An RNA-based tumor vaccine, preparation method, immune adjuvant and application

Abstract

The present invention provides an RNA-based tumor vaccine, a preparation method, an immune adjuvant, a vector and an application. The tumor vaccine can specifically act on tumors with positive expression of GPC3, such as hepatocellular carcinoma, ovarian clear cell carcinoma, yolk sac carcinoma, etc. The tumor vaccine includes circular RNA capable of encoding an immunogenic polypeptide. The circular RNA is configured to be capable of specifically encoding the immunogenic polypeptide GPC3. The circ-GPC3 in the present invention has a covalently closed-loop structure that can protect it from exonuclease-mediated degradation, has a longer half-life and higher stability compared to mRNA, and can ensure the continuous expression of immunogenic proteins. By artificially modifying and introducing a ribosome entry site (IRES) modification, the present invention can increase the translation efficiency of tumor-specific antigen RNA.

Description

Technical Field

[0001] The present invention relates to the technical field of antigen RNA in tumor treatment and drug preparation, and particularly relates to an RNA-based tumor vaccine, a preparation method, an immune adjuvant and an application thereof. Background Art

[0002] Tumor vaccines are an anti-tumor immunotherapy means with great application prospects. Among them, RNA vaccines are a new trend in the development of future tumor vaccines. It uses lipid nanoparticles (LNPs) to introduce mRNA into the body for rapid expression of antigen proteins, so as to stimulate the body to produce specific immune responses, achieve long-term regression of tumors and prevent metastasis, and avoid the risks of integration with the genome or T cell tolerance. So far, researchers have developed a variety of mRNA tumor vaccines, and some have entered the clinical trial stage in non-small cell lung cancer, melanoma and gastrointestinal tumors, and the safety and effectiveness are gradually evaluated. However, due to the limitations of the easy degradation characteristics, large molecular weight, charge properties, limited targeting ability, etc. of mRNA, the clinical benefits of mRNA vaccines in tumor treatment are limited. Circular RNA (circRNA) has a special circular structure, making it have high stability and RNase resistance, which makes up for the deficiencies of linear mRNA vaccines, and thus has a broader application prospect. Previous research reports have shown that circRNA vaccines against the new coronavirus have played a better immune protection role than mRNA, but currently no cancer vaccine based on circRNA has been successfully developed. Therefore, further research and development of novel circRNA tumor vaccines and their effects on treating hepatocellular carcinoma have important clinical significance and social benefits for improving the immunotherapy effect of liver cancer.

[0003] Toll-like receptor agonists are a class of drugs with great potential that can efficiently activate antigen-presenting cells to produce specific immune responses. However, the clinical treatment effects in the existing technologies are not very satisfactory. For example, small molecule drugs represented by imiquimod and resiquimod have been successively marketed, but the clinical efficacy is still not optimistic. PF-3512676 is a TLR9B agonist developed by Pfizer more than 10 years ago, but its development has been terminated due to the negative results of phase III trials. Another TLR7 agonist (GS-9620) showed no adverse reactions in the phase 1 / 2 study of the hepatitis B virus infection clinical trial, but lacked significant antiviral activity. For the newer generation of TLR8 agonists, although they show strong affinity for pathogen-associated molecular proteins, because they can induce abundant IL-12 and IL-18 production in liver monocytes and DC cells, a strong IFN-γ response will be generated in the liver, inducing a cytokine storm and resulting in severe adverse reactions in the body. The above research results all indicate that it is difficult to arouse effective immune responses by using TLR agonists alone, and the treatment doses generally have strong toxic and side effects, and serious systemic inflammatory side effects and even fatal drug side effects will occur after medication, restricting their wide application in clinical practice.

[0004] In addition, the low immunogenicity of hepatocellular carcinoma and the obstruction of the antigen presentation process are important factors leading to immune therapy resistance. Due to the existence of antigenic modulation, some tumor cells with stronger immunogenicity are cleared after inducing the body's anti-tumor immune response, and the tumor cells with weaker immunogenicity continue to proliferate after escaping the body's immune surveillance, ultimately leading to the weakening of tumor cell immunogenicity. In the liver cancer microenvironment, due to the infiltration of inhibitory immune cells, the activities of antigen-presenting cells such as DC and natural killer cells (NK) are weakened, and new antigens cannot be effectively presented, making it difficult to induce an immune response of sufficient intensity.

