 double-stranded siRNA conjugates, denoted as siRNA-ACC1-PIK3CB-1-L96, siRNA-ACC1-PIK3CB-2-L96, siRNA-ACC1-PIK3CB-3-L96, siRNA-ACC1-PIK3CB-4-L96, and siRNA-ACC1-PIK3CB-5-L96. The specific base sequences are shown in Table 1.

[0033] Table 1. Sequences of double-stranded siRNA conjugates

[0034] Example 2 (1) Human liver cancer HepG2 cells were taken and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution in an incubator at 37°C and 5% CO2 until the logarithmic growth phase. (2) The human liver cancer HepG2 cells obtained in step (1) were placed in 12-well cell culture plates containing complete culture medium and grouped accordingly: Blank control group (MOCK): cultured in complete culture medium for 24 h; Model group (OA): Add 2mM OA working solution and induce for 24h; Experimental group (siRNA-ACC1-PIK3CB-1 / 2 / 3 / 4 / 5-L96): After induction with 2mM OA working solution for 24h, 10nM siRNA-ACC1-L96 was added and treated for 24h. (3) After the culture was completed, total RNA was extracted from each group of cells using a commercial RNA extraction kit, and then cDNA was obtained by reverse transcription. (4) Real-time quantitative PCR uses cDNA as a template and amplifies human ACC1 and human PIK3CB with primers to detect the relative expression levels of ACC1 mRNA and PIK3CB mRNA. The internal reference gene is β-actin. The primer sequences are shown in Table 2 and the amplification system is shown in Table 3. Table 2 qPCR primer sequences

[0035] Table 3 qPCR amplification system

[0036] The results are as follows Figure 2 and Figure 3 As shown, compared with the blank control group, the mRNA expression levels of ACC1 and PIK3CB in the model group were significantly increased, while the mRNA expression of ACC1 and PIK3CB in the experimental group was significantly inhibited (*p<0.05). These results indicate that at the cellular level, all five synthesized siRNAs—ACC1-PIK3CB-L96—can effectively silence the ACC1 and PIK3CB genes. Among them, siRNA-ACC1-PIK3CB-1 showed the most significant inhibitory effect, and therefore this group of siRNAs was selected for subsequent experiments.

[0037] Example 3 Based on siRNA-ACC1-PIK3CB-1-L96, we explored the optimal way to conjugate siRNA with L96.

[0038] (1) Take the 5 pairs of siRNA synthesized and modified in step (1) of Example 1, wherein the 3' and 5' ends of the siRNA sense strand and the 3' and 5' ends of the antisense strand are covalently coupled to L96 to form four different conjugates, named siRNA-ACC1-PIK3CB-1(positive)-L96, siRNA-ACC1-PIK3CB-1(negative)-L96, L96-siRNA-ACC1-PIK3CB-1(positive), L96-siRNA A-ACC1-PIK3CB-1 (anti); (Note: positive represents the sense strand; negative represents the antisense strand; L96 at the tail end means L96 is coupled to the 3' end of the siRNA; L96 at the head end means L96 is coupled to the 5' end of the siRNA; where siRNA-ACC1-PIK3CB-1 (positive)-L96 is the same as siRNA-ACC1-PIK3CB-1-L96 in Examples 1 and 2, renamed here only to avoid confusion with the other three conjugates; (2) Take the four different conjugates from step (1) and purify and pair them according to steps (2)-(3) of Example 1 to form double-stranded siRNA conjugates; (3) Human liver cancer HepG2 cells were taken and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution in an incubator at 37°C and 5% CO2 until the logarithmic growth phase. (4) The human liver cancer HepG2 cells obtained in step (3) were placed in 12-well cell culture plates containing complete culture medium and grouped accordingly: Blank control group (MOCK): Cells were collected after culturing in complete culture medium for 48 hours; Model group (OA) and experimental groups (1 / 2 / 3 / 4): 2mM OA working solution was added, and after induction for 24h, 10nM of siRNA-ACC1-PIK3CB-1 (forward)-L96, siRNA-ACC1-PIK3CB-1 (reverse)-L96, L96-siRNA-ACC1-PIK3CB-1 (forward), and L96-siRNA-ACC1-PIK3CB-1 (reverse) were added to each experimental group, respectively. Cells were collected after 24h. (5) Extract total RNA from each group of cells, reverse transcribe it into cDNA, and perform real-time quantitative PCR. The operation steps are the same as in Example 2 (3)-(4).

[0039] The results are as follows Figure 4As shown, compared with the blank control group, the expression level of ACC1 mRNA in the model group was significantly increased, while the expression of ACC1 mRNA in the experimental group was significantly inhibited (*p<0.05), indicating that all four conjugates can effectively silence the ACC1 gene in HepG2 cells. Among them, the experimental group siRNA-ACC1-PIK3CB-1(positive)-L96 had the best effect. Figure 3 This also indicates that the conjugate can effectively silence the PIK3CB gene in HepG2 cells; therefore, among the four ways in which siRNA-ACC1-PIK3CB-1 is linked to L96, the method of covalently coupling the 3' end of the siRNA's positive strand with L96 is feasible.

