MRNA cancer vaccine for reversing malignant glioma immunosuppression microenvironment

Abstract

The invention belongs to the field of cancer mRNA (messenger Ribonucleic Acid) vaccines, and relates to an mRNA cancer vaccine for reversing a malignant glioma immunosuppression microenvironment. The vaccine comprises an mRNA molecule, the molecule comprises an open reading frame for coding an FAP antigen, the codon of the molecule is optimized, the vaccine can improve the expression quantity of the FAP antigen, the vaccine further comprises an open reading frame for coding a signal peptide SP and an open reading frame for coding a chemotactic factor hXCL1, and according to the preferable scheme, fusion protein can be guided out of cells through the signal peptide SP, so that the FAP antigen expression quantity can be improved. A chemotactic factor hXCL1 is targeted to DC cells to rapidly carry out antigen presentation, so that the immune response is improved, and the immunosuppression microenvironment of the malignant glioma is reversed.

Description

Technical Field

[0001] The present invention relates to an mRNA cancer vaccine, and particularly to an mRNA cancer vaccine for reversing the immunosuppressive microenvironment of malignant glioma. Background Art

[0002] Glioma is the most common tumor in the central nervous system, with an annual incidence of 8-10 per 100,000. It is difficult to remove surgically, has a high recurrence rate, a short survival period, and a poor prognosis, posing a great threat to human health. Gliomas can be divided into four grades from I to IV according to their malignancy. Grades I and II are low-grade gliomas (LGG), and grades III and IV are high-grade gliomas (HGG). The most malignant glioma is glioblastoma (GBM), a high-grade glioma with high invasiveness, high recurrence rate, and high lethality. The current standard treatment methods include surgery, radiotherapy, and chemotherapy, etc., and the median survival period is only 9 months. However, about 80-90% of glioblastoma patients will relapse within the range of the original lesion within half a year to one year after radiotherapy, which is the direct cause of death of patients with malignant glioma. With the in-depth study of medical research, some emerging therapies have emerged in recent years, such as proton therapy, electric field therapy, boron neutron therapy, CAR-T therapy, etc., providing new treatment options for patients with malignant glioma, but the curative effect is limited.

[0003] Oncolytic virus is a frontier topic in the treatment of malignant tumors. The applicant's scientific research team has successively developed two oncolytic viruses: ON-01 and TS-2021. Although the clinical trials have achieved encouraging results, like most clinical trials of glioma, the ultimate cause of death of the subjects is the recurrence of glioma. Therefore, exploring methods to prevent or delay tumor recurrence can buy time for the oncolytic virus treatment or other treatments of malignant glioma, effectively prolong the survival period of patients, and is crucial for other malignant glioma therapies such as oncolytic virus treatment.

[0004] An mRNA vaccine is a nucleic acid preparation that can, through a specific delivery system, transfer the gene sequence of an exogenous target antigen into cells and express a specific protein through transcription and translation. The transcribed protein activates the body's immune system, enabling immune cells to specifically recognize and target tumor cells, thereby achieving the purpose of preventing tumor recurrence, growth, or metastasis, etc. Among the cancer vaccines in clinical practice, most mRNA vaccines other than the HPV (cervical cancer) vaccine are therapeutic cancer vaccines.

[0005] The highly immunosuppressive microenvironment is an important feature of GBM, which leads to the insensitivity of GBM to immunotherapy. Cancer-associated fibroblasts (CAFs) are one of the most important components in the tumor microenvironment and play an essential role in the occurrence and development of tumors. CAFs can not only inhibit the functions of immune cells by secreting various cytokines or metabolites, and to a certain extent promote tumor development, invasion and metastasis; CAFs also have the function of shaping the tumor extracellular matrix and forming a penetration barrier for drugs or therapeutic immune cells, which can hinder the deep penetration of drugs (including oncolytic viruses) and immune cells into tumor tissues, thereby reducing the tumor treatment effect. Therefore, inhibiting tumors by regulating CAFs or overcoming their barrier effects is a new means of tumor treatment.

[0006] As a marker of CAFs, FAP participates in regulating the extracellular matrix and has many functions with pro-tumor activities. FAP (also known as fibroblast activation protein α or seprase) is a 97 kDa type II transmembrane serine oligopeptidase. The full length of human FAP consists of 760 amino acids and is mostly expressed during the growth and development of neonatal tissues and rarely expressed in fully differentiated tissues of healthy adults. However, at the remodeling sites of pathological tissues (precancerous lesions, hyperplasia, metaplasia, fibrosis, inflammation, regenerated and repaired tissues, etc.), especially on fibroblasts, the expression of FAP is highly upregulated. Therefore, vaccines targeting FAP are an important direction in current cancer treatment drug research. Vaccines that have been reported include DNA vaccines of FAP such as CN105879060B, but DNA vaccines have problems of low efficacy and poor safety. Thus, it is very necessary to develop mRNA vaccines targeting FAP. Summary of the Invention

[0007] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide an mRNA cancer vaccine for reversing the immunosuppressive microenvironment of malignant glioma.

[0008] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0009] In the first aspect of the present invention, an mRNA expression cassette is provided, including a DNA sequence encoding an FAP antigen as shown in Sequence 1 or Sequence 2 in the sequence listing.

[0010] Furthermore, the mRNA expression cassette further includes: a promoter located upstream of the DNA sequence encoding the FAP antigen, a 5'-UTR located downstream of the promoter and upstream of the DNA sequence encoding the FAP antigen, and a 3'-UTR located downstream of the DNA sequence encoding the FAP antigen;

[0011] Exemplarily, the promoter may be a T7 promoter; and / or, the nucleotide sequence of the 5'-UTR is as shown in Sequence 4 in the Sequence Listing, and / or, the nucleotide sequence of the 3'-UTR is as shown in Sequence 5 in the Sequence Listing.

[0012] Furthermore, the mRNA expression cassette further comprises: a DNA sequence encoding human leukocyte antigen (HLA) located between the 5'-UTR and the DNA sequence encoding the FAP antigen; and / or, a DNA sequence encoding MITD located between the DNA sequence encoding the FAP antigen and the 3'-UTR; and / or, a polyA tail located downstream of the 3'-UTR.