[0005] Through the above analysis, the problems and defects existing in the existing technologies are as follows: on the one hand, the existing clinical trial data suggest that the effect of TLR agonists in immunotherapy for tumor patients is limited, and the clinical benefit for advanced patients is poor. Moreover, using TLR agonists alone can activate the immune system in a non-specific manner, leading to dose-limiting toxicities and potential adverse reactions such as fever, flu-like symptoms, and inflammation, making it difficult to predict and control the immune response. On the other hand, the low immunogenicity of hepatocellular carcinoma and the obstruction of the antigen presentation process are important factors leading to immune therapy resistance.

[0006] Therefore, there is a need to provide a tumor vaccine that can both promote the antigen presentation efficiency of antigen cells and effectively control the toxic and side effects of agonists. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an RNA-based tumor vaccine, a preparation method, an immune adjuvant, a carrier and an application thereof, aiming at the problems existing in the background technology.

[0008] To solve the above technical problems, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides an RNA-based tumor vaccine, which can specifically act on tumors with positive expression of GPC3, such as hepatocellular carcinoma, ovarian clear cell carcinoma, yolk sac carcinoma, etc. The tumor vaccine includes circular RNA capable of encoding an immunogenic polypeptide.

[0010] Preferably, the circular RNA is configured to specifically encode the immunogenic polypeptide GPC3.

[0011] Preferably, the tumor vaccine introduces a ribosome entry site IRES modification to the circular RNA encoding the immunogenic polypeptide GPC3 (i.e., circ-GPC3), as Figure 2 shown. By artificially introducing a ribosome entry site (IRES) modification, the translation efficiency of tumor-specific antigen RNA can be increased.

[0012] The circular RNA capable of encoding an immunogenic polypeptide is preferably circ-GPC3. In the present invention, GPC3 encoded by the circular RNA, that is, circ-GPC3 has a covalently closed-loop structure, as Figure 2 shown, which can protect it from exonuclease-mediated degradation. Compared with mRNA, it has a longer half-life and higher stability, and can ensure the continuous expression of immunogenic proteins. In addition, the circRNA in the present invention can enhance the immunogenicity of tumor cells by interacting with immune cells and cytokines in the tumor microenvironment, and improve the reactivity to immunotherapy.

[0013] The gene sequence of the circ-GPC3 is shown as SEQ NO:1 in the sequence listing.

[0014] SEQ NO:1

[0015]

[0016] The amino acid sequence of the polypeptide encoded by the circ-GPC3 described above is as shown in SEQ NO:2 in the sequence listing.

[0017] SEQ NO:2

[0018] MAGTVRTACLLVAMLLGLGCLGQAQPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLTPQAFEFVGEFFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQMMNPGLPESVLDINECLRGARRDLKVFGSFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTRMWYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDKYWREYILSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNGGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDKKILKVAHVEHEETLSSRRRELIQKLKSFINFYSALPGYICSHSPVAENDTLCWNGQELVERYSQKAARNGMKNQFNLHELKMKGPEPVVSQIIDKLKHINQLLRTMSVPKGKVLDKSLDEEGLESGDCGDDEDECIGSSGDGMVKVKNQLRFLAELAYDLDVDDAPGNKQHGNQKDNEITTSHSVGNMPSPLKILISVAIYVACFFFLVH

[0019] Preferably, the tumor vaccine of the present invention further comprises a combined immune adjuvant, and the combined immune adjuvant is a Toll-like receptor agonist, and more preferably a Toll-like receptor agonist 4 (TLR agonist-4).

[0020] In a second aspect of the present invention, the present invention provides an application of a Toll-like receptor agonist in the preparation of an RNA-based tumor vaccine.

