A kind of mRNA, mRNA vaccine preparation and its preparation method and application

Abstract

The present invention provides an mRNA, an mRNA vaccine preparation, and a preparation method and application thereof, which belong to the field of biomedical technology. The amino acid sequence of the mRNA of the present invention is shown in SEQ ID NO.2. The present invention has found that the mRNA vaccine preparation prepared from the mRNA with the amino acid sequence as shown in SEQ ID NO.2 can effectively stimulate the maturation of dendritic cells and the secretion of proinflammatory cytokines, thereby activating effector T cells to kill prostate cancer. The method for preparing the mRNA vaccine preparation of the present invention is simple and can be used for industrial production. The present invention uses liposome nanoparticles as carriers, and the encapsulation rate of the mRNA vaccine preparation is high, so that the body can be effectively stimulated to produce an immune response, and the immunogenicity is high.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to mRNA, mRNA vaccine preparations, and preparation methods and applications thereof. Background Art

[0002] Prostate cancer is one of the most common cancers with the highest incidence and mortality rates among men worldwide. In the United States, prostate cancer has become the most serious malignant tumor that threatens men's health. With the aging of the population and the increase in the intake of Westernized diets, the incidence of prostate cancer has been growing rapidly in recent years and has become a major killer that threatens the health and quality of life of elderly men. Prostate cancer lacks specific clinical symptoms in the early stages and is relatively hidden. When obvious symptoms appear, the disease usually develops to the middle and late stages. The treatment of early prostate cancer mainly includes radical prostatectomy, androgen deprivation therapy (ADT), radiotherapy, etc., but patients may relapse after undergoing the above-mentioned local treatments. Moreover, prostate cancer is very prone to bone metastasis, and most patients lose the opportunity for radical surgical treatment at the time of initial diagnosis. For advanced prostate cancer, chemotherapy and endocrine therapy are the most commonly used treatments, but the clinical application of the above treatments is often limited by problems such as the patient's intolerance to chemotherapy side effects and resistance to endocrine therapy. Therefore, therapeutic tumor vaccines used in adjuvant therapy for preventing recurrence after radical prostatectomy for early-stage prostate cancer, and in advanced, inoperable metastatic hormone-sensitive prostate cancer (mHSPC) and metastatic castration-resistant prostate cancer (mCRPC) will be very promising.

[0003] Successful cancer vaccines must induce the recruitment of antigen-presenting cells (APCs), which, after taking up and activating antigens, transport the antigens to draining lymph nodes and activate and expand antigen-specific T cells. These T cells must be released from lymphoid organs and transported to the tumor microenvironment (TME) to exert their effector functions. After entering the tumor immune microenvironment, T cells face multiple challenges, including the matrix architecture, which results in many tumor-infiltrating lymphocytes being located around blood vessels rather than inside the tumor. T cells may also encounter physical and chemical barriers in the TME, including stromal cells, tumor cells, and immunosuppressive cells (such as myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells) that inhibit the activity of cytotoxic T cells by releasing immunomodulatory cytokines (such as TGFβ and IL-10) and expressing immune checkpoints and their ligands (such as PD-1 / PD-L1). These immunosuppressive cells can also release immunomodulatory metabolites, such as indoleamine 2,3-dioxygenase, which metabolizes tryptophan, which is essential for T cell survival, into uric acid, further inhibiting T cell activity. Successful cancer vaccines must rapidly self-amplify and overcome these barriers to generate anti-tumor immune responses that can inhibit cancer evolution.

[0004] mRNA vaccines are of great significance in the rapid development and prevention of SARS-CoV-2 infection during the COVID-19 epidemic, and this technology has considerable potential for cancer treatment. Compared with traditional anti-tumor vaccines based on proteins or peptides, mRNA vaccines are not restricted by the patient's own HLA typing, have engineering strategies to improve their stability and maintain their immunogenicity, and can induce complementary innate and adaptive immune responses. To date, although some phase I and II clinical trials have produced promising results, no mRNA tumor vaccine has been approved by regulatory authorities for formal clinical treatment.

[0005] In order to increase the production speed of mRNA vaccines, protect RNA from degradation in vivo, and accelerate the uptake of mRNA vaccines by antigen-presenting cells, carrier systems based on liposome nanoparticles (LNPs) have been widely used in the production of mRNA vaccines. LNPs are usually composed of ionizable lipids, auxiliary lipids, cholesterol, and polyethylene glycol. The diameter of LNPs is usually less than 200 nanometers to facilitate endocytosis and ensure the release of the payload before the fusion of endosomes and lysosomes and the degradation of the contents. LNPs usually include ionizable lipids to encapsulate negatively charged mRNA molecules, and auxiliary lipids to promote the delivery of payloads and endosomal release into the cytoplasm. Cholesterol is usually added to LNPs to improve the fluidity of the lipid membrane and increase transfection efficiency, and polyethylene glycol can prolong the stability of nanoparticles and prolong the circulation time. Iterative products of these technologies have been or are being used in the development of cancer vaccines. Traditional mRNA generated by in vitro transcription consists of a 5' cap, a 5' untranslated region (UTR) and a 3' UTR, an open reading frame (ORF), and a poly (A) tail. This artificial in vitro transcribed mRNA is easy to adjust, simple to synthesize, and can be mass-produced for the treatment of various diseases. Unlike DNA-based treatments, mRNA does not need to enter the cell nucleus, thus avoiding the risk of altering genetic material. However, for mRNA-based treatments, it is necessary to reduce immunogenicity and increase protein expression efficiency. By optimizing its structure, the stability and translation efficiency of mRNA can be enhanced. For example, by optimizing the 5' end and using autologous or synthetic 5' cap analogs, its stability and translation efficiency can be increased, and the immune response can be modulated.

