Self-replicating mRNA sequence and use thereof in preparation of Anti-HPV tumor drug

Abstract

Disclosed in the present invention are a self-replicating messenger ribonucleic acid (mRNA) sequence and a use thereof in preparation of an anti-human papillomavirus (HPV) tumor drug. A self-replicating mRNA base comprises a sequence as shown in at least one of SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 15. In the present invention, mutation and tandem design is carried out on the basis of E6 and E7 proteins, thereby improving the immunogenicity of E6 and E7 proteins expressed by an mRNA, and overcoming the pain point problem in the traditional technology of E6 and E7 proteins for vaccines being difficult to be effectively recognized by a human immune system.

Description

Self-replicating mRNA sequence and use thereof in the preparation of anti-HPV tumor drugs The present application claims priority to the Chinese patent application No. 202410860822.0, filed on June 28, 2024, and entitled "Self-replicating mRNA sequence and use thereof in the preparation of anti-HPV tumor drugs", the entire content of which is incorporated herein by reference. TECHNICAL FIELD The present application relates to the field of medical molecular biology, in particular to a self-replicating mRNA sequence and use of a preparation composition thereof in anti-HPV tumor drugs. BACKGROUND HPV (Human Papilloma Virus) related tumors refer to tumors caused by infection of certain high-risk types of HPV. HPV is a common sexually transmitted virus and is associated with the occurrence of various cancers, especially cervical cancer, vaginal cancer, vulvar cancer, anal cancer, oral cancer and throat cancer. For the treatment of HPV-related tumors, there are several drugs and treatments available. First, HPV vaccines: Preventing HPV infection is one of the most effective methods. HPV vaccination can reduce the risk of infection with high-risk HPV, thereby reducing the incidence of related tumors such as cervical cancer, oral cancer and throat cancer. Second, Immunotherapy Checkpoint Inhibitors: These drugs can help activate the immune system to more effectively attack and eliminate tumor cells. For example, PD-1 inhibitors (such as Pembrolizumab and Nivolumab) and PD-L1 inhibitors (such as Atezolizumab and Durvalumab) have been used to treat HPV-related oral cancer and throat cancer. Chemotherapy drugs are also an option: Chemotherapy can also be used to treat HPV-related tumors in some cases. Chemotherapy drugs such as Cisplatin and 5-Fluorouracil can be used to assist surgery, before and after radiotherapy, and to control advanced tumors. Surgical operation: For some HPV-related tumors, such as cervical cancer and oral cancer, surgical operation may be the preferred treatment, especially in the case of early diagnosis and local lesions. Finally, there is also radiotherapy: Radiotherapy uses high-energy rays to kill and control the growth of tumor cells. It can be used to reduce the size of the tumor, kill residual tumor cells or serve as postoperative adjuvant therapy. These drugs and treatments play a certain role in practical application, but still face some challenges and difficulties, such as HPV vaccines can only play a preventive role, chemotherapy and radiotherapy drugs have large side effects and high costs, and traditional treatment methods have poor specificity, high side effects, low efficiency and poor efficacy. According to the 2023 ICO / IARC China HPV and Related Disease Report, the incidence of cervical cancer among women aged 15-44 in China in 2020 ranked third among female tumors, with approximately 110,000 new cases and nearly 60,000 deaths, posing a serious threat to women's health. Therefore, for a country with a population of 1.4 billion, finding new treatment strategies and methods to improve the treatment of HPV-related tumors remains a daunting task. mRNA (messenger RNA) technology is a technology of gene expression in the process of genetic information transmission, which can use synthetic mRNA molecules to produce specific proteins. mRNA is a nucleic acid molecule transcribed from DNA, which carries the information of encoding proteins from genes, and ribosomes synthesize proteins in the cytoplasm using mRNA as a template. mRNA vaccine is just based on this technical principle, by providing mRNA sequences encoding specific antigens to cells, the antigen proteins produced by expression in cells are recognized by the recipient immune system, and humoral and cellular immune responses are generated. mRNA vaccines have the advantages of rapid development and production, and have achieved great success in the development of COVID-19 vaccines. This success case encourages researchers and technicians to apply mRNA technology to a wider field, especially in the field of HPV tumor treatment. Self-replicating mRNA is a new iteration of mRNA technology in the field of mRNA technology, which has the ability to self-replicate for a long time in recipient cells, can produce more mRNA molecules, and thus increase the expression of target proteins. Based on this mechanism, self-replicating mRNA has become the most potential drug carrier for antigen-specific immunotherapy, which can improve the efficacy of vaccines or drugs, while prolonging the in vivo action time of drugs, and exhibits the effect of long-term treatment with low injection frequency and low injection volume. The present application selects HPV tumor E6 and E7 protein targets, and designs and modifies point mutations of E6 and E7. The applicant designs a self-replicating mRNA molecule for treating HPV tumors