CN118141903A - A circular RNA vaccine for the treatment of canine melanoma - Google Patents

Abstract

The invention discloses a circular RNA vaccine for treating canine melanoma; the circular RNA vaccine comprises a circular RNA molecule encoding optimized canine tyrosinase, wherein the optimized canine tyrosinase comprises canine tyrosinase or a homologous sequence thereof, a connecting arm, and an enhancing sequence or a homologous sequence thereof. The invention stimulates the immune system to recognize the canine tyrosinase by optimizing and enhancing the immunogenicity of the canine tyrosinase, and simultaneously prepares the canine tyrosinase cyclic RNA vaccine by using a cyclic RNA technology and an LNP delivery technology, and the optimized canine tyrosinase is highly expressed in vivo, so that the immune system is activated to produce a therapeutic effect on canine melanoma.

Description

A circular RNA vaccine for canine melanoma

Technical Field

The invention relates to the technical field of vaccines for treating canine melanoma, and in particular to a circular RNA vaccine for treating canine melanoma.

Background technique

Canine melanoma is common in the oral cavity, skin, eyes and toes. It is one of the most common tumors in dogs and the main cause of death in elderly dogs. Currently, the clinical treatments for canine tumors mainly include surgery, chemotherapy and radiotherapy. Common chemotherapy drugs include cyclophosphamide, doxorubicin, vincristine and prednisolone, which all have disadvantages such as poor tumor specificity and large toxic side effects.

Tumor vaccine refers to the process of injecting tumor-associated antigens in tumor tissue into the body of a tumor patient in the form of a pharmaceutically acceptable carrier, activating the body's immune system to kill tumor cells in order to achieve the purpose of controlling and treating tumors.

Tyrosinase is the main catalytic enzyme in the process of melanin formation. Tyrosinase is expressed in compound melanocytic nevus, while its expression is reduced in the deep dermis, and it is highly expressed in malignant melanoma. Therefore, tyrosinase is often used as a marker and specific antigen for melanoma. Since tyrosinase is the body's own protein, it is easy to produce immunosuppression and tolerance during immunization, and cannot achieve the purpose of treating melanoma. Some foreign companies use xenogeneic antigens for immunization to break through the body's immunosuppression. For example, the ONCEPT vaccine launched by Merial is a DNA vaccine that forms human tyrosinase antigen in the form of plasmid DNA to treat canine melanoma. Subsequent retrospective studies have shown that the vaccine has limited therapeutic effects.

Summary of the invention

The purpose of the present invention is to provide a circular RNA vaccine for treating canine melanoma, and a preparation method and application thereof. The circular RNA expressing optimized canine tyrosinase is delivered into cells through liposomes, and the optimized canine tyrosinase is efficiently expressed in vivo, stimulating the body to produce an immune response, thereby achieving a therapeutic effect.

The objective of the present invention is achieved through the following technical solutions:

The present invention provides a circular RNA vaccine for treating canine melanoma. The circular RNA vaccine comprises a circular RNA molecule encoding an optimized canine tyrosinase. The optimized canine tyrosinase comprises canine tyrosinase or a homologous sequence thereof, a connecting arm, and an enhancing sequence or a homologous sequence thereof.

The amino acid sequence of the canine tyrosinase is shown in SEQ ID NO:1.

The nucleotide sequence of the canine tyrosinase is shown in SEQ ID NO:2.

The homologous sequences are sequences having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity.

As an embodiment of the present invention, the linker is a flexible polypeptide consisting of 2-20 flexible amino acids, and the flexible amino acids are selected from at least one of Gly, Ser, Ala and Thr. More preferably, the linker is (Gly-Gly-Gly-Gly-Ser)n, wherein n is an integer between 2 and 5 (for example, 2, 3, 4 or 5).

The preferred linker arm amino acid sequence is as follows: GGGGSGGGGSGGGGS (SEQ ID NO: 3).

The preferred linker arm nucleotide sequence is as follows:

GGCGGCGGGGGCAGCGGCGGCGGGGGATCTGGCGGAGGAGGCAGC (SEQ ID NO: 4).

As one embodiment of the present invention, the enhancing sequence is selected from the enhancing sequence shown in the amino acid sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11.

The enhancing sequence whose amino acid sequence is shown in SEQ ID NO:5 is recorded as enhancing sequence ConA; the nucleotide sequence of enhancing sequence ConA is shown in SEQ ID NO:6.