[0006] Previous Phase I / II clinical trials have shown that the mRNA vaccine CV9103, which encodes four prostate tumor-associated antigens, PSA, PSMA, PSCA, and STEAP1, has good tolerability and good immune activation in the treatment of prostate cancer. However, subsequent clinical trials of CV9104 containing two other antigens (PAP and Mucin-1) were terminated due to failure to improve overall survival of patients. These findings indicate that antigen selection is critical for activating antigen-presenting cells (APCs) and immune responses. In addition, as mentioned above, the selection of LNP types, the modification of LNPs to target APC cells, and the optimization of mRNA backbone structures are all important for generating cancer mRNA vaccines with transformation potential. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide an mRNA, an mRNA vaccine preparation, and a preparation method and application thereof. The mRNA vaccine preparation prepared from mRNA with an amino acid sequence as shown in SEQ ID NO.2 can effectively stimulate BMDC maturation and secrete proinflammatory cytokines, thereby inducing T lymphocytes to kill prostate cancer.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides an mRNA, the amino acid sequence of the mRNA is shown as SEQ ID NO.2.

[0010] The present invention provides an mRNA vaccine preparation, which includes the above-mentioned mRNA.

[0011] Preferably, the carrier of the mRNA vaccine is lipid nanoparticles.

[0012] The present invention provides a method for preparing an mRNA vaccine preparation, comprising the following steps:

[0013] Diluting the above mRNA to obtain an aqueous solution;

[0014] Mix SM-102, DSPC, cholesterol and DMG-PEG2000 to obtain a lipid phase solution;

[0015] The aqueous phase solution and the lipid phase solution are mixed to obtain an mRNA vaccine preparation.

[0016] Preferably, the dilution concentration of the mRNA is 90-95 μg / mL; the molar ratio of SM-102, DSPC, cholesterol and DMG-PEG2000 is (45-55): (9-11): (35-40): (1-2); and the volume ratio of the aqueous phase solution to the lipid phase solution is 1-6:1-3.

[0017] The present invention also provides the use of the above-mentioned mRNA, mRNA vaccine preparation or mRNA vaccine preparation obtained by the preparation method in the preparation of dendritic cells that mature and / or secrete pro-inflammatory cytokines.

[0018] The present invention also provides the use of the above-mentioned mRNA, mRNA vaccine preparation or the mRNA vaccine preparation obtained by the preparation method in the preparation of drugs for treating prostate cancer.

[0019] The present invention also provides a method for preparing dendritic cells that mature and / or secrete pro-inflammatory cytokines, characterized in that it includes the step of co-incubating the above-mentioned mRNA vaccine preparation or the mRNA vaccine preparation obtained by the preparation method with dendritic cells.

[0020] The present invention also provides a method for preparing effector T cells, comprising the step of co-incubating the dendritic cells obtained by the above preparation method with T lymphocytes.

[0021] The present invention also provides a use of the effector T cells obtained by the above preparation method in preparing a drug for treating prostate cancer.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides an mRNA, an mRNA vaccine preparation, and a preparation method and application thereof. The present invention has found that an mRNA vaccine preparation prepared from mRNA with an amino acid sequence such as SEQ ID NO.2 can effectively stimulate dendritic cell maturation and secrete proinflammatory cytokines, thereby activating effector T cells to kill prostate cancer cells. The method for preparing the mRNA vaccine preparation of the present invention is simple and can be used for industrial production. The present invention uses liposome nanoparticles as carriers to prepare an mRNA vaccine preparation with a high encapsulation rate, thereby effectively stimulating the body to produce an immune response, with strong targeting and good anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A in the figure shows CD80 after stimulation by different mRNA vaccines + CD86 + A is the proportion of BMDC; B is the content of TNF-α secreted by BMDC stimulated by different mRNA vaccines;

[0025] Figure 2 The killing efficiency of Tramp C1 target cells by BMDC activated T cells after stimulation with different mRNA vaccines;

[0026] Figure 3 CD80 after stimulation by different mRNA vaccines + CD86 + MoDC ratio;

[0027] Figure 4 A scheme for treating the Tramp C1 subcutaneous tumor model of prostate cancer in mice with mRNA vaccines;

[0028] Figure 5 A is a picture of Tramp C1 subcutaneous tumor of prostate cancer on the 40th day after treatment of different groups; B is the weight of Tramp C1 subcutaneous tumor of prostate cancer on the 40th day after treatment of different groups; C is the tumor growth curve after treatment of different groups; D is a schematic diagram of mouse weight after treatment of different groups;

[0029] Figure 6A shows the ratio of T lymphocytes infiltrating the spleen of C57BL / 6 mice after treatment with different groups; B shows the ratio of CD3 + CD44 high CD62L high The results of the central memory T lymphocyte ratio. DETAILED DESCRIPTION

[0030] The present invention provides an mRNA, the amino acid sequence of the mRNA is shown as SEQ ID NO.2.