[0013] Exemplarily, the DNA sequence encoding human leukocyte antigen (HLA) is as shown in Sequence 6 in the Sequence Listing, and the DNA sequence encoding MITD is as shown in Sequence 7 in the Sequence Listing;

[0014] Preferably, the mRNA expression cassette comprises: a T7 promoter, a 5'-UTR, a Kozak sequence, a first restriction site, a DNA sequence encoding human leukocyte antigen (HLA), a DNA sequence encoding the FAP antigen, a DNA sequence encoding MITD, a second restriction site, a 3'-UTR, and a polyA tail, which are sequentially connected from the 5'-end to the 3'-end.

[0015] The first restriction site and the second restriction site can be determined according to the plasmid vector into which the mRNA expression cassette is to be inserted, as long as these two restriction sites do not conflict with the restriction sites on the plasmid vector and cause the mRNA expression cassette to be unable to be inserted into the appropriate position. For example, when the plasmid vector is pcDNA3, the first restriction site may be a BamHI restriction site, and the second restriction site may be a SacI restriction site.

[0016] In the mRNA expression cassette of the present invention, an HLA sequence is preferably added. The HLA sequence is translated and co-expressed with the antigen, which can ensure that the antigen binds to HLA more efficiently, thereby improving the T cell recognition efficiency and enhancing the immune response. The added MITD sequence in the expression cassette can promote the endoplasmic reticulum loading and cell membrane presentation of antigen peptides after translation, thereby enhancing the CD8+ T cell-mediated immune response. These two sequences added to the mRNA expression cassette improve the antigen presentation efficiency and enhance the immune response.

[0017] Furthermore, the mRNA expression cassette further comprises: a DNA sequence encoding a signal peptide SP located between the 5'-UTR and the DNA sequence encoding the FAP antigen;

[0018] Preferably, the DNA sequence encoding the signal peptide SP is located between the 5'-UTR and the DNA sequence encoding human leukocyte antigen (HLA), and / or, the DNA sequence encoding the signal peptide SP is as shown in Sequence 8 in the Sequence Listing;

[0019] More preferably, the DNA sequence encoding the signal peptide SP is located between the first cleavage site and the DNA sequence encoding human leukocyte antigen (HLA).

[0020] Furthermore, the mRNA expression cassette further includes: a DNA sequence encoding chemokine hXCL1 located between the 5'-UTR and the DNA sequence encoding FAP antigen;

[0021] Preferably, the DNA sequence encoding chemokine hXCL1 is located between the DNA sequence encoding the signal peptide SP and the DNA sequence encoding FAP antigen, and / or, the DNA sequence encoding chemokine hXCL1 is as shown in Sequence 9 in the Sequence Listing;

[0022] More preferably, the DNA sequence encoding chemokine hXCL1 is located between the DNA sequence encoding human leukocyte antigen (HLA) and the DNA sequence encoding FAP antigen, or the DNA sequence encoding chemokine hXCL1 is located between the DNA sequence encoding the signal peptide SP and the DNA sequence encoding human leukocyte antigen (HLA);

[0023] Further preferably, the mRNA expression cassette includes: a T7 promoter, a 5'-UTR, a Kozak sequence, a first cleavage site, a DNA sequence encoding the signal peptide SP, a DNA sequence encoding human leukocyte antigen (HLA), a DNA sequence encoding chemokine hXCL1, a DNA sequence encoding FAP antigen, a DNA sequence encoding MITD, a second cleavage site, a 3'-UTR, and a polyA tail, which are sequentially connected from the 5'-end to the 3'-end.

[0024] In the mRNA expression cassette of the present invention, in order to enable the smooth transcription and expression of the DNA sequence encoding the fusion protein located between the 5'-UTR and the 3'-UTR (more specifically, between the first cleavage site and the second cleavage site), the DNA sequence encoding the fusion protein has exactly one stop codon. For example, the mRNA expression cassette does not contain the stop codon of the DNA sequence encoding FAP antigen as shown in Sequence 1 or Sequence 2 in the Sequence Listing, nor does it contain the stop codon of the DNA sequence encoding chemokine hXCL1 as shown in Sequence 9 in the Sequence Listing.

[0025] The first restriction site (such as BamHI restriction site) and the second restriction site (such as Sad restriction site) present in the preferred expression cassette of the present invention can be used for subsequent further modification and addition of specific sequences. In this expression cassette, the sequence to be translated into protein follows the Kozak sequence, so the first restriction site is optimally after the Kozak sequence, or immediately after the Kozak sequence.

[0026] The second aspect of the present invention provides a biomaterial selected from the following (1) or (2):

[0027] (1) a recombinant vector, comprising a plasmid vector and the mRNA expression cassette of the first aspect inserted into the plasmid vector; illustratively, the plasmid vector is pcDNA3;

[0028] (2) A host cell comprising the mRNA expression cassette described in the first aspect or the recombinant vector described above.

[0029] The third aspect of the present invention provides an mRNA molecule, comprising a product formed by transcription of the mRNA expression cassette described in the first aspect, wherein the mRNA molecule further comprises a 5' end cap structure.

[0030] The fourth aspect of the present invention provides an mRNA cancer vaccine, comprising the mRNA molecule described in the third aspect and an intracellular delivery system.

[0031] Furthermore, the intracellular delivery system is a lipid delivery system; preferably, the lipid delivery system comprises ionizable cationic lipids, auxiliary lipids, cholesterol and lipid-anchored polyethylene glycol.

[0032] The fifth aspect of the present invention provides the use of the mRNA expression cassette described in the first aspect, the biomaterial described in the second aspect, and the mRNA molecule described in the third aspect in the preparation of an mRNA vaccine for preventing and / or treating cancer; the cancer is a cancer in which FAP is relatively highly expressed in tumor cells;

[0033] Preferably, the cancer is selected from: malignant glioma, lung cancer or liver cancer.

[0034] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0035] 1. The present invention optimizes FAP codons to provide an mRNA expression cassette comprising a DNA sequence encoding a FAP antigen, thereby preparing an mRNA vaccine. Compared with wild-type human FAP codons, the FAP codons optimized by the present invention can increase the expression level of the FAP antigen.

[0036] 2. In a further embodiment, the present invention also adds a signal peptide (SP) before HLA-FAP in the expression cassette for preparing the mRNA vaccine to obtain the fusion gene SP-HLA-FAP. The expressed signal peptide can direct the fusion protein out of the cell, facilitating the diffusion of the fusion protein between cells.