[0021] In a third aspect of the present invention, the present invention provides a preparation method of an RNA-based tumor vaccine, and the preparation method is configured to be used for preparing the tumor vaccine described in the first aspect of the present invention, and comprises the following specific steps:

[0022] (1) Dissolve SM102, DMG-PEG2000, distearoyl phosphatidylcholine, and high-purity cholesterol CHO-HP in absolute ethanol, and prepare an organic phase containing lipid molecules according to a certain molar percentage as shown in Table 1;

[0023] Table 1

[0024] Component Mole percentage SM102 30-50％ DMG-PEG2000 1.5-2.5％ DSPC 10-20％ Cholesterol 38.5-47.5％

[0025] (2) Take the purified mRNA / circRNA and dilute it with a citric acid-sodium citrate buffer solution with pH = 4.5 - 5.5 to obtain an aqueous phase containing mRNA / circRNA nucleic acid molecules;

[0026] (3) Using microfluidic technology, fully mix the organic phase prepared in step (1) and the aqueous phase prepared in step (2);

[0027] (4) Remove the organic solvent by ultrafiltration method, and place it on a shaker at 2 - 10 °C overnight to obtain lipid nanoparticles loaded with mRNA / circRNA;

[0028] (5) Prepare a combined immune adjuvant according to a ratio. In the fourth aspect of the present invention, on the basis of the above technical solution, the present invention provides an application of an antigen RNA tumor vaccine, immune adjuvant and / or carrier in the immunotherapy of hepatocellular carcinoma.

[0029] In the fifth aspect of the present invention, on the basis of the above technical solution, the present invention provides an application of an antigen RNA tumor vaccine, immune adjuvant and / or carrier in the preparation of immunotherapeutic drugs for hepatocellular carcinoma.

[0030] The beneficial effects of the present invention are:

[0031] 1. The circular RNA in the present invention encodes a polypeptide with tumor immunotherapeutic activity. The coding sequence obtained by increasing the preferred modified nucleotide (IRES sequence) through RNA modification means further improves the translation protein efficiency of GPC3 while retaining its antigenic epitope. The present invention discovers for the first time that a preparation containing circular RNA molecules can significantly inhibit tumor growth in tumor patients and exert a significant tumor treatment effect in a tumor mouse model, providing a novel therapeutic drug with great application prospects for tumor immunotherapy.

[0032] 2. By optimizing the design of the GPC3-encoding circRNA sequence and constructing a novel RNA vaccine combined with a TLR4 agonist using an LNP drug delivery system, the present invention is expected to reshape the tumor-suppressive immune microenvironment, improve the efficiency of tumor antigen presentation, enhance the anti-tumor immune response, and provide a new strategy for the immunotherapy of hepatocellular carcinoma. In the present invention, the treatment regimen of the tumor vaccine combined with an adjuvant can accelerate the presentation of liver cancer-related tumor antigens, upregulate the expression of co-stimulatory molecules on the surface of dendritic cells, and promote the secretion of cytokines such as TNF-α and IFN-γ, driving the remodeling of the tumor microenvironment, thereby improving immunotherapy resistance. Moreover, its preparation method is simple, easy to repeat, and suitable for large-scale production, which can enhance the immunotherapy effect of liver cancer and has good application prospects.

[0033] 3. In the present invention, a TLR4 agonist is selected as the immune adjuvant for the tumor vaccine. Compared with other types of TLR agonists, it also has the following advantages:

[0034] (1) Strong immune activation. The TLR4 agonist can activate innate and adaptive immune responses, leading to the activation of antigen-presenting cells such as dendritic cells, and then stimulating T cell responses.

[0035] (2) Inducing Th1 response. The TLR4 agonist can bias the immune response towards Th1 response, which is crucial for controlling intracellular infections and anti-tumor immunity.

[0036] (3) Low toxicity. The TLR4 agonist has good safety, and the relevant results of in vitro and in vivo experiments all suggest that the use of this drug has good safety and few adverse reactions.

[0037] (4) Wide applicability. Other types of TLR agonists, such as TLR3 and TLR9 agonists, have significant limitations as immune adjuvants. For example, TLR3 agonists are mainly expressed in endosomes, so their ability to activate immune cells outside these compartments is limited. TLR9 agonists are mainly expressed in B cells and plasmacytoid dendritic cells, which limits their ability to activate other immune cells such as T cells. However, the TLR4 selected in the present invention is expressed in a variety of immune cells, including dendritic cells, macrophages, and B cells. The wide expression of TLR4 enables the TLR4 agonist to activate multiple immune pathways, thereby generating strong and diverse immune responses, showing promise in improving efficacy and overcoming immune tolerance to cancer.