[0031] In the present invention, three different mRNA vaccines are prepared by mRNAs with three different amino acid sequences. Studies have found that the mRNA vaccine preparation prepared by the mRNA shown in SEQ ID NO.2 can efficiently stimulate the body to produce an immune response, has strong targeting, and has good anti-tumor activity.

[0032] In the present invention, the present invention also provides an application of mRNA having an amino acid sequence as shown in SEQ ID NO.2 in the preparation of an mRNA vaccine preparation.

[0033] The present invention provides an mRNA vaccine preparation, which includes the above-mentioned mRNA.

[0034] In the present invention, the carrier of the mRNA vaccine is a lipid nanoparticle. The lipid nanoparticle comprises SM-102, DSPC, cholesterol and DMG-PEG2000. The molar ratio of SM-102, DSPC, cholesterol and DMG-PEG2000 is (45-55): (9-11): (35-40): (1-2), more preferably (48-52): (9.5-10.5): (37-39): (1.2-1.8), and more preferably 50:10:38.5:1.5.

[0035] The present invention provides a method for preparing an mRNA vaccine preparation, comprising the following steps:

[0036] Diluting the above mRNA to obtain an aqueous solution;

[0037] Mix SM-102, DSPC, cholesterol and DMG-PEG2000 to obtain a lipid phase solution;

[0038] The aqueous phase solution and the lipid phase solution are mixed to obtain an mRNA vaccine preparation.

[0039] In the above preparation method, the above mRNA is diluted to obtain an aqueous solution. The dilution concentration of the mRNA is 90-95 μg / mL, more preferably 90.5-93 μg / mL, and more preferably 91.75 μg / mL. The dilution method is to first dilute the mRNA to 180-185 μg / mL with water for injection, and then dilute it to 90-95 μg / mL with 90-110 mM citrate pH 4.0 buffer. The citrate pH 4.0 buffer is purchased from Nanjing Chemical Reagent Co., Ltd., item number C0132125124. The molar ratio of SM-102, DSPC, cholesterol and DMG-PEG200 is (45-55): (9-11): (35-40): (1-2), more preferably (48-52): (9.5-10.5): (37-39): (1.2-1.8), and more preferably 50:10:38.5:1.5. The SM-102 was purchased from Beijing Jiankai Technology Co., Ltd., item number S003; DSPC was purchased from Beijing Jiankai Technology Co., Ltd., item number S038; DMG-PEG200 was purchased from Beijing Jiankai Technology Co., Ltd., item number A3294. The volume ratio of the aqueous phase solution and the lipid phase solution is preferably 1~6:1~3. The present invention uses a microfluidic device with N / P=6:1, aqueous phase flow rate: ethanol phase flow rate=3:1, and a total flow rate of 12mL / min to mix the aqueous phase solution and the lipid phase solution evenly. In the present invention, lipids are soluble in the organic solvent ethanol, so the ethanol phase is the lipid phase.

[0040] The present invention also provides the use of the above-mentioned mRNA, mRNA vaccine preparation or mRNA vaccine preparation obtained by the preparation method in the preparation of dendritic cells that mature and / or secrete pro-inflammatory cytokines.

[0041] In the present invention, the dendritic cells include one or both of bone marrow-derived dendritic cells and human peripheral blood monocyte-derived dendritic cells.

[0042] The present invention also provides the use of the above-mentioned mRNA, mRNA vaccine preparation or the mRNA vaccine preparation obtained by the preparation method in the preparation of drugs for treating prostate cancer.

[0043] The present invention also provides a method for preparing dendritic cells that mature and / or secrete pro-inflammatory cytokines, characterized in that it includes the step of co-incubating the above-mentioned mRNA vaccine preparation or the mRNA vaccine preparation obtained by the preparation method with dendritic cells.

[0044] In the present invention, the incubation time is 20 to 26 hours, such as 22 or 24 hours. The concentration of the mRNA vaccine preparation is 8 to 12 μg / mL, and the density of the dendritic cells, based on a six-well plate, is 0.5 to 1.5×10 6 Pieces / hole.

[0045] The present invention also provides a method for preparing effector T cells, comprising the step of co-incubating the dendritic cells obtained by the above preparation method with T lymphocytes.

[0046] In the present invention, the ratio of the number of dendritic cells to T lymphocytes is 1:8-12, such as 1:10.

[0047] The present invention also provides a use of the effector T cells obtained by the above preparation method in preparing a drug for treating prostate cancer.

[0048] The present invention has found that the above-mentioned effector T cells can effectively kill prostate cancer cells.

