LYTAC molecule for targeted degradation of GPC3 as well as preparation method and application of LYTAC molecule

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an LYTAC molecule for targeted degradation of GPC3 and a preparation method and application of the LYTAC molecule. The LYTAC molecule is composed of a target TfR1 nucleic acid aptamer and a target GPC3 nucleic acid aptamer, wherein the nucleotide sequence of the targeted TfR1 nucleic acid aptamer is as shown in SEQ ID NO: 1; the nucleotide sequence of the targeted GPC3 nucleic acid aptamer is as shown in SEQ ID NO: 2. According to the present invention, the research results show that the GPC3 protein content is not affected by the single TfR1 nucleic acid aptamer or the GPC3 nucleic acid aptamer, and the LYTAC molecule can significantly reduce the GPC3 protein content on the surfaces of Hep3B and HepG2 cells. The LYTAC molecule induces GPC3 protein degradation through a lysosome way, then proliferation and migration of liver cancer cells are inhibited, liver cancer treatment is achieved, and the LYTAC molecule has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a LYTAC molecule that targets the degradation of GPC3, its preparation method, and its application. Background Technology

[0002] Glypican-3 (GPC3) is a heparan sulfate glycoprotein on the cell membrane surface. Anchored to the cell membrane by phosphatidylinositol, it is a multifunctional common receptor on the cell surface, playing a crucial role in mediating signal transduction. It can also influence signal transduction by binding to the extracellular matrix, growth factors, and proteases, playing a vital role in regulating tumor cell proliferation, differentiation, adhesion, and metastasis. Current research has found that GPC3 is almost not expressed in normal or benign liver disease hepatocytes, but it is specifically highly expressed in hepatocellular carcinoma (HCC) and is significantly correlated with tumor malignancy, metastasis, and poor prognosis. Therefore, GPC3 is considered a diagnostic biomarker and potential therapeutic target for liver cancer.

[0003] In 2020, Carolyn R. Bertozzi's team developed a lysosome-targeting chimera (LYTAC) that selectively introduces cell membrane or extracellular proteins into lysosomes for degradation, representing a breakthrough in membrane protein degradation. Although LYTAC is still in its early stages of development and faces many challenges, such as tissue specificity due to its reliance on cell surface lysosomal targeting receptors, it has shown great potential in the treatment of cancer, autoimmune diseases, and neurodegenerative diseases.

[0004] In 2025, Xin Zhou's team at Harvard Medical School developed a novel transferrin receptor-targeting chimera (TransTAC) that efficiently degrades a variety of membrane proteins through transferrin receptor 1 (TfR1)-mediated endocytosis. TfR1 is expressed at low levels in normal human cells but is significantly upregulated in cancer cells, exhibiting highly efficient and agonist-free targeted internalization.

[0005] In 2021, a new study reported a LYTAC molecule based on a bispecific nucleic acid aptamer. This molecule, composed of an IGFIIR nucleic acid aptamer and a target protein nucleic acid aptamer, can effectively induce the degradation of cell membrane surface receptors. The nucleic acid aptamer is a single-stranded DNA molecule with a specific spatial structure, and in this study, it was able to bind to the target protein with strong affinity and specificity.

[0006] Currently, there are no GPC3-targeted drugs on the market globally, nor are there any reports on the selective induction of GPC3 protein degradation using nucleic acid aptamers. However, several investigational drugs targeting GPC3 have entered clinical trials, including CAR-T, bispecific antibodies, and ADCs. Therefore, developing a novel GPC3-targeted degradation inducer holds promise for providing a new strategy for the treatment of hepatocellular carcinoma. Summary of the Invention

[0007] In view of this, the present invention proposes a LYTAC molecule for targeted degradation of GPC3, its preparation method, and its applications. The LYTAC molecule consists of a TfR1-targeting aptamer (TfR1ap) and a GPC3-targeting aptamer (GPC3ap). It effectively induces GPC3 degradation via endocytosis and lysosomal pathway, and this induction of GPC3 degradation inhibits the proliferation and migration of liver cancer cells.