[0038] (5) Adjuvant synergy. The TLR4 agonist can act synergistically with tumor antigens to further enhance immune activation and improve the vaccine effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is the map of the in vitro transcription template plasmid mRNA-GPC3 in the present invention.

[0040] Figure 2 It is the map of the in vitro transcription template plasmid circRNA-GPC3 in the present invention.

[0041] Figure 3 It is the result diagram of detecting the polypeptides encoded by mRNA-GPC3 and circRNA-GPC3 in two hepatoma cell lines respectively in Example 2.

[0042] Figure 4 It is the comparison diagram of fluorescence experiments of mRNA-GPC3-EGFP and circGPC3-EGFP in Example 3.

[0043] Figure 5 It is the comparison diagram of the expression of linear and circular Luciferase RNA in mice in Example 4.

[0044] Figure 6 It is the tumor growth curve of mice in the liver cancer model treated with the circRNA vaccine encoding GPC3 combined with various TLR agonists in Example 6.

[0045] Figure 7 It is the quantitative analysis diagram of the tumor mass of mice in the liver cancer model treated with the circRNA vaccine encoding GPC3 combined with various TLR agonists in Example 6.

[0046] Figure 8 It is the tumor growth curve diagram of mice in the liver cancer model treated with the circRNA vaccine encoding GPC3 combined with TLR4 agonist in Example 6.

[0047] Figure 9 It is the quantitative analysis diagram of the tumor mass of mice in the liver cancer model treated with the circRNA vaccine encoding GPC3 combined with TLR4 agonist in Example 6.

[0048] Figure 10 It is the scatter diagram of flow cytometry experiments to verify the activation of anti-tumor immune response (CD8+ T cells) by the treatment with the circRNA vaccine combined with TLR4 agonist in Example 6.

[0049] Figure 11 It is the quantitative result diagram of verifying the activation of anti-tumor immune response (CD8+ T cells) by the treatment with the circRNA vaccine combined with TLR4 agonist in Example 6.

[0050] Figure 12 It is the quantitative result diagram of the serum ALT of mice in evaluating the biological safety of the treatment regimen in Example 7.

[0051] Figure 13 It is the quantitative result diagram of the serum AST of mice in evaluating the biological safety of the treatment regimen in Example 7.

[0052] Figure 14 It is the HE pathological section diagram of the main organs of the mouse in the evaluation of the biosafety of the treatment plan in Example 7. Specific implementation manners

[0053] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement made to the methods, steps or conditions of the present invention shall fall within the scope of the present invention.

[0054] Unless otherwise specified, the test methods used in the following embodiments are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0055] In the embodiments of the present invention, the prevention and treatment of hepatocellular carcinoma are taken as specific application examples, but the implementation manners of the present invention are not limited thereto.

[0056] Example 1: Preparation of lipid nanoparticles loaded with RNA encoding antigen

[0057] This example provides a method for preparing lipid nanoparticles containing RNA nucleic acid molecules in the tumor vaccine of the present invention.

[0058] First, dissolve SM102, DMG-PEG2000, distearoyl phosphatidylcholine (DSPC), and high-purity cholesterol CHO-HP in absolute ethanol, and prepare a lipid ethanol solution for standby according to the molar percentages shown in Table 2. Then, take the purified mRNA / circRNA and dilute it with citric acid-sodium citrate buffer (pH = 4.5) for standby. Further, using microfluidic technology (TYD01-01(V3) type, Leadfluid), fully mix the organic phase containing lipid molecules (SM102, DMG-PEG2000, DSPC, cholesterol) and the aqueous phase containing nucleic acid molecules (diluted with citric acid-sodium citrate buffer (pH = 4.5)), and the mixing ratio is 1:1 - 1:10 (the preferred ratio is 1:3). Further, use the ultrafiltration method to remove organic solvents, and place it on a shaker at 4°C overnight to obtain lipid nanoparticles loaded with mRNA / circRNA.

[0059] Table 2

[0060] Component Mole percentage SM102 50％ DMG-PEG2000 1.5％ DSPC 10％ Cholesterol 38.5％

[0061] Example 2: Verification of the encoding of polypeptide GPC3 by mRNA / circRNA in liver cancer cells.