by using self-replicating mRNA technology. Through cell experiments and animal experiments, the results confirm that the sequence designed by the self-replicating mRNA molecule can efficiently and continuously express E6 and E7 proteins in the animal body, activate the immune system of the body, and play a specific role in activating cellular immunotherapy for tumors. SUMMARY The purpose of the present application is to provide a self-replicating mRNA sequence for treating HPV tumors with immune response persistence, specific treatment, direct activation of the immune system and high safety. To achieve the above purpose, the present application first provides an amino acid sequence, which comprises one or more of the following sequences: the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in SEQ ID NO: 1 in series before and after, the sequence shown in SEQ ID NO: 2, and the sequence shown in SEQ ID NO: 2 and the sequence shown in SEQ ID NO: 6 in series before and after. The present application also provides a self-replicating mRNA sequence, which comprises RNA of the coding region of the amino acid sequence of claim 1, and its stop codon is shown in SEQ ID NO: 5. Preferably, the self-replicating mRNA sequence comprises a base sequence as shown in at least one of SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 15. Further preferably, the self-replicating mRNA sequence comprises a base sequence as shown in any one of SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 15. The present application also provides use of the mRNA sequence in the preparation of an anti-HPV tumor drug. Preferably, the use is for the preparation of a composition comprising an immunologically effective amount of the construct of any one of the self-replicating mRNA sequences. In another aspect, the present application also provides a pharmaceutical composition comprising an immunologically effective amount of the construct of any one of the self-replicating mRNA sequences. Specifically, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Preferably, the pharmaceutically acceptable excipient is at least one selected from the group consisting of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, sugar syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate, and mineral oil. Further specifically, the dosage form of the pharmaceutical composition includes the dosage form of the medicine, including injections, injection powders, tablets, ointments, capsules, granules, aerosols, sprays, or powder inhalants. Specifically, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Preferably, the pharmaceutically acceptable excipient is one or more selected from the group consisting of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, bacteriostatic agents, suspending agents, suspending aids, solubilizers, thickening agents, stabilizers, sweeteners, and spices. In another aspect, the present application provides a method for treating a disease, comprising: administering to a subject in need a therapeutically effective amount of the construct of any one of the self-replicating mRNA sequences or the pharmaceutical composition. Specifically, the medicine is administered in a systemic or local administration manner. Further specifically, the systemic administration includes systemic administration in the form of subcutaneous injection, intramuscular injection, intravenous administration, oral administration, inhalation administration, or sustained release administration. The present application has the following advantages: 1. Screening of higher immunogenicity molecular design of target gene. The application carries out mutation and tandem molecular design on two proteins E6 and E7 of HPV, and codon optimization design on the tandem E6 and E7 proteins, increases the stability of mRNA secondary structure, and reduces the immunogenicity of mRNA molecules. Through experiments, a better and more optimal sequence design with higher immunogenicity is screened from 8 optimized base molecular designs. 2. Specific killing effect of activated specific CD8+ T cells on HPV-induced tumors. HPV-positive tumor cells can escape immune recognition through mechanisms such as down-regulation of MHC (Major Histocompatibility Complex) molecule expression. The product sequence of the application can deliver optimized target gene mRNA sequences in cells through self-replicating mRNA vectors for efficient and stable translation of E6 and E7 antigens, which are directly recognized by the immune system in the recipient cells and specifically activate CD8+ T cell response, thereby causing activated CD8+ T specific inhibition, killing and elimination of tumor cells. 3. Low injection frequency, low dose and long immune duration of drugs. The application activates the immune system to attack and eliminate HPV-related tumor cells using the principle of immunotherapy. The target gene can be expressed for more than 20 days after a single injection of 10-30 μg of mRNA composition, which is obviously superior to other traditional vaccines and non-self-replicating mRNA vaccines. The E6 and E7 protein molecular design screened in the application has higher immunogenicity, smaller toxic side effects and higher safety. BRIEF DESCRIPTION OF DRAWINGS Figure 1 is an electropherogram of the plasmid SEQ ID NO: 8 / Sal I + Xba I double enzyme digestion product. Figure 2 is an electropherogram of the plasmid SEQ ID NO: 9 / Sal I + Xba I double enzyme digestion product. Figure 3 is an electropherogram of the plasmid SEQ ID NO: 10 / Sal I + Xba I double enzyme digestion product. Figure 4 is an electropherogram of the plasmid