[0008] One of the objectives of this invention is to provide a LYTAC molecule that targets and degrades GPC3.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] LYTAC molecules that target and degrade GPC3, wherein the LYTAC molecules are composed of a TfR1-targeting aptamer and a GPC3-targeting aptamer.

[0011] Furthermore, the nucleotide sequence of the TfR1 aptamer is shown in SEQ ID NO:1; the nucleotide sequence of the GPC3 aptamer is shown in SEQ ID NO:2.

[0012] The LYTAC molecule (GPC3-LYTAC bispecific nucleic acid aptamer) provided by this invention can simultaneously bind to TfR1 protein and GPC3 protein on the cell membrane. It effectively utilizes the endocytosis of TfR1 protein to bring GPC3 protein from the cell membrane into the cell and induces GPC3 protein degradation through the lysosomal pathway, thereby inhibiting the proliferation and migration of liver cancer cells and achieving liver cancer treatment.

[0013] The second objective of this invention is to provide a method for preparing LYTAC molecules.

[0014] To achieve the above objectives, the present invention adopts the following technical solution:

[0015] The preparation method of LYTAC molecules includes the following steps:

[0016] 1) Target the 5' end of the GPC3 aptamer with phosphorylation;

[0017] 2) Mix the 5' phosphorylated GPC3 aptamer obtained in step 1) with the TfR1 aptamer and perform PCR annealing; the reaction product forms a bispecific aptamer under the action of T4 DNA ligase.

[0018] Furthermore, in step 1), 5' end phosphorylation is performed using PCR;

[0019] The PCR reaction system includes: 80-12 μL of 80-120 μM GPC3 aptamer, 0.5-2 μL of T4 Polynucleotide Kinase, 1-4 μL of Reaction Buffer A (10X), 0.5-2 μL of 8-12 mM ATP, and 20 μL of DEPC water.

[0020] The PCR reaction conditions are: incubation at 30-45℃ for 2-5 hours.

[0021] As a preferred option, the PCR reaction system is as follows: 10 μL of 100 μM GPC3 aptamer, 1 μL of T4 Polynucleotide Kinase (10 U / μL), 2 μL of Reaction Buffer A (10X), 1 μL of 10 mM ATP, and 6 μL of DEPC water to a final volume of 20 μL.

[0022] As a preferred option, the PCR reaction conditions are: incubation at 37°C for 3 hours.

[0023] Preferably, in step 1), after the PCR reaction is completed, the phosphorylase is inactivated at 90-100℃ for 4-10 min and then incubated at 2-8℃ for 10-20 min to obtain the 5' phosphorylated GPC3 aptamer.

[0024] Preferably, in step 1), after the PCR reaction is completed, the phosphorylase is inactivated at 95℃ for 5 min and then incubated at 4℃ for 15 min to obtain the 5' phosphorylated GPC3 aptamer.

[0025] Furthermore, in step 2), the concentration of the TfR1 nucleic acid aptamer is 40-60 μM, and the amount used is 15-25 μL;

[0026] The annealing procedure is set as follows:

[0027] (1) Denaturation: 90-100℃ for 4-10 minutes;

[0028] (2) Annealing: Cool to 20-30℃ at a rate of 0.05-0.2℃ / 8s and maintain for 80-100 minutes;

[0029] (3) Store at 2-8℃.

[0030] Preferably, in step 2), the concentration of the TfR1 nucleic acid aptamer is 50 μM and the amount used is 20 μL.

[0031] Preferably, in step 2), the annealing procedure is set as follows:

[0032] (1) Denaturation: 95℃ for 5 minutes;

[0033] (2) Annealing: Cool to 25°C at a rate of 0.1°C / 8s and maintain for 90 minutes;

[0034] (3) Store at 4℃.