[0049] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] In the following examples, the pDNA was purchased from Jiangsu Shenji Biotechnology Co., Ltd.; the WFI, whose Chinese name is water for injection, was purchased from Shanghai Lechun Biotechnology Co., Ltd., with the catalog number RW020-02; the restriction endonuclease buffer was purchased from Yisheng Biotechnology Co., Ltd., with the catalog number 10664ES92. The CAP analog was purchased from Jiangsu Shenji Biotechnology Co., Ltd., with the catalog number CAP3011.

[0051] Example 1

[0052] In this example, three mRNAs (named mRNA-1, mRNA-2 and mRNA-3) were designed. The amino acid sequence of mRNA-1 is:

[0053] MDAMKRGLCCVLLLCGAVFVSPSEAAAKEAAAKMWNLLHETDSAVATARRPRWVLAGGFFLLKKFLYNFTQIPHLAGTGGGGSGGGGSGGGGSNVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLEGGGGSGGGGSGGGGSARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGGGGGSGGGGSGGGGSSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKEGGGGSGGGGSGGGGSFYDPMFKYHLVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVGGGGSGGGGSGGGGSVLRMMNDQLMFLSHNKYAGESFPGIYDALFDIESKVDPSGGGGSGGGGSGGGGSAGLALQPGTALLCYSNCTQLGEQCWTARIRGGGGSGGGGSGGGGSDTDLCNASGAHALQPAAAILALLPALGLLLWGPGQLGGGGSGGGGSGGGGSPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYGGGGSGGGGSGGGGSLVYLPGVIAAIVQLLTRKQFGLLSFFFAVLHAIYSLSYPGGGGSGGGGSGGGGSIVGIVAGLAVLAVLAVLGAMVAVVMCRRKSSGGKGGSCSQAASSNSAQGSDVSLTA (SEQ ID NO.1).

[0054] The amino acid sequence of the mRNA-2 is as follows:

[0055] MDAMKRGLCCVLLLCGAVFVSPAKFVAAWTLKAAAMWNLLHETDSAVATARRPRWVLAGGFFLLKKFLYNFTQIPHLAGTGGGGSGGGGSGGGGSNVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLEGGGGSGGGGSGGGGSARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGGGGGSGGGGSGGGGSSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKEGGGGSGGGGSGGGGSFYDPMFKYHLVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVGGGGSGGGGSGGGGSVLRMMNDQLMFLSHNKYAGESFPGIYDALFDIESKVDPSGGGGSGGGGSGGGGSAGLALQPGTALLCYSNCTQLGEQCWTARIRGGGGSGGGGSGGGGSDTDLCNASGAHALQPAAAILALLPALGLLLWGPGQLGGGGSGGGGSGGGGSPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYGGGGSGGGGSGGGGSLVYLPGVIAAIVQLLTRKQFGLLSFFFAVLHAIYSLSYPGGGGSGGGGSGGGGSSQSTIPIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTAGGGGSGGGGSGGGGSIVGIVAGLAVLAVLAVLGAMVAVVMCRRKSSGGKGGSCSQAASSNSAQGSDVSLTA (SEQ ID NO.2).

[0056] The amino acid sequence of the mRNA-3 is as follows:

[0057] MRVTAPRTVLLLLSAALALTETWAAKFVAAWTLKAAAMWNLLHETDSAVATARRPRWVLAGGFFLLKKFLYNFTQIPHLAGTGGGGSGGGGSGGGGSNVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLEGGGGSGGGGSGGGGSARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGGGGGSGGGGSGGGGSSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKEGGGGSGGGGSGGGGSFYDPMFKYHLVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVGGGGSGGGGSGGGGSVLRMMNDQLMFLSHNKYAGESFPGIYDALFDIESKVDPSGGGGSGGGGSGGGGSAGLALQPGTALLCYSNCTQLGEQCWTARIRGGGGSGGGGSGGGGSDTDLCNASGAHALQPAAAILALLPALGLLLWGPGQLGGGGSGGGGSGGGGSPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYGGGGSGGGGSGGGGSLVYLPGVIAAIVQLLTRKQFGLLSFFFAVLHAIYSLSYPGGGGSGGGGSGGGGSSQSTIPIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTAGSGATNFSLLKQAGDVEENPGPMYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRmLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO.3).

[0058] The preparation of the above-mentioned mRNA-1, mRNA-2 and mRNA-3 is carried out as follows:

[0059] S1. Step of digesting the plasmid and preparing the linearized template

[0060] S1.1 Melt the plasmid linearization components in Table 1 on ice, calculate the dosage of each component based on the 40 μg plasmid linearization system, add each component into a 1.5 mL centrifuge tube in turn and mix well to obtain reaction solutions 01, 02 and 03 (i.e., reaction solutions containing SJ222-01, SJ222-02 or SJ222-03 plasmids, respectively).

[0061] Table 1 Plasmid linearization reaction system (400 μL)

[0062]

[0063] S1.2 Place the mixed reaction solutions 01, 02 and 03 into an oscillating reactor respectively, and set the parameters to 50°C, 300 rpm, and 2 h of reaction time.

[0064] S1.3 After the reaction is completed, the linearized plasmid reaction solution is purified. 400 μL of DNA magnetic beads are added to each tube of reaction solution for purification. Finally, 100 μL of injection water is added to each tube to elute and collect the sample to obtain SJ222-01, SJ222-02 and SJ222-03 linearized plasmids respectively.