The enhancing sequence whose amino acid sequence is shown in SEQ ID NO:7 is recorded as enhancing sequence T113G; the nucleotide sequence of enhancing sequence T113G is shown in SEQ ID NO:8.

The enhancing sequence whose amino acid sequence is shown in SEQ ID NO:9 is recorded as enhancing sequence K115T; the nucleotide sequence of enhancing sequence K115T is shown in SEQ ID NO:10.

The enhancing sequence whose amino acid sequence is shown in SEQ ID NO:11 is recorded as enhancing sequence L87T; the nucleotide sequence of enhancing sequence L87T is shown in SEQ ID NO:12.

As one embodiment of the present invention, the circular RNA comprises elements arranged in the order shown in any one of the following (a) to (d):

(a) the first exon, the second exon, the 5' spacer, the translation initiation element, the coding element and the 3' spacer;

(b) first exon, second exon, translation initiation element and coding element;

(c) translation initiation elements and coding elements;

(d) translation initiation elements, coding elements and insertion elements;

The coding element encodes the optimized canine tyrosinase.

As one embodiment of the present invention, the translation initiation element comprises one or more of the following sequences with translation initiation activity: an IRES sequence, a 5'UTR sequence, a Kozak sequence, a sequence comprising an m6A modification, and a complementary sequence of ribosomal 18S rRNA.

As one embodiment of the present invention, the insertion element comprises one or more sequences as shown below: a non-translated region sequence, a polyN sequence, an adapter sequence, a ribosome switch sequence, and a sequence that binds a transcriptional regulatory factor; wherein, in the polyN sequence, N is selected from at least one of A, T, G, and C.

As one embodiment of the present invention, the circular RNA molecule is obtained by transcribing a linear RNA from a recombinant nucleic acid molecule and further cyclizing it, and the coding region of the recombinant nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24.

The recombinant nucleic acid molecule comprises, from the 5' end to the 3' end, an intron fragment II, a translation initiation element truncated fragment II, a coding region, a polyAC element, a translation initiation element truncated fragment I and an intron fragment I; the coding region has a nucleotide sequence as shown in SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 24.

In some embodiments, the method for preparing the circular RNA molecule comprises the following steps:

S1, synthesizing a gene fragment including intron fragment II, translation initiation element truncated fragment II, coding region, polyAC element, translation initiation element truncated fragment I and intron fragment from 5' end to 3' end in sequence and cloning into pUC57 vector to obtain a DNA vector;

S2. The DNA vectors are transformed into DH5α competent cells to obtain engineered bacteria; the engineered bacteria are extracted by plasmid and sequenced to verify that the sequence is correct;

S3, the engineered bacterial plasmid with the correct sequence was verified, and then digested and purified to obtain a linearized plasmid;

S4, transcribing the linearized plasmid to obtain linear RNA;

S5. Circularization to obtain circular RNA.

In step S4, LiCl solution is added to the transcription product for precipitation, and a linear RNA precipitate is obtained by centrifugation, and the precipitate is resuspended in enzyme-free water to obtain a linear RNA aqueous solution; the linear RNA aqueous solution is cyclized, concentrated, HPLC purified, and ultrafiltered to obtain a circular RNA molecule.

As an embodiment of the present invention, the vaccine further comprises an ionizable cationic lipid carrier. By encapsulating the circular RNA into the lipid carrier, the uptake efficiency of the circular RNA by the body can be improved, and the storage stability of the circular RNA can be improved.

As an embodiment of the present invention, the ionizable cationic lipid carrier is a lipid nanoparticle, and the raw material composition of the lipid nanoparticle includes, by molar percentage, 45%-55% of cationic lipid, 35%-44% of cholesterol, 3%-10% of neutral lipid and 0.8%-1.8% of PEG-modified lipid; the cationic lipid is CMAX4; the cholesterol is 5-cholesten-3β-ol; the neutral lipid is DSPC or DOPE; the PEG-modified lipid is PEG-DMG, PEG-DSG or PEG-DPG.

As an embodiment of the present invention, the circular RNA vaccine for canine melanoma further comprises a cryoprotectant, wherein the cryoprotectant composition includes but is not limited to at least one of sucrose, trehalose and mannitol.

In the circular RNA vaccine, the content of the cryoprotectant is 5%-20% (w/v).