SEQ ID NO: 11 / Sal I + Xba I double enzyme digestion product. Figure 5 is an electropherogram of the plasmid SEQ ID NO: 12 / Sal I + Xba I double enzyme digestion product. Figure 6 is an electropherogram of the plasmid SEQ ID NO: 13 / Sal I + Xba I double enzyme digestion product. Figure 7 is an electropherogram of the plasmid SEQ ID NO: 14 / Sal I + Xba I double enzyme digestion product. Figure 8 is an electropherogram of the plasmid SEQ ID NO: 15 / Sal I + Xba I double enzyme digestion product. Figure 9 is a Western Blotting immunoblotting diagram. Figure 10 is a D19 E7 binding antibody detection result diagram. Figure 11 is a D29 E7 binding antibody detection result diagram. Figure 12 is a D35 CD4 / CD8 cell detection data diagram. DETAILED DESCRIPTION In order to better understand the present application, the following detailed description of the embodiments will be described in conjunction with the accompanying drawings. It should be understood that the embodiments are only used to illustrate the present application, and cannot limit the scope of the present application. Unless otherwise specified, the technical means adopted in the embodiments are conventional technical means in the art. The overall technical scheme of the present application is as follows: (I) Selection of target antigen The most important part of the self-replicating mRNA HPV tumor drug is the selection of tumor antigens. Only by selecting effective tumor antigens can the body's immune system be induced to produce an immune response against tumors, thereby inhibiting or eliminating HPV-related tumor cells. The reason why the present application selects E6 and E7 proteins as target antigens is as follows: The expression level of E6 and E7 proteins in most normal cells is low, while the expression level in HPV-related tumor cells is significantly increased. This specific expression makes it easier for the immune system to distinguish between normal cells and tumor cells and launch an immune attack on them. Therefore, E6 and E7 are selected as antigen molecules of HPV tumors. However, the E6 and E7 proteins in tumor cells caused by HPV infection are similar in structure to normal self-proteins, making it difficult for the immune system to recognize them as foreign bodies and trigger a sufficient immune response. In addition, long-term viral infection can lead to immune escape, allowing tumor cells to escape the attack of the immune system. Optimized E6 and E7 proteins are loaded by self-replicating mRNA and expressed in vivo, and as non-human self-proteins, they can effectively stimulate the body's immune response. When the immune system in the body recognizes these optimized E6 and E7 proteins, they will be regarded as foreign antigens, inducing specific cytotoxic T cell responses, especially CD8+ T cell responses, which can recognize and kill tumor cells that also express E6 and E7 proteins in the body. (II) Amino acid mutation of E6\E7 protein The optimized coding sequence can improve the immunogenicity of the protein. The present application makes three amino acid mutations of E6, E6C63G / E6C113G / E6I135T, the sequence is shown in SEQ ID NO: 1: Two amino acid mutations were made to E7, E7C24G / E7E36G, the sequence is shown in SEQ ID NO: 2: (III) Optimization of E6\E7 molecular design 1. Optimization of 5' end signal peptide To ensure the secretion of self-replicase and antigen, a signal peptide sequence is added to the 5' end sequence, preferably one of tPA signal peptide, tPA-1 signal peptide, IgE signal peptide, signal peptide sequence SP or a related combination; more preferably, the signal peptide tPA is added with a SAR mutation design; the sequence is SEQ ID NO: 3: 2. Optimization of target gene molecular design: ①Linker optimization: for E6, E7 and their mutant antigen protein sequences, a flexible Linker GGGGSGGGGSGGGGS (sequence SEQ ID NO: 4) is used. ② Codon optimization of target genes: By adjusting the codon sequence of the target gene, the expression level and translation efficiency in the host cell are optimized, which helps to improve the expression amount, stability and functionality of the target gene. According to the codon usage bias of the host cell, alternative common codons are selected to replace the codons in the original target gene that are not conducive to efficient translation. Delete or replace sequences in the target gene that may affect the stability of mRNA, such as RNA degradation-related sequences or poorly stable structural elements, to enhance the stability of mRNA. According to the GC content preference of the host cell, the GC content in the codon sequence of the target gene is adjusted to improve its expression level in the host cell. The average GC content of the optimized sequence is preferably 57%-61%; more preferably 58%-60%; more preferably 59%. ③ Termination codon overlap design To achieve precise control of protein translation, by changing the termination codon, the cell can be guided to terminate protein synthesis at a specific location, thereby producing the desired protein variant or avoiding the production of unnecessary proteins. The termination codon in the sequence is designed as SEQ ID NO: 5: TGATAATAGTGATAA. (IV) MITD design of transport domain protein: MITD (MHC class-Itrafficking domain, MHC class-Itrafficking domain) molecule presents endogenous antigen fragments on the cell surface, interacts with CD8+T cells, and participates in cellular immune response. The optimized MITD sequence is designed as SEQ ID NO: 6: (V) Construction of self-replicating RNA plasmid: The optimized target gene nucleotide sequence is cloned