[0065] S2. Steps for preparing mRNA samples by in vitro transcription (IVT)

[0066] S2.1 Melt the components of the IVT reaction system in Table 2 on ice, calculate the amount of each component based on the 20 μg IVT system, add each component into a 1.5 mL centrifuge tube in turn and mix well to obtain reaction solution a, reaction solution b and reaction solution c (i.e., the reaction solutions corresponding to the linearized plasmids of SJ222-01, SJ222-02 or SJ222-03, respectively).

[0067] Table 2 IVT reaction system

[0068]

[0069] In Table 2, Lin-pDNA indicates the linearized plasmid of SJ222-01, SJ222-02 or SJ222-03.

[0070] S2.2 The mixed reaction solution a, reaction solution b and reaction solution c were placed in an oscillating reactor respectively, and the parameters were set to 37°C, 300 rpm, and the reaction time was 2.5 h. After the reaction was completed, 40 μL DNase I was added to each tube of reaction solution, mixed evenly, and reacted at 37°C for 15 min to obtain IVT reaction solution a, IVT reaction solution b and IVT reaction solution c respectively.

[0071] S2.3 After the reaction is completed, IVT reaction solution a, IVT reaction solution b and IVT reaction solution c are purified respectively. 800 μL of RNA magnetic beads are added to each tube for purification. Finally, 1 mL of injection water is added to each tube to elute and collect the samples to obtain the original solution, that is, mRNA-1, mRNA-2 and mRNA-3.

[0072] S2.4 Take three samples of mRNA-1, mRNA-2 and mRNA-3 respectively to measure the concentration, and adjust the concentration to 1.0 mg / mL with water for injection, calculate the total amount and yield, and detect the integrity, capping rate and Poly A.

[0073] At the same time, sequencing showed that mRNA-1 with an amino acid sequence as shown in SEQ ID NO.1, mRNA-2 with an amino acid sequence as shown in SEQ ID NO.2, and mRNA-3 with an amino acid sequence as shown in SEQ ID NO.3 were successfully prepared.

[0074] Among them, the steps of capping rate detection are as follows:

[0075] (1) Preparation of reaction system

[0076] Thaw the mRNA and corresponding probe on ice, vortex to mix, centrifuge briefly, and prepare the enzyme digestion system as shown in the table below:

[0077] Table 3 Enzyme digestion system

[0078]

[0079] (2) Annealing: Perform annealing reaction on a PCR instrument: 95℃ for 5 min, 65℃ for 2 min, 55℃ for 2 min, 40℃ for 2 min, and 22℃ for 2 min.

[0080] (3) Pre-cleaning of magnetic beads: Take 100 μL SA Beads for each sample, remove the supernatant after adsorption on the magnetic stand, add 100 μL cleaning solution to wash 3 times, 100 μL Solution A to wash 2 times, and then add 100 μL Solution B to store for later use.

[0081] (4) Adsorb the magnetic beads, remove the supernatant, add the annealing product to resuspend, and leave at room temperature for 30 minutes.

[0082] (5) Add 5 μL RNase H to each sample, vortex and centrifuge, and incubate in a metal bath at 37°C, 1200 rpm for 2 h.

[0083] (6) Adsorb the magnetic beads, remove the supernatant, wash the magnetic beads three times with cleaning solution, and then wash them three times with DEPC water. Add 100 μL of 75% methanol solution to resuspend the magnetic beads, heat them on a metal bath at 80°C for 3 min, adsorb the magnetic beads while they are hot, and absorb the supernatant.

[0084] (7) Place the supernatant in a rotary evaporator and evaporate to dryness at room temperature. Add 25 μL of blank solution or DEPC water to reconstitute, place on the magnetic rack again to adsorb the magnetic beads, and absorb the supernatant to measure the product concentration.

[0085] (8) Analyze on a computer or store at -80°C for future use.

[0086] The steps for polyA tail detection are as follows:

[0087] ① Enzyme digestion reaction system: Use a pipette to take 100µg of mRNA sample, add 10μL RNase T1 and 10×RNase H reaction buffer (final concentration 1×), blow evenly, and place on a 37℃ constant temperature oscillator for reaction for 0.5 hours;

[0088] ② Take 500 μL of magnetic beads and transfer them to a centrifuge tube. After adsorption on the magnetic stand, remove the supernatant, add 100 μL of binding buffer, resuspend the magnetic beads, add the digested sample to the magnetic beads, mix thoroughly with a pipette, shake slowly on a shaker, and incubate at room temperature for 10 minutes to ensure that the polyA after digestion can bind to the magnetic beads.

[0089] ③After the incubation, remove the supernatant after the magnetic stand adsorption, adsorb the magnetic beads, remove the supernatant, wash the magnetic beads 3 times with magnetic bead washing solution, and then wash them 3 times with 100mM ammonium acetate solution. Add 100μL 75% methanol solution to resuspend the magnetic beads, heat them at 80℃ for 3min in a metal bath, adsorb the magnetic beads while they are hot, and absorb the supernatant.