[0037] 3. In a still further embodiment, the present invention also adds the human DC cell chemokine hXCL1 to the SP-HLA-FAP gene in the expression cassette for preparing the mRNA vaccine to obtain the fusion gene SP-HLA-hXCL1-FAP. The expressed chemokine hXCL1 enables the diffused fusion protein to target DC cells for rapid antigen presentation, enhancing the immune response.

[0038] In summary, the vaccine can increase the expression level of the FAP antigen. The preferred embodiment can also direct the fusion protein out of the cell through the signal peptide SP and target DC cells for rapid antigen presentation through the chemokine hXCL1, thereby enhancing the immune response and reversing the immunosuppressive microenvironment of malignant glioma. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a figure showing the expression results of the FAP gene before and after codon optimization in human HEK293T cells detected by Western Blot. Among them, A is the Western Blot result figure, and B is the statistical bar chart of the corresponding results.

[0040] Figure 2 It is a figure showing the expression results outside the cell after fusing the SP signal peptide before the gene HLA-FAP-P2 detected by Western Blot. Among them, A is the Western Blot result figure, and B is the statistical bar chart of the corresponding results.

[0041] Figure 3 It is a figure showing the expression results of the hXCL1 gene before and after optimization in human HEK293T cells detected by Western Blot. Among them, A is the Western Blot result figure, and B is the statistical bar chart of the corresponding results.

[0042] Figure 4Results of immunogenicity detection of LNP-mRNA vaccines. Among them, (a) shows the results of immunogenicity detection for the comparison of three groups: LNF-GFP (negative control), LNP-FAP-WT, and LNP-FAP-P2; (b) shows the results of immunogenicity detection for the comparison of three groups: LNF-GFP (negative control), LNP-FAP-P2, or LNP-SP-FAP-P2; (c) shows the results of immunogenicity detection for the comparison of four groups: LNF-GFP (negative control), LNP-SP-FAP-P2, LNP-SP-hXCL1-WT-FAP-P2, and LNP-SP-hXCL1-P-FAP-P2.

[0043] Figure 5 Results of detecting cell-specific T cell responses induced by LNP-mRNA vaccines using the Elispot technique. Among them, A is the result picture, and B is the statistical analysis histogram of the results of A.

[0044] Figure 6 Results of treating a mouse animal model of glioblastoma with LNP-SP-hXCL1-P-FAP-P2 (GV). Among them: A is the animal experiment procedure (tumor implantation is tumor transplantation, IVIS detection is IVIS detection, subcutaneous injection is subcutaneous injection); B is the observation of tumor conditions in the control group, LNP group, and GV group by small animal in vivo imaging on the 7th / 14th / 21st day; C is the quantitative analysis of the number of photons (photon flux) in tumors in each group, *P < 0.05 and **P < 0.01; D is the statistical analysis of the survival curves of mice in each group (survival rate is the survival rate, days after tumor implantation is the number of days after tumor transplantation), **P < 0.01; ns indicates no significant difference.

[0045] Figure 7 Structural diagrams of T7-pcDNA3-FAP series plasmids.

[0046] Figure 8 Structural diagram of the T7-pcDNA3-SP-FAP-P2 plasmid.

[0047] Figure 9 Structural diagrams of T7-pcDNA3-SP-hXCL1-FAP-P2 series plasmids. Detailed implementation manners

[0048] In order to more clearly understand the technical content of the present invention, the technical solutions of the present invention will be described in detail below in conjunction with the accompanying drawings and experimental examples. It should be understood that these specific examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0049] For the conditions and methods not specified in the following examples, they shall be carried out according to conventional conditions or the conditions recommended by the manufacturer. For example, reference can be made to the conditions described in "Molecular Cloning: A Laboratory Manual" edited by Sambrook et al., "Animal Cell Culture" (edited by R.I. Freshney, 1987), "Methods in Enzymology" (Academic Press, Inc.), "Handbook of Experimental Immunology" (edited by D.M. Weir and C.C. Blackwell), "Gene Transfer Vectors for Mammalian Cells" (edited by J.M. Miller and M.P. Calos, 1987), "Current Protocols in Molecular Biology" (edited by F.M. Ausubel et al., 1987), "PCR: The Polymerase Chain Reaction" (edited by Mullis et al., 1994), and "Current Protocols in Immunology" (edited by J.E. Coligan et al., 1991). For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained by purchasing in the market.

[0050] The following explanations are made for the terms used in the present invention:

[0051] mRNA (messenger RNA): It is any RNA that encodes (at least one) protein (a polymer of natural, non-natural or modified amino acids) and can be translated in vitro, in vivo, in situ or ex vivo to produce the encoded protein. Those skilled in the art will understand that unless otherwise stated, when the nucleic acid sequence described in this application represents a DNA sequence, T in the sequence is T, but in the case where the sequence represents RNA (e.g., mRNA), "T" will be replaced by "U". Therefore, any DNA disclosed and identified by a specific sequence identification number in this article also indirectly discloses the corresponding RNA (e.g., mRNA) sequence complementary to the DNA, where each "T" in the DNA sequence is replaced by "U".

[0052] The DNA sequence encoding a protein, i.e., CDS, is a continuous DNA that starts with a start codon and ends with a stop codon.

[0053] Open reading frame (ORF): A continuous segment of DNA or RNA that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG). An ORF typically encodes a protein.

[0054] When preparing a fusion gene, the connection position of the open reading frames of two adjacent genes is achieved by removing the stop codon (such as TAA) of the upstream gene without knocking out the stop codon of the downstream gene to keep the connection position of the open reading frame without a stop codon, thereby enabling the expression of the fusion gene.

[0055] In vitro transcription of RNA: The mRNA expression cassette described in the present invention can be transcribed using an in vitro transcription (IVT) system. In some embodiments, a recombinant plasmid containing the mRNA expression cassette is first prepared, and then the recombinant plasmid DNA is transfected into cells such as Escherichia coli. The transfected cells are cultured to replicate the recombinant plasmid DNA, and then the plasmid DNA is isolated and purified. Finally, the plasmid is linearized by restriction enzyme digestion, and the linearized plasmid is used as a template to obtain mRNA molecules through transcription by RNA polymerase.

[0056] The "5′ untranslated region" (UTR) refers to the region of an mRNA that does not encode a polypeptide and is located immediately upstream (i.e., 5′) of the start codon (i.e., the first codon of the mRNA transcript translated by ribosomes).