into a self-replicating vector according to the framework: signal peptide + mutated and optimized target gene + MITD using Sal I / Xba I double digestion to construct a self-replicating mRNA plasmid. (VI) Detection of the effectiveness of self-replicating RNA molecule design 1. In vitro cell experiment In vitro effectiveness is verified by Western Blotting of cell expressed proteins. HEK293 cells are selected, the sequence is transferred into the cells, and protein expression is detected by immunoblotting (Western Blotting), and the specific detection method is as follows: ① HEK293 cells are cultured in a microplate until the confluence of each well reaches 80%-90%; ② About 3 μg of mRNA stock solution prepared in Example 1 is diluted to 150 μL with Opti-MEM (serum-free medium) and mixed with 150 μL of diluted transformation reagent, and then incubated for 5 min. The incubated solution is added to the microplate. The microplate is incubated in a carbon dioxide incubator for 24 hours; ③ The microplate is removed and the culture medium is removed, and the cells are washed with ice PBS. Add 200 μL of lysis buffer to each well and lyse on ice for 5 min. Collect the lysate and centrifuge at 17000 g at 4°C for 30 min, and collect the supernatant; ④ Western Blotting is used to detect protein bands. 2. In vivo mouse animal experiment C57BL / 6 mice are selected for in vivo experiments. Drug administration is performed once a week, for a total of three times, and each administration is 15 μg. The empty LNP group is designed as a control group. Peripheral blood samples are taken from mice to detect binding antibodies. The binding antibody titer is used to confirm whether the mRNA transcribes and expresses the target gene in vivo to activate the immune response in vivo. Peripheral blood is taken and FACS (Fluorescence Activated Cell Sorting) technology is used for cell detection. The most important immune response in C57BL / 6 mice after E6 / E7 antigen stimulation is CD8+ T cells. Example One Self-replicating mRNA molecule design The amino acid sequence is shown in SEQ ID NO: 7. The optimized base sequence is shown in SEQ ID NO: 8. The plasmid identification map is shown in Figure 1. Example Two Self-replicating mRNA molecule design The amino acid sequence is shown in SEQ ID NO: 7. The optimized base sequence is shown in SEQ ID NO: 9: The plasmid identification map is shown in Figure 2. Example Three Self-replicating mRNA molecule design The amino acid sequence is shown as SEQ ID NO: 7. The optimised base sequence is shown as SEQ ID NO: 10: The plasmid identification map is shown in Figure 3. Example Four Self-replicating mRNA molecule design The amino acid sequence is shown as SEQ ID NO: 7. The optimised base sequence is shown as SEQ ID NO: 11: The plasmid identification map is shown in Figure 4. Example Five Self-replicating mRNA molecule design The amino acid sequence is shown as SEQ ID NO: 7. The optimised base sequence is shown as SEQ ID NO: 12: The plasmid identification map is shown in Figure 5. Example Six Self-replicating mRNA molecule design The amino acid sequence is shown as SEQ ID NO: 7. The optimised base sequence is shown as SEQ ID NO: 13: The plasmid identification map is shown in Figure 6. Example Seven Self-replicating mRNA molecule design The amino acid sequence is shown as SEQ ID NO: 7. The optimised base sequence is shown as SEQ ID NO: 14: The plasmid identification map is shown in Figure 7. Example Eight Self-replicating mRNA molecule design The amino acid sequence is shown as SEQ ID NO: 7: The optimised base sequence is shown as SEQ ID NO: 15: The plasmid identification map is shown in Figure 8. Example 9: Preparation of Self-Replicating mRNA 1. Plasmid preparation ① The designed drug sequence is inserted into a self-replicating plasmid vector, and plasmid DNA is synthesized chemically; ② The plasmid DNA sequence was introduced into E. coli cells by heat shock at a temperature of 42°C for 30-60 seconds. ③ After incubating the E. coli with the target plasmid in liquid culture medium at 37°C for about 1 hour, spread it on solid culture medium and place it in a constant temperature and humidity incubator at 37°C for about 24 hours. Select colonies with good colony morphology. ④ Add the selected colonies to a liquid synthetic medium and incubate at 37°C and 100-200 rpm for about 3 hours, then scale up the culture for 15 hours. Centrifuge at 4000 rpm for about 5 minutes to obtain the bacterial sludge. ⑤ Plasmid DNA was obtained by lysing and purifying the bacterial sludge using a plasmid kit. The prepared bacterial sludge was added to lysis buffer for alkaline lysis; the volume ratio of bacterial sludge to lysis buffer I was 4-6:250; the volume ratio of lysis buffer I: lysis buffer II: lysis buffer III was 5:5:7. The lysis mixture was centrifuged at 12000 rpm for 5-10 min, and the supernatant was added to a DNA adsorption column. The column was centrifuged at 12000 rpm for 1 min to remove waste liquid. After washing with washing buffer, the column was centrifuged at 12000 rpm for 1 min to remove waste liquid; elution buffer was added, and the column was incubated at 37℃ for 2 min, then centrifuged at 12000 rpm for 1 min to obtain the plasmid DNA solution. ⑥ Mix plasmid DNA, linearizing enzyme, and water, incubate at 50°C for 1 hour, and add magnetic beads to extract the linear plasmid DNA template. The purified linear plasmid DNA template sample was stored at -80℃. 2. Preparation of mRNA stock solution ① After mixing the linear plasmid DNA template, NTPs, buffer, and other substrates, add polymerase and react at 37°C for 2 hours in a PCR instrument;