Compared with the prior art, the present invention has the following beneficial effects:

1) There are no reports on canine melanoma mRNA antigens. The present invention optimizes and enhances the immunogenicity of canine tyrosinase, stimulates the immune system to recognize canine tyrosinase, and uses circular RNA technology and LNP delivery technology to prepare a canine tyrosinase circular RNA vaccine, which highly expresses the optimized canine tyrosinase in vivo, thereby activating the immune system to produce a therapeutic effect on canine melanoma.

2) Compared with the disclosed DNA vaccine human tyrosinase antigen, the canine melanoma vaccine prepared with the antigen of the present invention has more similar antigenic epitopes and T cell epitopes to the tyrosinase endogenously expressed in canine melanoma, and is more likely to activate specific T cells in vivo, resulting in better therapeutic effects and prolonging the survival of sick dogs.

3) The circular RNA vaccine of the present invention has a lower immunization dose, is safe for sick dogs, and has no side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

FIG1 is a capillary electrophoresis diagram of different circular RNAs synthesized in vitro;

FIG2 is a graph showing the cellular immunity intensity induced by dogs immunized with different circular RNA vaccines;

FIG. 3 is a graph showing the survival curves of dogs with melanoma treated with three circular RNA vaccines.

Detailed ways

The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

Example 1. Gene design and synthesis

Tyrosinase is a self-protein that is highly expressed during the growth and deterioration of melanoma. In order to break through the body's autoimmune barriers and make the circular RNA vaccine highly expressed in the body, the present invention designs different tyrosinase antigens and optimizes the mRNA sequence to achieve high expression of the circular RNA vaccine and break through autoimmunity, including canine tyrosinase dTYR, human tyrosinase hTYR, mouse tyrosinase mTYR, enhanced canine tyrosinase dTYR-ConA, enhanced canine tyrosinase dTYR-T113G, enhanced canine tyrosinase dTYR-K115T and enhanced canine tyrosinase dTYR-L87T.

The amino acid sequences and nucleotide sequences of the circular RNA coding regions corresponding to each antigen are as follows:

The amino acid sequence of canine tyrosinase dTYR is shown in SEQ ID NO: 1. The nucleotide sequence of canine tyrosinase dTYR is shown in SEQ ID NO: 2.

The amino acid sequence of human tyrosinase hTYR is shown in SEQ ID NO: 13. The nucleotide sequence of human tyrosinase hTYR is shown in SEQ ID NO: 14.

The amino acid sequence of mouse tyrosinase mTYR is shown in SEQ ID NO: 15. The nucleotide sequence of mouse tyrosinase mTYR is shown in SEQ ID NO: 16.

The amino acid sequence of the enhanced canine tyrosinase dTYR-ConA is shown in SEQ ID NO: 17. The nucleotide sequence of the enhanced canine tyrosinase dTYR-ConA is shown in SEQ ID NO:18.

The amino acid sequence of the enhanced canine tyrosinase dTYR-T113G is shown in SEQ ID NO: 19. The nucleotide sequence of the enhanced canine tyrosinase dTYR-T113G is shown in SEQ ID NO: 20.

The amino acid sequence of the enhanced canine tyrosinase dTYR-K115T is shown in SEQ ID NO: 21. The nucleotide sequence of the enhanced canine tyrosinase dTYR-K115T is shown in SEQ ID NO: 22.

The amino acid sequence of the enhanced canine tyrosinase dTYR-L87T is shown in SEQ ID NO: 23. The nucleotide sequence of the enhanced canine tyrosinase dTYR-L87T is shown in SEQ ID NO: 24.

The nucleotide sequences of the above-mentioned antigens are described in patent CN114438127A to form each antigen target coding region, and intron fragment II and translation initiation element truncation fragment II are added before each antigen target coding region; polyAC element, translation initiation element truncation fragment I and intron fragment I are added after each antigen target coding region to form a linear RNA for preparing circular RNA.

Among them, the nucleotide sequence of intron fragment II is shown in SEQ ID NO:25.

The nucleotide sequence of the translation initiation element truncated fragment II is as follows (SEQ ID NO: 26):

CTACTAAACTAGATATAGTTACATTTAAAACTCTTCTTTATATCATACAGTTGAATAGTAGAAAGAGAAA.

The polyAC nucleotide sequence is as follows (SEQ ID NO: 27):

AAAAAACAAAAAACAAAACAAAAAACAAAAAACAAAACAAAAAACAAAAA ACAAAAAAAAAACAAAAAACAAAAAACAAAAAACAAAAAACAAAACAAAAAACA AAAAACAAAA.

The nucleotide sequence of the translation initiation element truncated fragment I is shown in SEQ ID NO:28.