[0062] One night in advance, 1×10 5 liver cancer cells HCCLM3 and MHCC97H were seeded in a 6-well plate. The lipid nanoparticles loaded with mRNA / circRNA prepared by the method described in Example 1 were transfected into the cells. After 48 hours of cell transfection, the total protein in the cells was extracted. The protein encoded by mRNA / circRNA was verified by Western Blot experiment. The results showed that circGPC3 had the function of encoding polypeptide, and the expression level of the encoded polypeptide in liver cancer cells was significantly higher than that encoded by mRNA-GPC3, as Figure 3 shown.

[0063] Example 3: Verification of the expression of mRNA / circRNA in liver cancer cells.

[0064] One night in advance, 1×10 5 liver cancer cells HuH-7 were seeded in a 6-well plate. The mRNA-GPC3-EGFP@LNP or circGPC3-EGFP@LNP encoding green fluorescent protein EGFP prepared by the above method was transfected into the cells. After 24 hours of cell transfection, after washing 3 times with PBS, Hoechst was added to stain the nuclei for 10 minutes. The green fluorescence was observed using a laser confocal microscopy system. The results showed that green fluorescence could be successfully expressed in tumor cells using mRNA@LNP or circRNA@LNP, and the expression effect of circRNA was better than that of mRNA. It should be noted that after 96 hours, fluorescence could still be observed in the circRNA group, while the fluorescence in the mRNA group was very weak or even disappeared at this time. The verification of this example showed that circRNA was more stable and had a longer expression time compared to mRNA, indicating that the method prepared in Example 1 could be used to express specific antigen proteins in tumor cells and was preferably circRNA, as Figure 4 shown.

[0065] Example 4: Expression of linear mRNA and circular circRNA in tumors.

[0066] For hepa1-6 tumor model mice, nucleotide-modified linear mRNA and circular RNA encoding firefly luciferase were respectively injected into the tail vein at a dose of 10 μg. After 6 hours, potassium D-luciferin aqueous solution (15 mg / ml) was injected into the abdominal cavity of the mice at a concentration of 10 μl / g body weight. After 10 - 20 minutes of injection into the body, when the light signal reached the strongest stable plateau, in vivo fluorescence imaging was performed. The expression of linear and circular Luciferase RNA in vivo was analyzed by fluorescence intensity asFigure 5 as shown Figure 5 The left figure of Figure 5 shows the in vivo fluorescence imaging of mice injected with linear mRNA via the tail vein; Figure 5 The right figure of Figure 5 is the in vivo fluorescence imaging of mice injected with circular RNA via the tail vein. As Figure 5 can be seen, at the same dose, compared with linear mRNA, circular RNA is more stable in tumors and has more significant expression.

[0067] Example 6: Experiment on the administration of the polypeptide encoded by circ-GPC3 combined with TLR4 agonist.

[0068] Healthy male C57BL / 6 mice at 6-8 weeks old were used to construct a mouse liver cancer model. Hepa1-6 mouse liver cancer cells were diluted with PBS to 2×10 7 cells / mL, and 100 μl of the cell suspension was slowly injected into the right posterior axilla of the mice to establish a subcutaneous tumor model of mouse liver cancer. When the tumors of the mice grew to 50-100 mm 3 , the tumor-bearing mice were randomly divided into 7 groups and treated as follows: ① blank control group; ② CircGPC3@LNP + TLR3 agonist group; ③ CircGPC3@LNP + TLR4 agonist group; ④ CircGPC3@LNP + TLR5 agonist group; ⑤ CircGPC3@LNP + TLR7 agonist group; ⑥ CircGPC3@LNP + TLR8 agonist group; ⑦ CircGPC3@LNP + TLR9 agonist group. The TLR agonist was administered by intraperitoneal injection, and the CircGPC3@LNP vaccine was inoculated intratumorally at a dose of 10 μg circRNA / mouse. The tumor growth of the mice was observed and recorded every 2 days ( Figure 6 ). Calculation formula: Tumor volume = (length × width × width) / 2. 15 days after immunization, the mice were sacrificed by cervical dislocation, and the tumor tissues of the mice in each group were collected and weighed ( Figure 7 ). As Figure 5-6 can be seen, the treatment with CircGPC3@LNP combined with TLR agonist can effectively inhibit the tumor growth of mice. Among them, compared with other types of TLR agonists, after treatment with the TLR4 agonist, the tumor growth rate of the tumor-bearing mice was the slowest and the tumor burden was significantly reduced. Therefore, the combined treatment is preferably the TLR4 agonist.