[0035] As a preferred embodiment, in step 2), after PCR annealing, add 1-2 μL of T4 DNA ligase (800-1200 U / μL), 4-8 μL of 10×Ligation Buffer, and 3-6 μL of DEPC water to the reaction product, making a final volume of 50 μL; mix by pipetting and incubate overnight at room temperature to obtain bispecific nucleic acid aptamers.

[0036] As a preferred embodiment, in step 2), after PCR annealing, 1 μL of T4 DNA ligase (1000 U / μL), 5 μL of 10×Ligation Buffer, and 4 μL of DEPC water are added to the reaction product, with a final volume of 50 μL. The mixture is then blown and aspirated to mix, and ligated overnight at room temperature to obtain a bispecific nucleic acid aptamer.

[0037] The third objective of this invention is to provide a method for inducing the degradation of GPC3 protein in liver cancer cells.

[0038] To achieve the above objectives, the present invention adopts the following technical solution:

[0039] A method for inducing the degradation of GPC3 protein in liver cancer cells, the method being a non-disease diagnosis or treatment method; the method comprising: treating liver cancer cells with the aforementioned LYTAC molecule and / or LYTAC molecules prepared by the aforementioned method.

[0040] Furthermore, the concentration of the LYTAC molecules is ≥50nM, preferably 50-400nM; the treatment time is ≥12h, more preferably 12-48h.

[0041] The fourth objective of this invention is to provide an application of the aforementioned LYTAC molecule in the preparation of an anti-liver cancer drug.

[0042] To achieve the above objectives, the present invention adopts the following technical solution:

[0043] Application of LYTAC molecules in the preparation of anti-liver cancer drugs.

[0044] Preferably, the liver cancer includes hepatocellular carcinoma.

[0045] The fifth objective of this invention is to provide an application of the aforementioned LYTAC molecule in the preparation of a GPC3-targeted degradation inducer, a liver cancer cell proliferation inhibitor, and / or a liver cancer cell migration inhibitor for the treatment of liver cancer.

[0046] The sixth objective of this invention is to provide the application of a TfR1-targeting nucleic acid aptamer combined with a GPC3-targeting nucleic acid aptamer in the preparation of a drug for treating liver cancer.

[0047] To achieve the above objectives, the present invention adopts the following technical solution:

[0048] Application of TfR1-targeting nucleic acid aptamers combined with GPC3-targeting nucleic acid aptamers in the preparation of drugs for the treatment of liver cancer.

[0049] Preferably, the nucleotide sequence of the TfR1 aptamer is shown in SEQ ID NO:1; and the nucleotide sequence of the GPC3 aptamer is shown in SEQ ID NO:2.

[0050] Preferably, the treatment of liver cancer includes improving the degradation efficiency of GPC3 protein and / or inhibiting the proliferation and migration of liver cancer cells. The beneficial effects of the present invention are as follows:

[0051] 1. This invention provides a LYTAC molecule based on a bispecific nucleic acid aptamer to target liver cancer cells and degrade GPC3 protein via endocytosis and lysosomal pathway. This LYTAC molecule effectively induces GPC3 degradation in a time- and concentration-dependent manner via endocytosis and lysosomal pathway, and effectively inhibits the proliferation and migration of liver cancer cells by inducing GPC3 degradation. This invention provides a novel strategy for the treatment of liver cancer.

[0052] 2. The bispecific nucleic acid aptamer GPC3-LYTAC of the present invention has the specificity to induce GPC3 protein degradation. Experiments have shown that GPC3-LYTAC can significantly reduce the GPC3 protein content on the surface of Hep3B and HepG2 cells, while the GPC3 protein content on the cell membrane is not affected by TfR1ap or GPC3ap alone. Attached Figure Description

[0053] Figure 1 This is a schematic diagram illustrating the GPC3 protein degradation induced by the GPC3-LYTAC bispecific nucleic acid aptamer.

[0054] Figure 2 This is a diagram showing the preparation and verification results of the GPC3-LYTAC bispecific nucleic acid aptamer in Example 1; where, Figure 2 -A is a schematic diagram illustrating the preparation principle of the GPC3-LYTAC bispecific nucleic acid aptamer; Figure 2-B is the gel electrophoresis detection result of the GPC3-LYTAC bispecific nucleic acid aptamer in Example 1.