The nucleotide sequence of intron fragment I is as follows (SEQ ID NO: 29):

TAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGG GAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTACCGTCAGTTGCTC ACTGTGCATCAGATT.

The above gene fragments were commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize and clone into the pUC57 vector, and the recombinant plasmids P-dTYR, P-hTYR, P-mTYR, P-dTYR-ConA, P-dTYR-T113G, P-dTYR-K115T, and P-dTYR-L87T were obtained, which were verified to be correct by gene sequencing. Linear RNA was obtained by transcription from the vector DNA, and circular RNA was formed from the linear RNA according to the following mechanism: ribozyme recognized the connection position of translation initiation element truncated fragment I (IRES fragment I) and intron fragment I to first generate a break, releasing intron fragment I; then ribozyme recognized the connection position of translation initiation element truncated fragment II (IRES fragment II) and intron fragment II to generate a break, releasing intron fragment II. The 3' end of translation initiation element truncated fragment I and the 5' end of translation initiation element truncated fragment II are connected to form a circular molecule.

Example 2. Preparation of circular RNA

(a) Preparation of strains: The DNA vectors synthesized in Example 1 were transformed into DH5α competent cells, respectively, and spread on LB medium plates containing 100 μg/ml ampicillin, and cultured at 37°C. When the colonies on the plates were clearly visible, single colonies were picked and placed in 3 ml LB liquid medium containing 100 μg/ml ampicillin, and cultured at 37°C. 1 ml of the bacterial solution was added to glycerol with a final concentration of 8%, and stored at -80°C to obtain engineered bacteria, numbered DH5α-dTYR, DH5α-hTYR, DH5α-mTYR, DH5α-dTYR-ConA, DH5α-dTYR-T113G, DH5α-dTYR-K115T, and DH5α-dTYR-L87T, respectively. They were used as seed bacteria for subsequent experiments.

(b) Plasmid extraction: The engineered bacteria prepared in the previous step were activated at 37°C/220 rpm for 3-4 h; the activated bacterial solution was taken for expansion culture, and the culture conditions were: shaking culture at 37°C/220 rpm overnight; plasmids were extracted using a commercial kit (Tiangen Endotoxin-Free Plasmid Extraction Kit), and the sequence was verified to be correct by sequencing.

(c) Plasmid digestion and purification: The plasmid was digested by restriction endonuclease BsaI (nearshore protein)/BsaI (Takara), and the digestion system was as follows: 10× buffer: 100 μl, plasmid: 5 mg, BsaI: 150 μl (5000 U), water was added to 1000 μl, and digestion was carried out at 37°C overnight. After digestion, the digestion product was purified by column using a commercial DNA recovery kit (TIANGEN) to obtain a linearized plasmid.

(d) In vitro transcription: The transcription system is configured as shown in Table 1 below:

Table 1

Reaction conditions: Constant temperature shaking reaction at 37°C/220rpm for 2-4h, then add 2ml of deoxyribonuclease I (DNaseI) to the transcription system, and constant temperature shaking reaction at 37°C/220rpm for 15min.

(e) Precipitation of RNA of the transcription product: LiCl solution (final concentration 2.5 M) was added to the transcription product obtained above, and the mixture was placed at -20°C for overnight precipitation. The overnight treatment solution was centrifuged to obtain RNA precipitate, which was then dried at room temperature and resuspended in enzyme-free water to obtain a linear RNA aqueous solution.

(f) RNA cyclization and concentration: The cyclization system and conditions are shown in Table 2 below:

Table 2

The above solutions were mixed thoroughly and heated at 55°C for 15 min. The circularized RNA product was concentrated by ultrafiltration tube centrifugation, and the filtrate was discarded; and the ultrafiltration liquid was transferred to a new centrifuge tube.

(g) Circular RNA HPLC purification: Circular RNA was separated by SEC-1000 (SEPAX) column in 150 mM phosphate buffer mobile phase, and the main peak component was collected.

(h) Ultrafiltration and concentration of purified circular RNA: The circular RNA is concentrated by centrifugation in an ultrafiltration tube and the filtrate is discarded; enzyme-free water is added to the ultrafiltration tube and the circular RNA is washed twice; the ultrafiltration liquid is transferred to a new centrifuge tube.

(i) Various circular RNA molecules were successfully prepared (see Table 3), numbered as cmRNA-dTYR, cmRNA-hTYR, cmRNA-mTYR, cmRNA-dTYR-ConA, cmRNA-dTYR-T113G, cmRNA-dTYR-K115T, cmRNA-dTYR-L87T, and RNA tests were performed as follows:

1) Identification: Each RNA was sequenced successfully and the sequence was consistent with the target sequence.

