A template DNA molecule and its application in the preparation of mRNA and vaccines

Abstract

The present invention relates to a template DNA molecule for in vitro transcription, the mRNA obtained by transcribing according to the template DNA molecule, its preparation method and the application of the mRNA in disease prevention and treatment, wherein the template DNA molecule sequentially includes a promoter, a 5'-untranslated region (5'UTR), a Kozak sequence, a polypeptide or protein coding region, a 3'-untranslated region (3'UTR) and a polyadenylic acid (polyA) sequence; through the promoter, the 5'-untranslated region, the Kozak sequence, the polypeptide or protein coding region, the 3'-untranslated region and the polyadenylic acid sequence can be co-transcribed to produce a co-transcript. The template DNA molecule or mRNA molecule provided by the present invention can be used to prepare vaccines and drugs for treating diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a template DNA molecule and its application in preparing mRNA and vaccines. Background Art

[0002] The design of the transcription template for mRNA vaccines is one of the key conditions for their successful production. Key design points include the upstream untranslated region (5'UTR) and downstream untranslated region (3'UTR) of the antigen coding region, as well as the polyadenylic acid (polyA) tail. Currently, patents for mRNA vaccine transcription template design are primarily held by foreign mRNA vaccine companies, and the polyA tail is often over 100 nucleotides long, making it very susceptible to recombination loss during template amplification using plasmids.

[0003] mRNA is unstable and susceptible to degradation. When designing mRNA vaccines, it is crucial to ensure the subsequent mRNA is highly stable. The structure of mRNA is closely related to its intracellular stability and expression efficiency. The untranslated text (UTRs) flanking the coding region are key regions in mRNA structure. The 5' UTR and its cap structure are crucial factors influencing mRNA stability and translation efficiency. The cap structure protects mRNA from nuclease degradation and participates in the binding of the transcription initiation complex in eukaryotes, thereby affecting translation efficiency. The 5' UTR primarily participates in the recognition and binding of the transcription initiation complex, affecting mRNA translation efficiency (Gray NK et al. 1998). Most of the instability of mRNA is located in the 3' UTR. The sequence and structure of the 3' UTR determine mRNA stability, localization, and expression (Schlake T et al., 2011; Goodarzi H et al., 2012). Besides the 5' cap, the 3' poly A tail is the most important factor in mRNA stability; most mRNA degradation begins at the poly A tail.

[0004] At present, the transcription templates of the new coronavirus mRNA vaccine internationally mostly use the UTRs of alpha and beta globin or UTRs modified based on them. BioNTech uses two tandem beta globin 3'UTRs and a polyA tail with 120 adenosine residues in between to enhance the stability of mRNA and has applied for related patents (US2019 / 0062762A1).

[0005] However, the above mRNA expression vectors are not stable enough, and excessively long polyA is prone to recombination loss. Summary of the invention

[0006] The present invention provides a new design strategy for mRNA vaccine transcription templates. Through this strategy, high-level mRNA expression can be achieved, plasmid amplification is stable, and the polyA tail is not easily recombinantly lost. Specifically,

[0007] In the first aspect of the present invention, there is provided a template DNA molecule, which includes a 5'-untranslated region (5'UTR), a coding region for a target polypeptide or protein, a 3'-untranslated region (3'UTR), and a polyadenylic acid (polyA) region, and the UTR sequence is derived from a UTR with translation function in the human genome.

[0008] Preferably, the length of the 5'UTR sequence is 20-70bp, and its sequence structure should be loose and not easily form a secondary structure.

[0009] Preferably, the length of the 5'UTR sequence is any range or any integer value within the range of 20-70bp, such as 22-68bp, 25-65bp, 28-65bp, 20, 22, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, etc.

[0010] Preferably, the 3'UTR sequence includes 50-400bp, and its sequence structure should avoid AU, AUUUA, or GU-rich sequences.

[0011] More preferably, the length of the 3'UTR sequence is any range or any integer value within the range of 50-400bp, such as 60-380bp, 70-350bp, 90-300bp, etc., such as 50, 55, 60, 67, 75, 90, 106, 130, 150, 175, 200, 220, 249, 275, 300, 320, 360, 375, 390, 400bp, etc.