[0057] The "3′ untranslated region" (UTR) refers to the region of an mRNA that does not encode a polypeptide and is located immediately downstream (i.e., 3′) of the stop codon (i.e., the codon of the mRNA transcript that signals the termination of translation).

[0058] "Poly(A) tail" is a region located downstream, such as immediately downstream (i.e., 3′), of the 3′UTR of mRNA that contains multiple consecutive adenosine monophosphates. For example, the poly(A) tail may contain 10 to 300 adenosine monophosphates. For example, the poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, the poly(A) tail contains 50 to 250 adenosine monophosphates. "Poly(A) tail" can also be a segmented polyA, which is less likely to lose A compared to consecutive adenosine monophosphates, ensuring expression efficiency. Specifically, the segmented polyA can have several other nucleotides, such as TGCAT, etc., arranged between two segments of consecutive adenosine monophosphates. In relevant biological environments (e.g., in cells, in vivo), the poly(A) tail is used to protect mRNA from, for example, enzymatic degradation in the cytoplasm, assist in transcriptional termination, and help with the export and translation of mRNA from the nucleus. The addition of the poly(A) tail can be achieved by using an E. coli poly(A) polymerase kit (#M0276L, New England Biolabs, USA) to add it to the already transcribed mRNA sequence, or by directly adding a polyA tail to the mRNA expression cassette for co-transcription with the fusion gene.

[0059] In the present invention, the meaning of the term "link" can indicate that the two sequences or structural units before and after are directly linked, or it can also mean that there are other sequences or structural units (such as a linker or a restriction site) arranged between the two sequences or structural units before and after, which can represent either direct connection or indirect connection between the two. For the subsequent requirements of vector modification, any feasible restriction site can be added at the appropriate position of the nucleic acid sequence provided in the present invention to facilitate the insertion of other required nucleic acid sequences. Whether the various sequences or structural units are directly or indirectly linked, as long as the desired fusion protein or amino acid sequence can be expressed after transcription and translation. For the present invention, the fusion protein obtained after transcription and translation of the nucleic acid sequence should be able to exert the extracellular guiding function of the signal peptide SP, the function of stimulating the body to produce an immune response by the FAP antigen, and the function of encoding the chemokine hXCL1 to target DC cells for rapid antigen presentation.

[0060] When constructing a recombinant vector using the mRNA expression cassette of the present invention, the plasmid vector can be selected from pcDNA3, pcDNA3.1, CET 1019HS-puro, pIRES, or pRL-SV40, and is further preferably pcDNA3.

[0061] The host cell for expanding and propagating the recombinant plasmid can be a prokaryotic cell or a eukaryotic cell; further preferably, the prokaryotic cell includes DH5α, JM109 or BL21, and more preferably DH5α; the eukaryotic cell includes yeast cells, Marco-145, PK-15, Hela or insect cells, and more preferably Marco-145.

[0062] The application of the mRNA expression cassette, biomaterial, and mRNA molecule provided by the present invention in the preparation of an mRNA vaccine for preventing and / or treating cancer; the cancer is a cancer with relatively high expression of FAP in tumor cells, such as cancers like lung cancer, liver cancer, and malignant glioma, etc. Relatively high expression of FAP means that the expression level of FAP in tumor tissue or cells is higher than that of FAP in normal tissues of the human or animal body, or it can be said that the expression level of FAP is up-regulated.

[0063] The mRNA vaccine provided by the present invention can also be used in combination with other drugs for preventing and / or treating cancer, such as existing immunotherapies like oncolytic viruses, CAR-T, PD-1 inhibitors, small molecule inhibitors, etc.

[0064] The preparation and efficacy verification of the mRNA cancer vaccine of the present invention will be described in detail through specific examples below.

[0065] Preparation and expression verification of an mRNA vaccine capable of expressing the fusion protein HLA-FAP-MITD in Example 1

[0066] 1.1 Preparation of the mRNA vaccine

[0067] (1) Construction of the recombinant plasmid

[0068] (a) Using the pcDNA3 plasmid with the T7 promoter as the basic plasmid, the pCDNA3 vector was digested with the restriction enzymes Bgl II and Pvu I. The digestion product was run on an agarose gel to recover the large fragment, and then the single-stranded sticky ends were filled in to become blunt ends using Klenow enzyme. It was run on an agarose gel again to recover the vector, and then it was ligated with T4 ligase and transformed. The new vector obtained after transformation deleted the Amp resistance and only carried the Kana resistance. Deleting the Amp resistance can avoid its adverse effects on mRNA packaging.

[0069] (b) Using the HindIII and XbaI restriction enzyme sites in the multiple cloning site, a pre-synthesized nucleic acid sequence that is directly ligated in sequence is added after the T7 promoter: HindIII restriction enzyme site, 5'UTR (such as the sequence 4 in the sequence listing), kozak sequence (GCCACC), BamHI, random base sequence (ACCAG) and SacI restriction enzyme site, 3’UTR (such as the sequence 5 in the sequence listing), and polyA tail (30 adenosine monophosphates + TGCAT + 70 adenosine monophosphates), BspQI restriction enzyme site, XbaI restriction enzyme site. Positive clones are selected and verified by sequencing. The resulting recombinant plasmid is named T7-pcDNA3, and its partial sequence structure is shown in Figure 7 .

[0070] (c) Synthesize the fusion target gene according to the sequence. The fusion target gene sequence is: a DNA sequence encoding human leukocyte antigen (HLA) (such as the sequence 6 in the sequence listing), a DNA sequence encoding FAP antigen, and a DNA sequence encoding MITD (such as the sequence 7 in the sequence listing), which are directly ligated in sequence from the 5’ end to the 3’ end. A BamHI restriction enzyme site is added to the 5’ end, and a SacI restriction enzyme site is added to the 3’ end. Among them, there are three DNA sequences encoding FAP antigen: wild-type FAP is represented by FAP-WT, which is a human FAP gene, and its nucleotide sequence is shown in the sequence listing as sequence 3; the optimized FAP coding gene sequences are represented by FAP-P1 and FAP-P2, and their DNA sequences are respectively shown in the sequence listing as sequences 1 and 2. The above-mentioned artificially synthesized fusion target gene has a stop codon only at the 3’ end of the DNA sequence encoding MITD, that is, the CDS of the FAP antigen gene needs to remove the stop codon and then fuse with MITD (Major histocompatibility complex class I transport signal).