2) Concentration analysis: The concentration of RNA was measured by UV spectrophotometer. The results are shown in Table 3.

3) Integrity analysis: The purity of RNA was determined by capillary gel electrophoresis (CGE), which was no less than 80%. The results are shown in Table 3 and Figure 1.

table 3

Circular RNA molecules purity/% Concentration/mg/ml cmRNA-dTYR 89 2.6 cmRNA-hTYR 93 2.3 cmRNA-mTYR 96 2.4 cmRNA-dTYR-ConA 92 2.9 cmRNA-dTYR-T113G 94 2.8 cmRNA-dTYR-K115T 92 3.0 cmRNA-dTYR-L87T 90 3.1

Example 3. Preparation of canine melanoma circular RNA vaccine

(1) Preparation of solution I: cationic lipid CMAX4, cholesterol (5-cholesten-3β-ol), neutral lipid DSPC (distearoylphosphatidylcholine), and PEG-modified lipid PEG-DMG (polyethylene glycol-dimyristyl glyceride) were dissolved in ethanol at a lipid molar ratio of 50:38.5:10:1.5 to obtain solution I.

Among them, the cationic lipid CMAX4 (4-[(3-{[3-({3-[bis(3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino]propyl}(methyl)amino)propyl](3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino}propoxy)oxy]butyl 2-octanoate) was purchased from Suzhou Coremade Biopharmaceutical Technology Co., Ltd., and cholesterol (5-cholesten-3β-ol), neutral lipid DSPC (distearoylphosphatidylcholine), and PEG-modified lipid PEG-DMG (polyethylene glycol-dimyristylglycerol) were purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.

(2) Preparation of Solution II: The circular RNA prepared in Example 2 was dissolved in a 10 mM citric acid buffered saline solution at pH 4.0 and diluted to a final concentration of 200 μg/mL to obtain Solution II.

(3) Using microfluidics technology, solution I and solution II were quickly mixed in a volume ratio of 1:3, and the buffer environment was replaced with PBS with a pH of 7.4 using tangential flow technology to remove ethanol, thereby preparing LNP-mRNA.

(4) Adding cryoprotectant: Use Quant-iT ^™ RiboGreen ^™ RNA Assay Kit (Invitrogen ^™ R11490) to determine the mRNA concentration in the prepared LNP-mRNA, calculate the encapsulation efficiency, dilute the LNP-mRNA solution to 50 ug/ml, and add sucrose to a final concentration of 8% (w/v). After sterile filtration, the solution was divided and frozen to obtain the circular RNA vaccines V-dTYR, V-hTYR, V-mTYR, V-dTYR-ConA, V-dTYR-T113G, V-dTYR-K115T, and V-dTYR-L87T, respectively.

(5) The particle size, polydispersity index (PDI), and surface potential of the LNP particles of the seven circular RNA vaccines were measured using a dynamic light scattering method on a Zeta potential-laser particle size analyzer Malvern Zetasizer Nano-ZEN 3600 (Malvern). The results are shown in Table 4:

Table 4

serial number Particle size (nm) PDI Surface potential (mV) Encapsulation rate (%) V-dTYR 102 0.13 -7.3 92 V-hTYR 106 0.11 -7.5 89 V-mTYR 95 0.16 -7.1 95 V-dTYR-ConA 98 0.12 -6.9 90 V-dTYR-T113G 91 0.19 -6.7 91 V-dTYR-K115T 99 0.14 -7.1 92 V-dTYR-L87T 96 0.12 -7.2 93

Example 4, Canine Immunization and Effect Detection

7-8 month old beagles were randomly divided into 8 groups, 3 in each group, and the circular RNA vaccine prepared in Example 3 was immunized with muscle, the vaccination dose was 50ug/dog, PBS was used as negative control, and immunization was performed at 0 weeks and 3 weeks, respectively. Blood was collected on the 35th day after immunization, PBMC in the blood was separated using lymphocyte separation fluid (Biyuntian), and the number of antigen-specific T cells was detected using a canine IFN-gamma ELISPOT kit (R&D systems). The polypeptide pool is a canine tyrosinase 10aa, 5aa overlap polypeptide fragment, which is synthesized by GenScript Bio. The polypeptide pool stimulation concentration is 5ug/well. The PBMC cell plating concentration is 1*10E5/well, and the reaction color is developed after 72h of polypeptide stimulation culture. The ELISPOT detection plate is automatically collected, counted and analyzed by an immunospot analyzer, and the results are shown in Figure 2. As a result, the enhanced canine tyrosinase antigen optimized by the present invention has a higher specific T cell activation level. During the immunization period, each group of experimental dogs grew well without toxic side effects.