[0114] ② Add water and lithium chloride to the solution after the reaction. The volume ratio of reaction solution:H2O:LiCl is 1:1.5:1.5. Precipitate RNA with lithium chloride, centrifuge at 12000g for 15min at 4℃ to remove waste liquid, add 70% ethanol to wash impurities, and then add nuclease-free water to release RNA from LiCl to obtain the mRNA stock solution. The purified mRNA stock solution sample was stored at -80℃. 3. Preparation of mRNA composition ① Water phase preparation: mRNA stock solution was taken out from -80℃ ultra-low temperature refrigerator and thawed in cold water bath, and then diluted with citric acid buffer salt to a concentration of about 0.2 mg / mL, which was mRNA working solution. Organic phase preparation: the mass ratio of cationic lipids (MC3, EMA-1, DC-Chol, DOTAP): cholesterol: PEG: phospholipid compounds (PC, PE, PS) was about 6:1.5:1.2:2.7; Organic phase: water phase (mRNA working solution) volume ratio was 1:3, and nanoparticles were prepared by a collision device; ② After harvesting the nanoparticles, 1xPBS was mixed with them to increase the volume by 8 times. Ultrafiltration centrifuge tubes with 100-300KD were used for concentration at 2-8℃, 1500-3000g. After concentration to a volume of 1-2mL, 8 times volume of 1xPBS was added for dilution, and the concentration was continued to the expected concentration volume. After filtration with a 0.2μm membrane, the sample was obtained. The sample was stored at -20℃. Case Ten Verification and Screening of Self-replicating mRNA Molecule Effect 1. In vitro cell experiment ① HEK293 cells were cultured in a microplate until the confluence of each well reached 80%-90%; ② About 3μg of self-replicating RNA stock solution prepared in Case Nine was diluted to 150μL with Opti-MEM (serum-free medium) and mixed with 150μL of diluted transformation reagent. After standing for 5min, the solution was added to the microplate. The microplate was incubated in a carbon dioxide incubator for 24 hours; ③ The microplate was removed and the culture solution was removed, and the cells were washed with ice PBS. 200μL of lysis solution was added to each well and lysed on ice for 5min. The lysate was collected and centrifuged at 17000g at 4℃ for 30min, and the supernatant was collected; ④ Western Blotting was used to detect protein bands. The detection results are shown in Figure 9. Figure 9 shows that SEQ ID NO:7, SEQ ID NO:12 and SEQ ID NO:14 appear target bands at corresponding positions, indicating that SEQ ID NO:7, SEQ ID NO:12 and SEQ ID NO:14 express target proteins in cells. 2. In vivo mouse animal experiment The self-replicating RNA composition samples prepared in Example 9 were used for mouse immunization experiments, with 15 μg per mouse per administration, and blood samples were taken to detect the binding antibody titers. The results are shown in Figures 10 and 11. E7 antibodies were detected in most of the sequences in the animals in vivo, and the E7 antibody titer of the SEQ ID NO: 14 group was the highest. High-titer E6 antibodies were only detected in the SEQ ID NO: 12 group in vivo. In summary, SEQ ID NO: 7, SEQ ID NO: 12 and SEQ ID NO: 14 were selected for subsequent self-replicating RNA drug research. The SEQ ID NO: 7, SEQ ID NO: 12 and SEQ ID NO: 14 groups were selected, and peripheral blood was taken for cell detection using FACS technology. The most important immune response in C57BL / 6 mice after E6 / E7 antigen stimulation was CD8+T cells, and the effect of the drugs designed with different sequences was determined by comparing the CD8+T cell fold detection values. The CD8+CD25+T cell fold of the SEQ ID NO: 14 group was the highest. The cell detection results are shown in Figure 12.