[0055] Figure 3 This is a graph showing the results of Western blotting analysis of GPC3 protein degradation in Hep3B cells under different treatment conditions in Example 2. Figure 3 -A is an electrophoresis image showing the degradation of GPC3 protein in Hep3B cells under different treatment conditions as detected by Western blotting. Figure 3 -B is Figure 3 -A quantization result graph.

[0056] Figure 4 This is a graph showing the results of Western blotting analysis of GPC3 protein degradation in HepG2 cells under different treatment conditions in Example 2. Figure 4 -A is an electrophoresis image showing the degradation of GPC3 protein in HepG2 cells under different treatment conditions as detected by Western blotting. Figure 4 -B is Figure 4 -A quantization result graph.

[0057] Figure 5 This is a graph showing the results of immunofluorescence assays on Hep3B cells in Example 2. Figure 5 -A is the immunofluorescence image of Hep3B cells; Figure 5 -B is Figure 5 -A quantization result graph.

[0058] Figure 6 This is a graph showing the results of immunofluorescence assays on HepG2 cells in Example 2. Figure 6 -A is the immunofluorescence image of HepG2 cells; Figure 6 -B is Figure 6 -A quantization result graph.

[0059] Figure 7 This is a graph showing the results of Western blotting detection of GPC3 protein degradation at different GPC3-LYTAC concentrations in Example 3. Figure 7 -A is an electrophoresis diagram showing the degree of GPC3 protein degradation at different GPC3-LYTAC concentrations as detected by Western blotting; Figure 7 -B is Figure 7 -A quantization result graph.

[0060] Figure 8 This is a graph showing the results of Western blot analysis of GPC3 protein degradation at different treatment times with GPC3-LYTAC in Example 3. Figure 8 - Electrophoretic image of GPC3 protein degradation degree under different GPC3-LYTAC treatment times as detected by Western blot of protein A; Figure 8 -B is Figure 8 -A quantization result graph.

[0061] Figure 9 This is a graph showing the results of Western blotting analysis of GPC3 protein degradation under GPC3-LYTAC, GPC3-LYTAC+Chloroquine, and GPC3-LYTAC+MG132 treatments in Example 4. Figure 9 -A is an electrophoresis diagram showing the degradation of GPC3 protein under different treatment conditions as detected by Western blotting. Figure 9 -B is Figure 9 -A quantization result graph.

[0062] Figure 10 This is a graph showing the detection results of the cell proliferation experiment in Example 5.

[0063] Figure 11 This is a graph showing the detection results of the cell scratch assay in Example 5, where... Figure 11 -A shows microscopic images of liver cancer cells treated with GPC3-LYTAC from 0 to 48 hours. Figure 11 -B is a graph showing the wound closure rate of liver cancer cells treated with GPC3-LYTAC from 0 to 48 hours. Detailed Implementation

[0064] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0065] In this embodiment of the invention, the main instruments and equipment are shown in Table 1.

[0066] Table 1. Key Instruments, Equipment, and Reagents

[0067]

[0068]

[0069] In this embodiment of the invention, the TfR1 nucleic acid aptamer and the GPC3 nucleic acid aptamer involved were both synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0070] Example 1. Preparation of GPC3-LYTAC bispecific nucleic acid aptamers

[0071] The preparation principle of the GPC3-LYTAC bispecific nucleic acid aptamer is as follows: A complementary sequence is designed and added to the 5' end of both the TfR1 and GPC3 nucleic acid aptamers to form a 13bp linker. After phosphorylation of the 5' end of the GPC3-targeting aptamer, it is combined with the TfR1-targeting aptamer using T4 DNA ligase to form a bispecific nucleic acid aptamer. See details... Figure 2 -A. The nucleotide sequence (5'-3') targeting the TfR1 aptamer (TfR1ap) is shown in SEQ ID NO:1; the nucleotide sequence (5'-3') targeting the GPC3 aptamer (GPC3ap) is shown in SEQ ID NO:2. The first 13 bases at the 5' end of the TfR1ap and GPC3ap sequences constitute the linker portion.