Example 5. Therapeutic effect of canine melanoma circular RNA vaccine on canine melanoma

Twenty dogs with melanoma were screened, with an average age of 9 years (7-15 years), including Labrador Retriever, Golden Retriever, Shepherd, Husky, Samoyed, and Alaskan dog. According to the TNM tumor staging, all dogs were in stage II-III. All dogs were divided into four groups, 5 in each group, and intramuscularly injected with circular RNA vaccines V-dTYR, V-hTYR, and V-dTYR-K115T, 50ug/dog/time, and PBS, 1ml/dog/time, once a month for 6 consecutive times. After the end of the administration, the dogs were observed for one year. During the experiment, the tumor size was measured once a month by computed tomography to evaluate the tumor burden, and the complete remission rate and objective remission rate were compared; complete remission (CR): no tumor was detected by computed tomography; partial remission (PR): the tumor diameter was reduced by more than 30%. Complete remission rate = dogs with complete remission/total dogs × 100%; objective remission rate = dogs with complete remission + dogs with partial remission/total dogs × 100%. The treatment effect is shown in Table 5. In addition, the survival of all sick dogs was counted, and the survival rate of all groups was counted at the end of the experiment. The survival rate of sick dogs is shown in Figure 3. The treatment results show that the optimized antigen of the present invention is more effective in tumor inhibition during the treatment period and the subsequent observation period, with less tumor recurrence and longer survival.

table 5

The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims (10)

1. A circular RNA vaccine for treating canine melanoma, characterized in that the circular RNA vaccine contains a circular RNA molecule encoding an optimized canine tyrosinase, wherein the optimized canine tyrosinase includes canine tyrosinase or a homologous sequence thereof, a linker, and an enhancing sequence or a homologous sequence thereof. 2. The circular RNA vaccine according to claim 1, characterized in that the connecting arm is a flexible polypeptide composed of 2-20 flexible amino acids, and the flexible amino acids are selected from at least one of Gly, Ser, Ala and Thr. 3. The circular RNA vaccine according to claim 1 or 2, characterized in that the connecting arm is (Gly-Gly-Gly-Gly-Ser)n, wherein n is an integer between 2 and 5. 4. The circular RNA vaccine according to claim 3, characterized in that the amino acid sequence of the connecting arm is shown in SEQ ID NO: 3. 5. The circular RNA vaccine according to claim 1, characterized in that the enhancing sequence is selected from the enhancing sequence shown in the amino acid sequence such as SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11. 6. The circular RNA vaccine according to claim 1, characterized in that the amino acid sequence of the optimized canine tyrosinase is shown in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23. 7. The circular RNA vaccine according to claim 1, characterized in that the circular RNA molecule comprises elements arranged in the order shown in any one of the following (a) to (d): (a) the first exon, the second exon, the 5' spacer, the translation initiation element, the coding element and the 3' spacer; (b) first exon, second exon, translation initiation element and coding element; (c) translation initiation elements and coding elements; (d) translation initiation elements, coding elements and insertion elements; The coding element encodes the optimized canine tyrosinase. 8. The circular RNA vaccine according to claim 7, characterized in that the translation initiation element comprises one or more of the following sequences with translation initiation activity: IRES sequence, 5'UTR sequence, Kozak sequence, sequence containing m6A modification, complementary sequence of ribosomal 18S rRNA; the insertion element comprises one or more of the following sequences: untranslated region sequence, polyN sequence, aptamer sequence, ribosomal switch sequence, sequence binding transcriptional regulatory factor; wherein, the polyN sequence, wherein N is selected from at least one of A, T, G, and C. 9. The circular RNA vaccine according to claim 1 is characterized in that the circular RNA molecule is obtained by transcribing a recombinant nucleic acid molecule to obtain a linear RNA, which is further cyclized, and the coding region of the recombinant nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 24. 10. The circular RNA vaccine according to claim 1, characterized in that the circular RNA vaccine further comprises a cryoprotectant, wherein the cryoprotectant includes but is not limited to at least one of sucrose, trehalose, and mannitol.