[0090] ④ Place the supernatant in a rotary evaporator and evaporate to dryness at room temperature. Add 25 μL of blank solution or DEPC water to reconstitute, place it on the magnetic stand again to adsorb the magnetic beads, absorb the supernatant, and determine the product concentration.

[0091] ⑤Analyze on machine or store at -80℃ for future use.

[0092] The test results are shown in Table 4.

[0093] Table 4 Results of concentration, total amount, yield, integrity, capping rate and polyA of different mRNA samples

[0094]

[0095] The results in Table 4 show that the three prepared mRNAs have a high concentration, all higher than 1050 ng / μL, which is suitable for subsequent liposome encapsulation and application. The yield of mRNA samples is between 186 and 192 times, with high production efficiency and small differences, and the method is stable and reliable. The integrity of mRNA samples is between 91.1% and 94.4%, which means that most of the mRNA maintains a complete chain structure during the preparation process, with less damage or degradation, which is conducive to subsequent functional research and application. The capping rate of mRNA samples is between 96.3% and 96.7%, with high capping efficiency, which is crucial for improving mRNA stability and translation efficiency. The polyA tail length of mRNA samples ranges from 31-33A or 30-34A (short chain) and 71-85A or 71-82A (long chain), indicating that the samples are more consistent in the distribution of polyA tail length, which is conducive to improving the stability and translation efficiency of mRNA.

[0096] Comparative Example 1

[0097] A method for preparing an mRNA vaccine, comprising the following steps:

[0098] (1) Aqueous solution

[0099] The mRNA-1 prepared in Example 1 was diluted to 183.5 μg / mL with water for injection, and then diluted to 91.75 μg / mL with 100 mM citrate pH 4.0 buffer to obtain an aqueous phase solution.

[0100] (2) Lipid phase solution

[0101] The lipid phase solution was prepared in a molar ratio of SM-102: DSPC: cholesterol: DMG-PEG2000 = 50:10:38.5:1.5, i.e., the concentration of SM-102 was 5.00 mM; the concentration of DSPC was 1.00 mM; the concentration of cholesterol was 3.85 mM; and the concentration of DMG-PEG2000 was 0.15 mM.

[0102] (3) Mixing of water phase and lipid phase

[0103] According to the N / P (molar ratio of nitrogen to phosphorus, i.e. the ratio of the molar number of nitrogen atoms to phosphorus atoms) = 6:1, the water phase flow rate: ethanol phase flow rate = 3:1, and the total flow rate of 12 mL / min, 9 mL of the water phase solution and 3 mL of the lipid phase solution were mixed using a microfluidic device to obtain the mRNA vaccine, which was recorded as mRNA LNP-1.

[0104] (4) Sample solution replacement

[0105] The mRNA vaccine sample obtained in step (3) is replaced with a preservation solution by ultrafiltration.

[0106] Preparation of the storage solution: weigh 10 mM Tris buffer (1.21 g), 0.1 g NaCl, 80 g sucrose, and dissolve in ddHO. 2 O to a final volume of 1 L, and finally adjust the pH value to 7.4 and mix well.

[0107] Example 2

[0108] A method for preparing an mRNA vaccine, comprising the following steps:

[0109] (1) Aqueous solution

[0110] The mRNA-2 prepared in Example 1 was diluted to 183.5 μg / mL with water for injection, and then diluted to 91.75 μg / mL with 100 mM citrate (Cit) pH 4.0 buffer to obtain an aqueous phase solution.

[0111] (2) Lipid phase solution

[0112] The lipid phase solution was prepared in a molar ratio of SM-102: DSPC: cholesterol: DMG-PEG2000 = 50:10:38.5:1.5, i.e., the concentration of SM-102 was 5.00 mM; the concentration of DSPC was 1.00 mM; the concentration of cholesterol was 3.85 mM; and the concentration of DMG-PEG2000 was 0.15 mM.

[0113] (3) Mixing of water phase and lipid phase

[0114] 9 mL of aqueous solution and 3 mL of lipid phase solution were mixed using a microfluidic device at N / P (molar ratio of nitrogen to phosphorus) = 6:1, aqueous phase flow rate: ethanol phase flow rate = 3:1, and a total flow rate of 12 mL / min to obtain an mRNA vaccine, recorded as mRNA LNP-2.

[0115] (4) Sample solution replacement

[0116] The mRNA vaccine sample obtained in step (3) is replaced with a preservation solution by ultrafiltration.

[0117] Preparation of the storage solution: weigh 10 mM Tris buffer (1.21 g), 0.1 g NaCl, 80 g sucrose, and dissolve in ddHO. 2 O to a final volume of 1 L, and finally adjust the pH value to 7.4 and mix well.

[0118] Comparative Example 2

[0119] A method for preparing an mRNA vaccine, comprising the following steps:

[0120] (1) Aqueous solution

[0121] The mRNA-3 prepared in Example 1 was diluted to 183.5 μg / mL with water for injection, and then diluted to 91.75 μg / mL with 100 mM citrate pH 4.0 buffer to obtain an aqueous phase solution.