[0072] The advantages of this design are as follows: First, the sequences of TfR1ap and GPC3ap in this invention, after SELEX screening and optimization, can achieve excellent targeting of TfR1 and GPC3. Connecting the two aptamers with a short 13bp double-linker maintains the original secondary structures of TfR1ap and GPC3ap while avoiding interference between the two aptamers at their binding sites. Second, the design of the 5' phosphorylation and complementary sequences makes the ligation reaction efficient and targeted, minimizing the formation of multimers.

[0073] The preparation method of GPC3-LYTAC bispecific nucleic acid aptamers specifically includes the following steps:

[0074] (1) After centrifuging the purchased nucleic acid aptamers, open the cap and dissolve them in DPBS (containing 5mM magnesium ions) to a final concentration of 100uM. Take 10ul of 100uM GPC3 nucleic acid aptamers, add 1ul of T4 Polynucleotide Kinase (10U / μl), 2ul of Reaction Buffer A (10X), 1ul of 10mM ATP, and add 6ul of DEPC water to a final volume of 20ul. Mix well by pipetting and incubate at 37℃ for 3h in a BIO-RAD PCR instrument. After the reaction, inactivate the phosphorylase at 95℃ for 5min and incubate at 4℃ for 15min (rapid cooling to 4℃ after high-temperature denaturation can prevent the single-stranded nucleic acid from reforming the secondary structure, ensuring the efficiency and specificity of subsequent ligation) to obtain 5' phosphorylated GPC3 nucleic acid aptamers.

[0075] (2) Add 20 μL of 50 μM TfR1 nucleic acid aptamer to the 5' phosphorylated GPC3 nucleic acid aptamer obtained in step (1), mix well by pipetting and annealing in a PCR instrument. The annealing program is shown in Table 2.

[0076] Table 2. Annealing Procedure Table

[0077]

[0078]

[0079] (3) Add 1ul of T4 DNA ligase (1000U / μl), 5ul of 10×Ligation Buffer, and 4ul of DEPC water (final volume 50ul) to the product obtained in step (2). Mix well by pipetting and incubate overnight at room temperature to obtain the final product.

[0080] The successful preparation of the bispecific nucleic acid aptamers was further verified by 2% agarose gel electrophoresis (0.5×TBE, 160V, 30min). The results are as follows: Figure 2 As shown in -B, the TfR1 aptamer and the GPC3 aptamer were successfully coupled.

[0081] Example 2. Specificity study of GPC3 protein degradation induced by GPC3-LYTAC bispecific nucleic acid aptamer

[0082] This embodiment further investigates the specificity of GPC3 protein degradation induced by the GPC3-LYTAC bispecific nucleic acid aptamer prepared in Example 1.

[0083] I. Experimental Methods

[0084] (1) Cell Culture

[0085] Two liver cancer cell lines, Hep3B and HepG2, were cultured in DMEM containing 10% FBS and digested with trypsin during passage.

[0086] (2) Cell modeling

[0087] Hep3B and HepG2 cells were cultured in 6-well plates (3×10^5 cells / well) until the cell density reached about 70%, and then cultured for 72 h in medium containing TfR1ap, GPC3ap, and GPC3-LYTAC, with a final aptamer concentration of 200 nM for each cell.

[0088] (3) Cell protein extraction

[0089] After cell modeling is completed, remove the cells from the incubator and observe cell growth under a microscope. Discard the culture medium, place the cells on ice, and wash them three times with 1×PBS. After removing the PBS, add cell lysis buffer containing PMSF (RIPA:PMSF 100:1) and lyse thoroughly on ice. Scrape the cells and transfer them to 1.5ml centrifuge tubes. Centrifuge at 13500 rpm for 15 minutes at 4°C, then collect the supernatant and mix it with 5×loading buffer at a 4:1 ratio. Boil at 100°C for 8 minutes, cool on ice, and store at -20°C for later use.