[0071] (d) Insert the fusion target gene synthesized in step (c) into the recombinant plasmid obtained in step (b) through the BamHI and SacI restriction enzyme sites, and a new recombinant plasmid is obtained through identification.

[0072] (e) Transform the recombinant plasmid obtained in step (d) into the host cell DH5α to obtain a transformant. After the transformant proliferates, a large amount of recombinant vector is extracted. The three recombinant plasmids are respectively named: T7-pcDNA3-FAP-WT, T7-pcDNA3-FAP-P1, and T7-pcDNA3-FAP-P2, and their partial structures are shown in Figure 7 .

[0073] (2) Synthesis of mRNA

[0074] The three extracted recombinant plasmids, T7-pcDNA3-FAP-WT, T7-pcDNA3-FAP-P1, and T7-pcDNA3-FAP-P2, were linearized using the restriction enzyme BspQ1, and T7 RNA polymerase was used to transcribe mRNA with the linearized plasmids as templates. The mRNA was capped using an m7G capping kit and 2′-O-methyltransferase. Subsequently, the m7G-mRNA was purified using an RNA purification kit, and finally, mRNA containing 5’cap 1, 5’UTR, the ORF of HLA-FAP-MITD, 3’UTR, and a polyA tail was prepared. All mRNA was analyzed by agarose gel electrophoresis and stored frozen at -80°C.

[0075] (3) Preparation of RNA-LNP

[0076] The organic phase was prepared by mixing and dissolving the cationic amino lipid SM102, distearoyl phosphatidylcholine, cholesterol, and PEG in ethanol at a molar ratio of 50:10:38.5:1.5. A citrate buffer solution with a pH of 4.0 was prepared using citric acid and sodium citrate to dissolve the mRNA to prepare the aqueous phase. The aqueous phase and the organic phase were synthesized into an mRNA-LNP solution at a volume ratio of 3∶1 through a microfluidic device, and an mRNA-LNP preparation, that is, an mRNA vaccine, was obtained after dialysis. Three vaccines corresponding to the three recombinant plasmids were named LNP-FAP-WT, LNP-FAP-P1, and LNP-FAP-P2, respectively. The potential and particle size of the mRNA-LNP preparation were measured using a Zetasizer laser particle size analyzer, and the detection results were +43.3 mV and 101.3 nm, respectively. The encapsulation efficiency was detected to be 88.5%.

[0077] 1.2 Expression verification of LNP-mRNA in human HEK293T cells

[0078] LNP-FAP-WT, LNP-FAP-P1, and LNP-FAP-P2 were obtained using the above LNP packaging process. After quantification, the same mass of the above LNP-mRNA was transfected into human HEK293T cells to detect the expression of FAP protein. Specifically:

[0079] Twenty-four hours before transfection, 2.5×10 5HEK293T cells were used. When the cell density reached 60% - 70%, the transfection experiment was started. Before transfection, the cell culture medium (the basal medium was DMEM containing 10% FBS) and serum-free Opti-MEM medium were preheated in a 37°C water bath. During transfection, 1 μg of LNP-FAP-WT, LNP-FAP-P1, and LNP-FAP-P2 were respectively added to 200 μL of serum-free Opti-MEM, mixed well, and then left to stand at room temperature for 20 minutes to obtain the transfection system. The cells to be transfected were replaced with fresh cell culture medium, and then gently added to the above transfection system and gently shaken. The cells were placed back in the cell incubator and cultured for 6 hours, then the fresh cell culture medium was changed, and the cells were harvested after 24 hours.

[0080] Western Blot was used to detect the expression of the FAP gene in HEK293T cells. The cells were collected and 60 μL of 0.5% NP40 lysis buffer containing PMSF or Cocktail protease inhibitor was added. The cells were resuspended thoroughly and lysed by rotating at 4°C for 30 minutes. The lysate was centrifuged at 12000 rpm at 4°C for 10 minutes, and the supernatant was collected into a new 1.5 mL EP tube, and the precipitate was discarded. According to the actual volume of the sample, 5×SDS-PAGE protein loading buffer was added, mixed well, and the sample was heated in a 100°C air bath for 10 minutes, and then Western Blot was immediately carried out and detected using the FAP antibody, with β-actin as the internal reference control. The results are shown in Figure 1 A and Figure 1 B, showing that the expression of FAP in the LNP-FAP-P2 group was higher than that in the LNP-FAP-WT or LNP-FAP-P1 group, and the expression level of FAP in the LNP-FAP-P1 group was higher than that in the LNP-FAP-WT group, indicating that the mRNA translation of FAP in the LNP-FAP-P2 group was better and the codon optimization of this group was the best.

[0081] Preparation and expression verification of an mRNA vaccine capable of expressing the fusion protein SP-HLA-FAP-MITD in Example 2

[0082] 2.1 Preparation of the mRNA vaccine

[0083] (1) Construction of the recombinant plasmid T7-pcDNA3-SP-FAP-P2

[0084] Except for changing the sequence of the fusion target gene, the other construction methods are the same as those for constructing the recombinant plasmid in Example 1. In this example, the following fusion target gene sequence was artificially synthesized: the DNA sequence encoding the signal peptide SP (shown as Sequence 8 in the Sequence Listing), the DNA sequence encoding human leukocyte antigen (HLA) (shown as Sequence 6 in the Sequence Listing), the DNA sequence encoding the FAP-P2 antigen (shown as Sequence 2 in the Sequence Listing), and the DNA sequence encoding MITD (shown as Sequence 7 in the Sequence Listing), which were directly connected in sequence from the 5'-end to the 3'-end. A BamH I restriction site was added to the 5'-end, and a Sac I restriction site was added to the 3'-end. The above-mentioned artificially synthesized fusion target gene has a stop codon only at the 3'-end of the DNA sequence encoding MITD, that is, the stop codon of the FAP antigen gene CDS needs to be removed before fusing with MITD.

[0085] Partial structures in the T7-pcDNA3-FAP-P2 plasmid and the T7-pcDNA3-SP-FAP-P2 plasmid are shown in Figure 8 。

[0086] (2) The synthesis of mRNA and the preparation of RNA-LNP were the same as the corresponding parts in Example 1. The mRNA packaged by LNP was LNP-SP-FAP-P2.

[0087] 2.2 Verification of the expression of LNP-SP-FAP-P2 outside human HEK293T cells

[0088] After quantification, the same mass of LNP-FAP-P2 and LNP-SP-FAP-P2 were transfected into human HEK293T cells respectively, and a negative control (Control) was set at the same time. The expression of FAP protein in the supernatant and cells was detected (the transfection method and the Western Blot procedure were the same as those in Example 1).