[0122] (2) Lipid phase solution

[0123] The lipid phase solution was prepared in a molar ratio of SM-102: DSPC: cholesterol: DMG-PEG2000 = 50:10:38.5:1.5, i.e., the concentration of SM-102 was 5.00 mM; the concentration of DSPC was 1.00 mM; the concentration of cholesterol was 3.85 mM; and the concentration of DMG-PEG2000 was 0.15 mM.

[0124] (3) Mixing of water phase and lipid phase

[0125] 9 mL of aqueous solution and 3 mL of lipid phase solution were mixed using a microfluidic device at N / P (molar ratio of nitrogen to phosphorus) = 6:1, aqueous phase flow rate: ethanol phase flow rate = 3:1, and a total flow rate of 12 mL / min to obtain an mRNA vaccine, recorded as mRNA LNP-3.

[0126] (4) Sample solution replacement

[0127] The mRNA vaccine sample obtained in step (3) is replaced with a preservation solution by ultrafiltration.

[0128] Preparation of the storage solution: weigh 10 mM Tris buffer (1.21 g), 0.1 g NaCl, 80 g sucrose, and dissolve in ddHO. 2 O to a final volume of 1 L, and finally adjust the pH value to 7.4 and mix well.

[0129] Example 3

[0130] Ⅰ. The particle size, polydispersity index (PDI), encapsulation efficiency, total mRNA concentration and free mRNA concentration of mRNA LNP-1 prepared in Comparative Example 1, mRNA LNP-2 prepared in Example 2 and mRNA LNP-3 prepared in Comparative Example 2 were measured. The results are shown in Table 5.

[0131] Table 5 Characterization results of different mRNA vaccines

[0132]

[0133] The results in Table 5 show that the preparation method of the present invention can successfully obtain nano-scale mRNA vaccines with high encapsulation rates of 96.28%, 93.28% and 95.4%, respectively.

[0134] II. Verification of the immune response effects of mRNA LNP-1 prepared in Comparative Example 1, mRNA LNP-2 prepared in Example 2, and mRNA LNP-3 prepared in Comparative Example 2

[0135] (1) Add 1×10 6 C57BL / 6 mouse bone marrow-derived dendritic cells (BMDCs) were cultured and 10 μg / mL mRNA LNP-1, 10 μg / mL mRNA LNP-2, 10 μg / mL mRNA LNP-3 and PBS were added to the culture medium containing BMDCs for 24 h. The expression of costimulatory molecules CD80 and CD86 on the surface of BMDCs was detected by flow cytometry. The experimental results are shown in Figure 1 A in the figure; ELISA assay was used to detect the TNF-α content secreted by BMDC in the culture medium. The experimental results are shown in Figure 1 B in.

[0136] Figure 1 The results showed that PBS, mRNA LNP-1, mRNA LNP-2, and mRNA LNP-3 stimulated the generation of CD80 + CD86 + The proportions of BMDC were 16.2%, 60.9%, 84.7% and 74.9%, respectively, and the levels of TNF-α stimulated by BMDC were 565.0pg / mL, 781.1pg / mL, 777.3pg / mL and 718.4pg / mL, respectively. mRNA LNP-2 had the strongest effect in stimulating BMDC maturation and secretion of proinflammatory cytokines.

[0137] (2) The BMDC stimulated in step (1) above was co-cultured with C57BL / 6 mouse spleen T lymphocytes at a ratio of 1:10 for 72 h. The mouse spleen T lymphocytes after co-culture were co-cultured with the mouse prostate cancer cell line Tramp C1 expressing PSMA, PSCA, and STEAP1 (purchased from the ATCC cell bank in the United States) at a cell ratio of 20:1 for 24 h, and the lactate dehydrogenase (LDH) content in the supernatant of the co-culture system culture medium was detected, and the killing rate of T lymphocytes induced by different treatment groups against Tramp C1 was calculated. LDH detection was performed using a lactate dehydrogenase cytotoxicity detection kit (purchased from Beyotime, catalog number: C0017), and the cell killing rate (%) = (target cell effector cell mixed OD value - effector cell control well OD value - target cell control well OD value) ÷ (target cell maximum enzyme activity control well OD value - target cell control well OD value) × 100%.

[0138] Figure 2The results showed that the killing rates of lymphocytes induced by PBS, mRNA LNP-1, mRNA LNP-2, and mRNA LNP-3 groups against Tramp C1 were 16.7%, 19.7%, 41.7%, and 27.4% respectively when the number ratio was 20:1. The effector T cells induced by mRNA LNP-2 had the best killing effect on target cells (prostate cancer cells).

[0139] (3) Induction of human peripheral blood monocyte-derived dendritic cells (MoDC):

[0140] A1. Use fresh human platelet-rich buffy coat (taken from 400 mL of whole blood, with a volume of about 40-45 mL) to separate peripheral blood mononuclear cells from healthy donors using Ficoll density gradient centrifugation. In a 15 mL centrifuge tube, add 5 mL of buffy coat to the upper layer of 5 mL of Ficoll separation solution and centrifuge at 700 g for 25 min; gently aspirate the middle mononuclear cell layer and wash twice in PBS.