[0090] (4) Western blot (WB)

[0091] Total protein was separated on a 10% SDS-PAGE gel (80V for 30 min, 120V for 40 min) and transferred to a 0.45 μm NC membrane. The membrane was blocked with 5% skim milk for 1 h and then incubated overnight with anti-GPC3 antibody (Proteintech; 1:5000 dilution). After incubation, HRP-conjugated secondary antibody (Proteintech; 1:10000 dilution) was added and incubated at room temperature for 1 h. After washing with TBST for 40 min, the membrane was developed using ultrasensitive ECL chemiluminescence buffer. After development, antibodies were removed using primary and secondary antibody removal buffer. The membrane was then incubated overnight with anti-GAPDH antibody (1:6000 dilution) followed by secondary antibody incubation and development.

[0092] (5) Cell immunofluorescence

[0093] Climbing slides were placed in 35 mm cell culture dishes, and Hep3B and HepG2 cell suspensions were seeded at appropriate densities. After cell adhesion, the culture medium was replaced with medium containing TfR1ap, GPC3ap, and GPC3-LYTAC, with a final aptamer concentration of 200 nM for 72 h. After treatment, cells were fixed with 4% paraformaldehyde at room temperature and blocked with 5% BSA at 4 °C for 4 h. Cells were then incubated overnight at 4 °C with anti-GPC3 antibody (Proteintech; 1:800 dilution). After washing three times in PBS buffer, fluorescent dye antibody was incubated with the sample at room temperature in the dark for 1 h. After washing three times, slides were mounted with mounting medium containing DAPI, and cells were photographed under a fluorescence microscope at a scale bar of 25 μm.

[0094] II. Experimental Results

[0095] See the results of Western blot analysis for protein immunoblotting. Figure 3 and Figure 4 .like Figure 3 As shown, compared with the control group, approximately 55% of the GPC3 protein in Hep3B cells was degraded after treatment with a final concentration of 200 nM GPC3-LYTAC for 72 h, while no protein degradation was observed in either the TfR1ap or GPC3ap treatment groups alone. Figure 4 As shown, compared with the control group, approximately 62% of GPC3 protein in HepG2 cells was degraded after treatment with a final concentration of 200 nM GPC3-LYTAC for 72 h. No protein degradation was observed in either the TfR1ap or GPC3ap treatment groups alone.

[0096] Cell immunofluorescence results as follows Figure 5 , Figure 6As shown, the content of GPC3 protein on the cell membrane is not affected by TfR1ap or GPC3ap alone, while GPC3-LYTAC significantly reduced the content of GPC3 protein on the surface of Hep3B and HepG2 cells.

[0097] The above results fully demonstrate that the bispecific nucleic acid aptamer GPC3-LYTAC prepared in this invention has the specificity to induce GPC3 protein degradation.

[0098] Example 3. Concentration- and time-dependent study of GPC3 protein degradation induced by GPC3-LYTAC bispecific nucleic acid aptamer.

[0099] This embodiment further investigates the concentration- and time-dependent degradation of GPC3 protein induced by the bispecific nucleic acid aptamer prepared in Example 1.

[0100] I. Experimental Methods

[0101] (1) Hep3B cells were cultured and modeled according to steps (1) and (2) in Example 2. The difference was that when the cell density reached about 70%, this step used a medium containing GPC3-LYTAC bispecific nucleic acid aptamer for 72 h, and the final concentrations of GPC3-LYTAC were 0, 25 nM, 50 nM, 100 nM, 200 nM and 400 nM.

[0102] (2) Hep3B cells were cultured and modeled according to steps (1) and (2) in Example 2. The difference was that when the cell density reached about 70%, this step used a culture medium containing a final concentration of 200 nM GPC3-LYTAC bispecific nucleic acid aptamer. The GPC3-LYTAC treatment time was grouped as 0, 12h, 24h, 48h, and 72h.