[0089] The experimental results are shown in Figure 2 A and Figure 2 B, showing that the expression level of FAP in the supernatant of the LNP-SP-FAP-P2 group was significantly higher than that of the negative control group and the LNP-FAP-P2 group, indicating that the SP signal peptide effectively led to the exocytosis of FAP protein.

[0090] Preparation and expression verification of LNP-mRNA capable of expressing the fusion protein HLA-hXCL1-MITD in Example 3

[0091] 3.1 Preparation of LNP-mRNA

[0092] (1) Construction of the recombinant plasmid T7-pcDNA3-hXCL1

[0093] Except for changing the fused target gene sequence, the other construction methods are the same as those for constructing the recombinant plasmid in Example 1. In this example, the following fused target gene sequence was artificially synthesized: a DNA sequence encoding human leukocyte antigen (HLA) (shown as Sequence 6 in the sequence listing), a DNA sequence encoding hXCL1, and a DNA sequence encoding MITD (shown as Sequence 7 in the sequence listing), which were directly connected in sequence from the 5'-end to the 3'-end. A BamH I restriction site was added to the 5'-end, and a Sac I restriction site was added to the 3'-end. The above artificially synthesized fused target gene only has a stop codon at the 3'-end of the DNA sequence encoding MITD, that is, the CDS of the hXCL1 gene needs to remove the stop codon before fusing with MITD. The DNA sequences encoding hXCL1 include two types. One is the wild-type human-derived hXCL1-WT, whose nucleotide sequence is shown as Sequence 10 in the sequence listing, and the other is the codon-optimized hXCL1-P, whose nucleotide sequence is shown as Sequence 9 in the sequence listing. Recombinant plasmids T7-pcDNA3-hXCL1-WT and T7-pcDNA3-hXCL1-P were obtained using the two DNA sequences encoding hXCL1 respectively.

[0094] (2) The synthesis of mRNA and the preparation of RNA-LNP were the same as the corresponding parts in Example 1. The mRNA packaged by LNP was LNP-hXCL1-WT and LNP-hXCL1-P.

[0095] 3.2 Expression verification of LNP-hXCL1-WT and LNP-hXCL1-P in human HEK293T cells

[0096] After quantification, the same mass of LNP-hXCL1-WT and LNP-hXCL1-P were transfected into human HEK293T cells respectively, and a negative control (Control) was set up to detect the expression of hXCL1 protein. The results are shown in Figure 3 A and Figure 3 B, showing that the optimized hXCL1 was translated better in human cells, and there was a significant improvement after codon optimization compared with before optimization.

[0097] Preparation of an mRNA vaccine capable of expressing the fusion protein SP-HLA-hXCL1-FAP-MITD in Example 4

[0098] To enhance the antigen presentation ability of the expressed protein, the applicant added hXCL1, a ligand of the DC cell-specific XCR1 receptor, to the fusion gene.

[0099] (1) Construction of the recombinant plasmid T7-pcDNA3-SP-hXCL1-FAP-P2

[0100] Except for changing the sequence of the fusion target gene, the other construction methods are the same as those for constructing the recombinant plasmid in Example 1. In this example, the following fusion target gene sequence was artificially synthesized: The DNA sequence encoding the signal peptide SP (shown as Sequence 8 in the sequence listing), the DNA sequence encoding human leukocyte antigen (HLA) (shown as Sequence 6 in the sequence listing), the DNA sequence encoding hXCL1, the DNA sequence encoding the FAP-P2 antigen (shown as Sequence 2 in the sequence listing), and the DNA sequence encoding MITD (shown as Sequence 7 in the sequence listing) were directly connected in sequence from the 5'-end to the 3'-end. A BamH I restriction site was added to the 5'-end, and a Sac I restriction site was added to the 3'-end. The above-mentioned artificially synthesized fusion target gene has a stop codon only at the 3'-end of the DNA sequence encoding MITD, that is, the stop codons of the DNA sequences encoding hXCL1 and the FAP antigen gene CDS need to be removed. There are two kinds of DNA sequences encoding hXCL1. One is the wild-type human-derived hXCL1-WT, and its nucleotide sequence is shown as Sequence 10 in the sequence listing. The other is the codon-optimized hXCL1-P, and its nucleotide sequence is shown as Sequence 9 in the sequence listing. The recombinant plasmids T7-pcDNA3-SP-hXCL1-WT-FAP-P2 and T7-pcDNA3-SP-hXCL1-P-FAP-P2 were obtained by using the two DNA sequences encoding hXCL1 respectively. See Figure 9 for the partial structures of the two plasmids.

[0101] (2) The synthesis of mRNA and the preparation of RNA-LNP were the same as the corresponding parts in Example 1. The mRNAs packaged by LNP were LNP-SP-hXCL1-WT-FAP-P2 and LNP-SP-hXCL1-P-FAP-P2.

[0102] Example 5 Detection of the energy efficiency of LNP-mRNA

[0103] 5.1 Immunogenicity detection

[0104] 5.1.1 Experimental animals: 18 male C57BL6 mice at 6 - 8 weeks old were randomly divided into three groups, with 6 mice in each group, namely the LNF-GFP negative control group, the LNP-FAP-WT group, and the LNP-FAP-P2 group. Among them, LNF-GFP was prepared according to the method in Example 1, and only the nucleic acid sequence of FAP was replaced with the nucleic acid sequence of GFP.

[0105] Drug administration process: Three drug administrations were carried out on the 0th day, the 5th day, and the 10th day respectively, by subcutaneous injection, 2 μg each time. Then, about 100 μL of orbital blood was taken on the 14th day (i.e., 14 days after the first injection) for blood flow cytometry detection. The change in the number of CD45+CD8+ cells in the peripheral blood of the mice was detected to detect the immunogenicity of the cancer vaccine.

[0106] The results are shown in Figure 4 (a), which shows the ratio of CD45+CD8+ cells in the peripheral blood of three groups of mice detected by flow cytometry 14 days after injection. The ratios of CD45+CD8+ cells in the peripheral blood of mice in the LNF-GFP negative control group, the LNP-FAP-WT group, and the LNP-FAP-P2 group were 3.4%, 6.3%, and 7.5% respectively. This indicates that the immune response induced by LNP-FAP-P2 is the strongest.