Claims

1. An amino acid sequence comprising one or more of the following sequences: the sequence shown in SEQ ID NO: 1, the sequence tandemly connected to SEQ ID NO: 3 and SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2, and the sequence tandemly connected to SEQ ID NO: 2 and SEQ ID NO:

6.

2. A self-replicating mRNA sequence comprising the coding region of the amino acid sequence of claim 1, and having a stop codon as shown in SEQ ID NO:

5.

3. The self-replicating mRNA sequence of claim 2, comprising at least one of the sequences shown in SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO:

15.

4. The self-replicating mRNA sequence as described in claim 3, wherein the sequence is shown in any one of SEQ ID NO: 8, SEQ ID NO: 13, or SEQ ID NO:

15.

5. Use of the mRNA sequence as described in any one of claims 2-4 in the preparation of an anti-HPV tumor drug.

6. The use as described in claim 5, for the preparation of a composition, said composition comprising an immunologically effective amount of a construct of the mRNA sequence of any one of claims 2-4.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the amino acid sequence of claim 1 or the self-replicating mRNA sequence of any one of claims 2-4.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, characterized in that, The dosage form of the pharmaceutical composition includes injections, powders for injection, tablets, ointments, capsules, granules, aerosols, sprays, or powder inhalers.

10. The use of the mRNA sequence according to any one of claims 2-4 and / or the pharmaceutical composition according to any one of claims 7-9 in the treatment of HPV tumors.

11. A method for treating HPV tumors, characterized in that, Administer to a patient the mRNA sequence according to any one of claims 2-4 and / or the pharmaceutical composition according to any one of claims 7-9.

12. The method according to claim 11, characterized in that, The methods of administration include: subcutaneous injection, intramuscular injection, intravenous administration, oral administration, inhalation administration, or sustained-release administration.

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