[0103] (3) After processing, extract total protein according to steps (3) and (4) in Example 2 for protein immunoblotting experiment.

[0104] II. Experimental Results

[0105] See the results of the Western blot for protein immunoblotting. Figure 7 and Figure 8 .like Figure 7 As shown, the concentration-dependent hydrolysis of GPC3 protein by the GPC3-LYTAC bispecific nucleic acid aptamer begins at a GPC3-LYTAC concentration of 50 nM, and the degree of GPC3 protein degradation increases with increasing GPC3-LYTAC concentration. Figure 8As shown, the GPC3-LYTAC bispecific nucleic acid aptamer degrades GPC3 protein in a time-dependent manner; after 12 hours of treatment, the GPC3 protein content decreased by 41%. These results clearly demonstrate that the prepared bispecific nucleic acid aptamer GPC3-LYTAC induces GPC3 protein degradation in a concentration- and time-dependent manner.

[0106] Example 4. Study on GPC3-LYTAC-induced GPC3 protein degradation via endocytosis-lysosomal pathway

[0107] This embodiment further explores the mechanism by which bispecific nucleic acid aptamers induce GPC3 protein degradation, verifying whether it induces GPC3 protein degradation through the endocytosis-lysosomal pathway. Specific methods and results are as follows:

[0108] I. Experimental Methods

[0109] (1) Hep3B cells were cultured and modeled according to steps (1) and (2) in Example 2. The difference was that when the cell density reached about 70%, this step was carried out using culture media containing GPC3-LYTAC (200 nM), GPC3-LYTAC (200 nM) combined with Chloroquine (20 μM), and GPC3-LYTAC (200 nM) combined with MG132 (500 nM) for 72 h.

[0110] (2) After processing, extract total protein according to steps (3) and (4) in Example 2 for protein immunoblotting experiment.

[0111] II. Experimental Results

[0112] Western blot results for proteins are as follows Figure 9 As shown, when the lysosomal inhibitor Chloroquine was combined with GPC3-LYTAC, the GPC3-LYTAC-induced GPC3 degradation was alleviated, while the proteasome inhibitor MG132 combined with GPC3-LYTAC did not show this effect. This indicates that the bispecific nucleic acid aptamer GPC3-LYTAC induces GPC3 protein degradation through the endocytic lysosomal pathway. A detailed schematic diagram is shown below. Figure 1 As shown, the GPC3-LYTAC bispecific nucleic acid aptamer can simultaneously bind to TfR1 and GPC3 proteins on the cell membrane, effectively utilizing the endocytosis of TfR1 protein to bring GPC3 protein from the cell membrane into the cell and induce GPC3 protein degradation through the lysosomal pathway.

[0113] Example 5. Study on the inhibitory effect of GPC3-LYTAC on liver cancer cells by inducing GPC3 degradation.

[0114] (1) Cell proliferation experiment

[0115] To investigate whether the bispecific nucleic acid aptamer prepared in Example 1 induced GPC3 protein degradation and inhibited the proliferation of liver cancer cells, a CCK8 assay was performed to measure cell proliferation. The specific procedures are as follows:

[0116] 1) Culture Hep3B cells according to step (1) in Example 2.

[0117] 2) Cell modeling

[0118] After cell counting, cells were seeded into 96-well plates at a density of 5000 cells / well. When the cell density reached 60%, medium containing 200 nM GPC3-LYTAC was added and the cells were cultured for 72 hours.

[0119] 3) After the treatment is completed, take out the 96-well plate, add 10 μL of CCK8 reagent in the dark and shake gently. Incubate at 37°C for 3 hours and measure the absorbance at 450 nm using an ELISA reader.

[0120] The absorbance statistics are as follows Figure 10 As shown, GPC3-LYTAC-induced GPC3 protein degradation can inhibit the proliferation of Hep3B cells, with an inhibition rate of over 50%.