[0107] 5.1.2 Experimental animals: 18 male C57BL6 mice, 6-8 weeks old, were randomly divided into three groups of 6 mice each, namely the LNF-GFP negative control group, the LNP-FAP-P2 group, and the LNP-SP-FAP-P2 group. The administration procedure was the same as in 5.1.1.

[0108] The results are shown in Figure 4 (b), which shows the ratio of CD45+CD8+ cells in the peripheral blood of three groups of mice detected by flow cytometry 14 days after injection. The ratios of CD45+CD8+ cells in the peripheral blood of mice in the LNF-GFP negative control group, the LNP-FAP-P2 group, and the LNP-SP-FAP-P2 group were 3.5%, 7.2%, and 11.6% respectively. This indicates that the immune response induced by LNP-SP-FAP-P2 is the strongest.

[0109] 5.1.3 Experimental animals: 24 male C57BL6 mice, 6-8 weeks old, were randomly divided into four groups of 6 mice each, namely the LNF-GFP negative control group, the LNP-SP-FAP-P2 group, the LNP-SP-hXCL1-WT-FAP-P2 group, and the LNP-SP-hXCL1-P-FAP-P2 group. The administration procedure was the same as in 5.1.1.

[0110] The results are shown in Figure 4 (c), which shows the ratio of CD45+CD8+ cells in the peripheral blood of four groups of mice detected by flow cytometry 14 days after injection. The ratios of CD45+CD8+ cells in the peripheral blood of mice in the LNF-GFP (negative control), LNP-SP-FAP-P2, LNP-SP-hXCL1-WT-FAP-P2, and LNP-SP-hXCL1-P-FAP-P2 groups were 4.1%, 13.9%, 17.8%, and 24.9% respectively. This indicates that the immune response induced by LNP-SP-hXCL1-P-FAP-P2 is the strongest.

[0111] 5.2 Inducing cell-specific T cell responses

[0112] Stimulate with the PEP-3 polypeptide of FAP, and use the FAP wild-type scanning peptide library as an irrelevant control for the Elispot assay of splenic lymphocytes of mice after vaccination.

[0113] Mice (6 - 8 week - old male C57BL6 mice, 5 mice per group) were immunized three times subcutaneously with LNP - SP - hXCL1 - P - FAP - P2 at days 0, 5, and 10. The injection dose for each mouse was 2 μg per time. Then, on the 14th day after the first injection, the spleens of each group of mice were dissected and placed in PBS solution containing heparin. The spleens were ground into a cell state, and impurities such as connective tissue were filtered out through a filter screen. The cell solution was centrifuged at 1500 rpm for 3 min. The supernatant was discarded, and the remaining precipitate was dispersed by shaking method and then 2 mL of erythrocyte lysate was added to lyse at room temperature for 1 min. Immediately after lysis, 10 mL of PBS was added to terminate the lysis process. Then all samples were centrifuged at 1500 rpm for 3 min. The supernatant was discarded, and the cells were washed once with 5 mL of PBS solution. The second centrifugation was at 1500 rpm for 3 min. After discarding the supernatant, 5 mL of inactivated 10% FBS 1640 medium was added, and the precipitate was resuspended. After cell counting, the mononuclear cell suspension was obtained by resuspending the precipitate.

[0114] Take out the pre - coated plate strips for detection from the Elispot kit, wash them 4 times with sterile PBS, 200 μL per well per time. After the last wash, discard the liquid and pat dry on the absorbent paper. Add 200 μL of 1640 medium to each well and incubate at room temperature for 30 min. Discard the medium in the wells of the plate and pat dry on the absorbent paper. According to the method recommended by the kit, add 2.5×10 5 such mononuclear cells to each well. At the same time, add 2 μg / well of the polypeptide library (the polypeptide library is a mixture of full - length polypeptide libraries of FAP antigen sequences) solution to the wells in the experimental group for mixing; nothing is added to the negative control group (blank). Irrelevant peptide refers to a non - murine antigen epitope that is not related to the target, namely the human CMV PP65 epitope. Incubate in a 37 °C cell culture incubator for 18 h; the next day, stain and wash the samples according to the operation procedure of the kit, finally develop color for 5 min, count the number of spots in the Elispot and compare the data.

[0115] See Figure 5 A and Figure 5 B. The results showed that LNP - SP - hXCL1 - P - FAP - P2 could significantly stimulate specific T - cell responses. This indicates that the hXCL1 chemokine can effectively induce the binding of antigen molecules to specific immune cells, thus greatly enhancing the effect of antigen molecule cross - presentation, and finally enabling hXCL1 to induce a stronger specific immune response to antigen molecules. The designed LNP - SP - hXCL1 - P - FAP - P2 in this project is the combination of fusion gene sequences with the strongest current immune effect. Figure 5 It shows that the immune response detected by the Elispot experiment is caused by the injection of FAP protein.

[0116] 5.3 Detection of the experimental efficacy of glioma in situ animal model

[0117] Obtaining glioma in situ animal model:

[0118] Mouse glioma cells GL261 (containing luciferase gene, with the number of cells being 1×10 5 cells) were implanted into the caudate nucleus of C57 mice. Specifically: C57BL / 6 mice at 6 - 8 weeks old were continuously anesthetized with gas. After anesthesia, they were fixed on a stereotactic apparatus, and the scalp on the head was disinfected with Anerdian II. Then, a 0.5 - cm - long skin incision was made longitudinally in the middle of the top of the mouse's head to expose the skull. The skull was wiped with hydrogen peroxide to expose structures such as the anterior fontanelle, sagittal suture, and coronal suture. Then, at 0.5 mm in front of the coronal suture and 2.5 mm to the right of the sagittal suture, a cranial drill with a diameter of 1.0 mm was used to drill through the skull to the dura mater. The glioma cells were respectively prepared into cell suspensions. 5 μL of cells (10 5 glioma cells) were vertically injected into the bone hole by a micro - syringe for 4.0 mm, then retracted 1.0 mm, and then the inner core was slowly and uniformly advanced within 20 min to implant the glioma cells into the right caudate nucleus of the mouse. The needle was slowly withdrawn, and the bone hole was sealed with bone wax. After suturing the scalp with silk thread, the mouse was left to wake up naturally. One week later, small - animal in - vivo imaging or nuclear magnetic resonance was used to detect the formation of tumors, and thus the glioma in situ animal model was obtained.