[0040] Example 4 (1) Eight-week-old SPF-grade C57BL / 6 mice were randomly divided into the following groups: Control group (Mock): fed with standard maintenance diet; Model group (NAFLD): NAFLD was induced by feeding a high-fat diet for 16 weeks; Treatment group (siRNA-ACC1-PIK3CB-1-L96): After being fed a high-fat diet for 16 weeks, siRNA-ACC1-PIK3CB-1-L96 was injected via the tail vein once a week for 5 consecutive weeks; the dose of siRNA-ACC1-PIK3CB-1-L96 was 5 nmol / animal / dose, and the total injection dose per animal was 25 nmol; (2) Record the daily changes in mouse body weight from the first administration date; 3 weeks after the last injection, anesthetize the mice, separate the liver, observe the morphology of the liver, take a portion of liver tissue, fix it with 4% paraformaldehyde for histopathological analysis. (3) After the fixed liver was dehydrated, embedded in paraffin, and sectioned (3 μm thick), it was stained with HE, mounted with neutral resin, and observed under a microscope. At the same time, after the fixed liver was dehydrated by a sucrose solution gradient, it was embedded in OCT, frozen sectioned (20 μm thick), stained with Oil Red O, mounted with glycerol gelatin, and the lipid deposition was observed.

[0041] The results are as follows Figure 5 As shown, using the body weight of the blank control group mice as the normal baseline, the model group mice showed significant weight changes after being fed a high-fat diet, with a significant increase in weight in the later stages, exhibiting an obese pattern; while in the treatment group, after injection of siRNA-ACC1-PIK3CB-1-L96, the body weight gradually decreased and tended to normal levels. The gross morphology of the liver also showed the same trend as the weight changes, with the liver morphology of the treatment group showing significant improvement compared to the model group. Figure 6 ).

[0042] After HE staining, the hepatocytes in the blank control group were tightly arranged with no obvious vacuoles; the liver tissue in the model group showed obvious steatosis, severe vacuolation, and irregular arrangement. After intervention, the hepatocyte vacuolation in the treatment group was significantly improved, and the fat vacuoles were significantly smaller than those in the model group. Figure 7 After Oil Red O staining of liver tissue, no significant lipid accumulation was observed in the livers of mice in the blank control group, while a large number of lipid droplets appeared in the livers of mice in the model group. In the treatment group, the number of lipid droplets in hepatocytes was significantly reduced after intervention. Figure 8 ).

[0043] Example 5 (1) Take liver tissue from each group in Example 4, separate hepatocytes, extract total RNA from each group of liver tissue using a commercial RNA extraction kit, and then reverse transcribe to obtain cDNA; (2) Design mouse-derived ACC1-F and ACC1-R primers, PIK3CB F and PIK3CB R primers respectively, and use mouse-derived β-actin as an internal reference gene for real-time quantitative PCR detection. The primer sequences are shown in Table 4, and the amplification system is the same as in Table 3.

[0044] Table 4 qPCR primer sequences

[0045] The results are as follows Figure 9 and Figure 10 As shown, after a high-fat diet induced NAFLD in C57BL / 6 mice, the expression of ACC1 mRNA and PIK3CB mRNA in hepatocytes was significantly increased; after treatment with siRNA-ACC1-PIK3CB-1-L96, the expression of ACC1 mRNA and PIK3CB mRNA in hepatocytes was significantly downregulated. This result indicates that siRNA-ACC1-PIK3CB-1-L96 can silence the ACC1 and PIK3CB genes in hepatocytes, thereby improving hepatocyte steatosis and thus being used to treat non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

Claims

1. A bipolar targeted small nucleic acid drug NAS01, characterized in that: The core effector molecule in the bipolar targeted small nucleic acid drug NAS01 is a siRNA, which is selected from any one of the following five siRNAs composed of sense and antisense RNA: (1) siRNA composed of the sense strand RNA shown in SEQ ID NO:1 and the antisense strand RNA shown in SEQ ID NO:2; (2) siRNA composed of the sense strand RNA shown in SEQ ID NO:3 and the antisense strand RNA shown in SEQ ID NO:4; (3) siRNA composed of the sense strand RNA shown in SEQ ID NO:5 and the antisense strand RNA shown in SEQ ID NO:6; (4) siRNA composed of the sense strand RNA shown in SEQ ID NO:7 and the antisense strand RNA shown in SEQ ID NO:8; (5) siRNA consisting of the sense strand RNA shown in SEQ ID NO:9 and the antisense strand RNA shown in SEQ ID NO:

10.

2. The bipolar targeted small nucleic acid drug NAS01 according to claim 1, characterized in that: The bipolar targeted small nucleic acid drug NAS01 can simultaneously downregulate the expression of ACC1 and PIK3CB genes.

3. The bipolar targeted small nucleic acid drug NAS01 according to claim 1 or 2, characterized in that: The siRNA was chemically modified.

4. The bipolar targeted small nucleic acid drug NAS01 according to claim 3, characterized in that: The chemical modification is phosphodiester bond modification and / or ribose 2' modification.

5. The bipolar targeted small nucleic acid drug NAS01 according to claim 4, characterized in that: The phosphodiester bond modification is that one or more of the phosphodiester bonds at positions 1 and 2 of the sense strand, and positions 1, 2, and the last two phosphodiester bonds of the antisense strand are modified with thiophosphate; the ribose 2' modification is that one or more of the 2' positions of the nucleotides at positions 7, 9, 10, and 11 of the sense strand, and positions 2, 6, 14, and 16 of the antisense strand are modified with fluorine, and the 2' position of the remaining nucleotides is modified with methoxy groups.

6. The bipolar targeted small nucleic acid drug NAS01 according to any one of claims 1-5, characterized in that: The siRNA is conjugated with the conjugate.

7. The bipolar targeted small nucleic acid drug NAS01 according to claim 6, characterized in that: The conjugate is GalNAc or a GalNAc derivative.

8. The bipolar targeted small nucleic acid drug NAS01 according to claim 6, characterized in that: The siRNA and the conjugate are connected by covalently linking the conjugate to the 3' end of the positive strand of the siRNA.

9. The use of NAS01, a bipolar targeted small nucleic acid drug as described in any one of claims 1-8, in the preparation of a drug for the treatment and / or prevention of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

10. The application according to claim 9, characterized in that: The drug includes one or more pharmaceutically acceptable carriers.

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