[0121] (2) Cell scratch test

[0122] To investigate whether the bispecific nucleic acid aptamer prepared in Example 1 could inhibit the degradation of GPC3 protein by induced cell carcinoma cell migration, a cell scratch assay was performed in this example. The specific procedures are as follows:

[0123] 1) Culture Hep3B cells and establish the model according to steps (1) and (2) in Example 2. The difference is that, when the cell density reaches 90%, this step uses the same 200 μL pipette tip to gently and slowly scrape the cells vertically from bottom to top, forming a cross. Gently rinse with PBS to remove detached cells. Add culture medium containing a final concentration of 200 nM GPC3-LYTAC for further culture.

[0124] 2) At 0h, 12h, 24h, 36h and 48h after GPC3-LYTAC treatment, the cells were photographed under a microscope and the images were saved. The relative cell migration rate between 0 and 48h was analyzed using ImageJ software.

[0125] The migration area statistics are as follows Figure 11 As shown, compared with the control group, Hep3B cell migration was inhibited after 24 hours of GPC3-LYTAC treatment. The migration ability decreased with the extension of time. After 36 hours of GPC3-LYTAC treatment, the inhibition rate reached about 50%.

[0126] The above results indicate that the GPC3-LYTAC prepared in this invention can inhibit the proliferation and migration of liver cancer cells by inducing GPC3 degradation, thereby exerting an anti-tumor effect.

Claims

1. A LYTAC molecule that targets and degrades GPC3, characterized in that, The LYTAC molecule consists of a TfR1-targeting aptamer and a GPC3-targeting aptamer.

2. The LYTAC molecule according to claim 1, characterized in that, The nucleotide sequence of the TfR1 aptamer is shown in SEQ ID NO:1; the nucleotide sequence of the GPC3 aptamer is shown in SEQ ID NO:

2.

3. The method for preparing the LYTAC molecule according to claim 1, characterized in that, Includes the following steps: 1) Target the 5' end of the GPC3 aptamer with phosphorylation; 2) Mix the 5' phosphorylated GPC3 aptamer obtained in step 1) with the TfR1 aptamer and perform PCR annealing; the reaction product forms a bispecific aptamer under the action of T4 DNA ligase.

4. The preparation method according to claim 3, characterized in that, In step 1), 5' end phosphorylation is performed by PCR; the PCR reaction system includes: 80-120uM GPC3 aptamer 8-12ul, T4 polynucleotide kinase 0.5-2ul, Reaction Buffer A (10X) 1-4ul, 8-12mM ATP 0.5-2ul, and DEPC water to make up to 20ul. The PCR reaction conditions are: incubation at 30-45℃ for 2-5 hours.

5. The preparation method according to claim 3, characterized in that, In step 2): the concentration of TfR1 aptamer is 40-60 μM, and the amount used is 15-25 μL; The annealing procedure is set as follows: (1) Denaturation: 90-100℃ for 4-10 minutes; (2) Annealing: Cool to 20-30℃ at a rate of 0.05-0.2℃ / 8s and maintain for 80-100 minutes; (3) Store at 2-8℃.

6. A method for inducing the degradation of GPC3 protein in liver cancer cells, characterized in that, The method is a non-disease diagnosis or treatment method; the method includes: treating liver cancer cells with LYTAC molecules according to any one of claims 1 to 2 and / or LYTAC molecules prepared by the method according to any one of claims 3 to 5.

7. The method according to claim 6, characterized in that, The concentration of the LYTAC molecules is ≥50 nM; the treatment time is ≥12 h.

8. The use of the LYTAC molecule according to any one of claims 1 to 2 in the preparation of an anti-liver cancer drug.

9. The use of the LYTAC molecule according to any one of claims 1 to 2 in the preparation of a GPC3-targeted degradation inducer, a liver cancer cell proliferation inhibitor, and / or a liver cancer cell migration inhibitor for the treatment of liver cancer.

10. Application of TfR1-targeting nucleic acid aptamers combined with GPC3-targeting nucleic acid aptamers in the preparation of drugs for the treatment of liver cancer.

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