[0119] Experimental process and results:

[0120] The tumor - bearing experimental animals were randomly divided into 3 groups, with 5 animals in each group, and the following treatments were carried out respectively: 1) Control group (control), subcutaneous injection of PBS with the same volume as the LNP group on the 3rd / 6th / 9th day respectively; 2) LNP group, subcutaneous injection of LNP on the 3rd / 6th / 9th day respectively, 2 μg per mouse each time. LNP is a liposome without encapsulated mRNA and is obtained according to the method for preparing RNA - LNP in Example 1, with the organic phase being the same as above but the aqueous phase not containing mRNA; 3) LNP - SP - hXCL1 - P - FAP - P2 (GV) group, subcutaneous injection of LNP - SP - hXCL1 - P - FAP - P2 (GV) on the 3rd / 6th / 9th day respectively, 2 μg per mouse each time. The animal experiment process is shown in Figure 6 A.

[0121] On the 7th / 14th / 21st day after the first injection, small - animal in - vivo imaging was used to detect the intracranial tumor situation. The results of observing the tumor situations of the control group, LNP group, and GV group by small - animal in - vivo imaging on the 7th / 14th / 21st day are shown in Figure 6 B. Quantitative analysis of the number of photons (tumor cell activity) in the tumors of each group by small - animal in - vivo imaging ROI, and the results are shown in Figure 6 C. Statistical analysis of the survival curves of the mice in each group, and the results are shown in Figure 6 D.Figure 6 Both B-6D showed that the injection of GV significantly inhibited the growth of intracranial gliomas and prolonged the survival time of experimental animals.

Claims

1. An mRNA expression cassette comprising a DNA sequence encoding a FAP antigen as shown in Sequence 1 or Sequence 2 in the sequence listing.

2. The mRNA expression cassette according to claim 1, characterized in that Also includes: A promoter located upstream of a DNA sequence encoding a FAP antigen, a 5'-UTR located downstream of the promoter and upstream of the DNA sequence encoding the FAP antigen, and a 3'-UTR located downstream of the DNA sequence encoding the FAP antigen; Preferably, the promoter is a T7 promoter, and / or the nucleotide sequence of the 5'-UTR is as shown in Sequence 4 in the sequence listing, and / or the nucleotide sequence of the 3'-UTR is as shown in Sequence 5 in the sequence listing.

3. The mRNA expression cassette according to claim 2, characterized in that Also includes: a DNA sequence encoding human leukocyte antigen located between the 5'-UTR and the DNA sequence encoding the FAP antigen; and / or, a DNA sequence encoding MITD located between the DNA sequence encoding the FAP antigen and the 3'-UTR; and / or, a polyA tail located downstream of the 3′-UTR; Preferably, the DNA sequence encoding human leukocyte antigen is shown as Sequence 6 in the sequence listing, and the DNA sequence encoding MITD is shown as Sequence 7 in the sequence listing; Preferably, the mRNA expression cassette comprises: T7 promoter, 5'-UTR, Kozak sequence, first restriction site, DNA sequence encoding human leukocyte antigen, DNA sequence encoding FAP antigen, DNA sequence encoding MITD, second restriction site, 3'-UTR and polyA tail connected sequentially from 5' end to 3' end.

4. The mRNA expression cassette according to claim 3, characterized in that Also includes: a DNA sequence encoding a signal peptide SP located between the 5'-UTR and the DNA sequence encoding the FAP antigen; Preferably, the DNA sequence encoding the signal peptide SP is located between the 5'-UTR and the DNA sequence encoding human leukocyte antigen, and / or, the DNA sequence encoding the signal peptide SP is as shown in Sequence 8 in the sequence listing; More preferably, the DNA sequence encoding the signal peptide SP is located between the first restriction site and the DNA sequence encoding human leukocyte antigen.

5. The mRNA expression cassette according to claim 4, characterized in that Also includes: a DNA sequence encoding the chemokine hXCL1 located between the 5′-UTR and the DNA sequence encoding the FAP antigen; Preferably, the DNA sequence encoding the chemokine hXCL1 is located between the DNA sequence encoding the signal peptide SP and the DNA sequence encoding the FAP antigen, and / or, the DNA sequence encoding the chemokine hXCL1 is as shown in Sequence 9 in the sequence listing; More preferably, the DNA sequence encoding the chemokine hXCL1 is located between the DNA sequence encoding the human leukocyte antigen and the DNA sequence encoding the FAP antigen, or the DNA sequence encoding the chemokine hXCL1 is located between the DNA sequence encoding the signal peptide SP and the DNA sequence encoding the human leukocyte antigen; Further preferably, the mRNA expression cassette comprises: a T7 promoter, a 5'-UTR, a Kozak sequence, a first restriction enzyme cleavage site, a DNA sequence encoding a signal peptide SP, a DNA sequence encoding a human leukocyte antigen, a DNA sequence encoding a chemokine hXCL1, a DNA sequence encoding a FAP antigen, a DNA sequence encoding a MITD, a second restriction enzyme cleavage site, a 3'-UTR and a polyA tail connected in sequence from the 5' end to the 3' end.

6. Biological material selected from the following (1) or (2): (1) A recombinant vector comprising a plasmid vector and the mRNA expression cassette according to any one of claims 1 to 5 inserted into the plasmid vector; preferably, the plasmid vector is pcDNA3; (2) A host cell comprising the mRNA expression cassette according to any one of claims 1 to 5 or the recombinant vector according to claim 6.

7. An mRNA molecule comprising a product formed by transcription of the mRNA expression cassette according to any one of claims 1 to 5, wherein the mRNA molecule further comprises a 5' end cap structure.

8. An mRNA cancer vaccine comprising the mRNA molecule of claim 7 and an intracellular delivery system.

9. The mRNA cancer vaccine according to claim 8, characterized in that The intracellular delivery system is a lipid delivery system; preferably, the lipid delivery system comprises ionizable cationic lipids, auxiliary lipids, cholesterol and lipid-anchored polyethylene glycol.

10. Use of the mRNA expression cassette according to any one of claims 1 to 5, the biomaterial according to claim 6, and the mRNA molecule according to claim 7 in the preparation of an mRNA vaccine for preventing and / or treating cancer; the cancer is a cancer in which FAP is relatively highly expressed in tumor cells; Preferably, the cancer is selected from: malignant glioma, lung cancer or liver cancer.

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