[0069] Furthermore, another subcutaneous tumor model of mouse liver cancer was constructed by the same method and the experimental groups were set: ① blank control group; ② TLR4 agonist group; ③ CircGPC3@LNP group; ④ CircGPC3@LNP + TLR4 agonist group. The TLR4 agonist was administered by intraperitoneal injection, and the CircGPC3@LNP vaccine was inoculated intratumorally. The tumor growth of the mice was observed and recorded every 2 days (attached Figure 8)。Fifteen days after immunization, the mice were sacrificed by cervical dislocation, and the tumor tissues of the mice in each group were collected and weighed (Appendix Figure 9 )。After weighing, the tumor tissues were made into sections or single-cell suspensions and the infiltration of T cells was observed (Appendix Figure 10-11 )。

[0070] Example 7: Safety assessment.

[0071] Fresh tissues were taken from the main organs of the mice in the control group and each treatment group after treatment. After being fixed with paraformaldehyde for 24 - 72 h, they were dehydrated and cleared with 75% ethanol. Then the cleared tissue blocks were embedded in melted paraffin to form wax blocks. Further, the wax blocks were fixed on a microtome and cut into thin slices 5 - 8 microns thick. Then the slices were flattened in warm water and pasted onto glass slides. The glass slides were placed in an oven for baking to make the tissue sections adhere to the slides. After further dewaxing and washing with water, they were stained using the Hematoxylin-Eosin staining method. The stained sections were dehydrated with absolute alcohol and then cleared with xylene. Neutral gum was dropped onto the cleared sections and a coverslip was placed on top for sealing, thus obtaining the HE pathological sections of the main organs of the mice for result interpretation. In addition, blood was taken from the orbits of the mice, and the serum was obtained by centrifugation and stored frozen for routine biochemical assessment. The pathological conditions of the main organs were analyzed by the H&E staining method and the biological safety of the protocol in Example 6 was evaluated by serum ALT / AST. The results showed that this method was safe and feasible. (As Figure 12-14 shown)

[0072] The present invention has been described exemplarily above in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited thereto. As long as various improvements are made by adopting the method concept and technical solution of the present invention, or the concept and technical solution of the present invention are directly applied to other occasions without improvement, they are all within the protection scope of the present invention.

Claims

1. An RNA-based hepatocellular carcinoma-specific tumor vaccine, characterized in that, The tumor vaccine includes circular RNA capable of encoding the immunogenic polypeptide GPC3; the circular RNA is configured to specifically encode the immunogenic polypeptide GPC3; the tumor vaccine introduces a ribosome entry site IRES modification to the RNA sequence encoding the immunogenic polypeptide GPC3; the gene sequence capable of encoding GPC3 after modification is as shown in SEQ ID NO.1 in the sequence listing, and also includes a combined immune adjuvant, and the combined immune adjuvant is a TLR4 agonist.

2. The RNA-based hepatocellular carcinoma-specific tumor vaccine according to claim 1, characterized in that, The content ratio of the circular RNA to the immune adjuvant is 0.75-1.25:0.75-1.

25.

3. A preparation method of an RNA-based hepatocellular carcinoma-specific tumor vaccine, characterized in that, The preparation method is configured to prepare the tumor vaccine according to claim 1 or 2, and the preparation method includes the following specific steps: (1) After dissolving SM102, DMG-PEG2000, distearoyl phosphatidylcholine, and high-purity cholesterol CHO-HP in absolute ethanol, an organic phase containing lipid molecules is configured according to a certain molar percentage; (2) Take the purified circRNA and dilute it with a citric acid-sodium citrate buffer solution to obtain an aqueous phase containing circRNA nucleic acid molecules; (3) Using microfluidic technology, fully mix the organic phase prepared in step (1) and the aqueous phase prepared in step (2); (4) Remove the organic solvent by ultrafiltration method, and place it on a shaker at 2-10°C overnight to obtain lipid nanoparticles loaded with circRNA; (5) Prepare the combined immune adjuvant according to the ratio.

4. Use of an RNA-based hepatocellular carcinoma-specific tumor vaccine according to claim 1 in the preparation of a hepatocellular carcinoma-specific tumor immunotherapy drug.

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