[0141] A2. Disperse the obtained PBMCs into 1640 medium containing 1% FBS and place them in a culture flask for adherent culture for 2 hours.

[0142] A3. Gently shake the culture flask to remove the non-adherent cells (human peripheral blood T lymphocytes). Add 1640 complete medium containing 10% FBS, 200 ng / mL GM-CSF, and 100 ng / mL IL-4 to the remaining adherent cells. Culture for 6 to 8 days, and replace half of the medium every other day to supplement with fresh cytokines.

[0143] A4. Plate MoDC cells in a 6-well plate, with 3×10 cells per well. 6 pcs, 2 mL of culture medium, the content of cytokines in the culture medium was maintained.

[0144] Add 1×10 6 MoDCs were cultured and 10 μg / mL mRNA LNP-1, 10 μg / mL mRNA LNP-2, 10 μg / mL mRNA LNP-3, and PBS were added to the MoDC culture medium for 24 h. The expression of costimulatory molecules CD80 and CD86 on the surface of MoDCs was detected by flow cytometry. The experimental results are shown in Figure 3 .

[0145] Figure 3 The results showed that PBS, mRNA LNP-1, mRNA LNP-2 and mRNA LNP-3 stimulated the production of CD80 + CD86 + The proportions of MoDC are 1.14%, 17.2%, 83.5% and 24% respectively.

[0146] Example 7

[0147] Validation of therapeutic subcutaneous tumor-bearing model in mice. Animal model selection and treatment time are shown in Figure 4 , the specific process is as follows:

[0148] The mRNA vaccines used in this example are mRNA LNP-1 prepared in Comparative Example 1, mRNA LNP-2 prepared in Example 2, and mRNA LNP-3 prepared in Comparative Example 2.

[0149] mRNA vaccine treatment of subcutaneous prostate cancer tumor model in mice: 20 6-8 week old male C57BL / 6 mice (purchased from Beijing Biotechnology Co., Ltd.) were selected and divided into 4 groups, 5 mice in each group. Each group was injected intramuscularly with PBS (140 μL), mRNA LNP-1 (20 μg), mRNA LNP-2 (20 μg), and mRNA LNP-3 (20 μg). 3×10 6 / Tramp C1 cells were used as day 0, and mRNA vaccine and PBS were given intramuscularly twice on days 14 and 28. The weight and tumor volume of mice were recorded every 5 days during the experiment. Mice were killed on day 40, and the tumor size and weight on day 40 were recorded. At the same time, the spleen and lymph nodes of mice were taken for detection of immune-related indicators, including immunohistochemistry, flow cytometry, Elispot and LDH detection.

[0150] according to Figure 5 The results showed that mRNA LNP-2 had the best tumor inhibition effect, with the mRNA LNP-2 group having the lowest tumor volume and mass, and no significant difference in body weight between mouse groups. Figure 6 The results showed that the proportion of infiltrating T lymphocytes and central memory T lymphocytes in the spleen of mice in the mRNA LNP-2 group was the largest.

[0151] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An mRNA, characterized in that The amino acid sequence of the mRNA is shown in SEQ ID NO.

2.

2. An mRNA vaccine preparation, characterized in that: The mRNA vaccine preparation comprises the mRNA of claim 1.

3. The mRNA vaccine preparation according to claim 2, characterized in that The carrier of the mRNA vaccine is lipid nanoparticles.

4. A method for preparing an mRNA vaccine preparation, characterized in that: The following steps are involved: Diluting the mRNA according to claim 1 to obtain an aqueous solution; Mix SM-102, DSPC, cholesterol and DMG-PEG2000 to obtain a lipid phase solution; The aqueous phase solution and the lipid phase solution are mixed to obtain an mRNA vaccine preparation.

5. The preparation method according to claim 4, characterized in that: The dilution concentration of the mRNA is 90-95 μg / mL; the molar ratio of SM-102, DSPC, cholesterol and DMG-PEG2000 is (45-55): (9-11): (35-40): (1-2); and the volume ratio of the aqueous phase solution to the lipid phase solution is 1-6:1-3.

6. Use of the mRNA described in claim 1, the mRNA vaccine preparation described in claim 2 or 3, or the mRNA vaccine preparation obtained by the preparation method described in claim 4 in the preparation of dendritic cells that mature and / or secrete proinflammatory cytokines.

7. Use of the mRNA according to claim 1, the mRNA vaccine preparation according to claim 2 or 3, or the mRNA vaccine preparation obtained by the preparation method according to claim 4 in the preparation of drugs for treating prostate cancer.

8. A method for preparing dendritic cells that mature and / or secrete pro-inflammatory cytokines, characterized in that: The method comprises the step of co-incubating the mRNA vaccine preparation according to claim 2 or 3 or the mRNA vaccine preparation obtained by the preparation method according to claim 4 with dendritic cells.

9. A method for preparing effector T cells, characterized in that: The method comprises the step of co-incubating the dendritic cells obtained by the preparation method according to claim 8 with T lymphocytes.

10. Use of the effector T cells obtained by the preparation method according to claim 9 in preparing drugs for treating prostate cancer.

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