[0012] More preferably, the 5'UTR sequence is as shown in any one of SEQ ID NO:3, 9-14, and the 3'UTR sequence is as shown in any one of SEQ ID NO:6, 15-23.

[0013] In a specific embodiment, the 5'UTR sequence is as shown in SEQ ID NO:3, and the 3'UTR sequence is as shown in any one of SEQ ID NO:6, 15-23.

[0014] In a specific embodiment, the 5’UTR sequence is as shown in any one of SEQ ID NO:3, 9-14, and the 3’UTR sequence is as shown in SEQ ID NO:6.

[0015] Preferably, the polyA is no more than 100bp. Any value of the polyA no more than 100bp, such as no more than 100, 95, 90, 85, 80, 79, 78, 76, 75, 72, 70, 68, 65, 63, 60, 58, 56, 54, 52, 50, 49, 48, 46, 45, 43, 41, 40, 39, 37, 35, 33, 31, 30, 29, 28, 25, 23, 20, 17, etc.

[0016] Preferably, the target polypeptide or protein is derived from any organism.

[0017] More preferably, the target polypeptide or protein is derived from a virus, archaea, prokaryote, eukaryote; even more preferably, the target polypeptide or protein is derived from a virus, such as varicella-zoster virus (VZV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rabies virus, respiratory syncytial virus, influenza virus, or the target polypeptide or protein is derived from an antigen related to animal or human diseases, such as allergen protein, autoantigen, tumor-specific antigen, etc.

[0018] In a specific embodiment, the target polypeptide or protein is the gE protein of varicella-zoster virus or its mutant; or the S protein of severe acute respiratory syndrome coronavirus 2 or its mutant, the G protein of rabies virus CTN-1 strain (RVG) or its variant, the F protein of respiratory syncytial virus or its mutant, the HA protein of influenza virus or its mutant, allergen protein, autoantigen, tumor-specific antigen, etc.

[0019] In a specific embodiment, the coding sequence of the gE protein of varicella-zoster virus is as shown in SEQ ID NO:25, or a variant of SEQ ID NO:25, and the variant has the function of encoding the gE protein.

[0020] In a specific embodiment, the coding sequence of the S protein of severe acute respiratory syndrome coronavirus 2 is as shown in SEQ ID NO:26, or a variant of SEQ ID NO:26, and the variant has the function of encoding the S protein of severe acute respiratory syndrome coronavirus 2.

[0021] Preferably, the coding region further includes a reporter gene. More preferably, the reporter gene includes but is not limited to chloramphenicol acetyltransferase gene (CAT), human growth hormone gene (hGH), secreted alkaline phosphatase gene (SEAP), red fluorescent protein gene (RFP), green fluorescent protein gene (GFP), enhanced green fluorescent protein gene (EGFP), β-galactosidase gene (β-Gal), or firefly luciferase gene. Even more preferably, the reporter gene is EGFP or GFP.

[0022] Preferably, the DNA molecule further includes a 5' cap structure. More preferably, the 5' cap structure sequence is a standard cap structure m7G(5')ppp(5')G or a 3'-O-Me-m7G(5')ppp(5')G anti-reverse cap structure or a modified cap structure, and the modified cap structure includes m7G(5')ppp(5')(2'OMeA)pG, m7G(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeG)pG, m7G(5')ppp(5')(2'OMe,m6A)pG, m7G(3'OMeG)(5')ppp(5')(2'OMeG)pG, etc.

[0023] Preferably, the template DNA molecule further includes a Kozak sequence. More preferably, the Kozak sequence is located upstream of the polypeptide or protein coding region. Even more preferably, the Kozak sequence includes GCCACC.

[0024] In the second aspect of the present invention, an mRNA molecule is provided. The mRNA molecule includes a 5' untranslated region (5'UTR), a polypeptide or protein coding region, a 3' untranslated region (3'UTR), and a polyadenylate (polyA) region, and the UTR sequence is derived from a UTR with translational function in the human genome.

[0025] Preferably, the mRNA molecule is transcribed from the above template DNA molecule.

[0026] Preferably, the mRNA molecule can be natural or modified RNA; more preferably, the RNA can be modified by partially or completely replacing natural uridine with a modified uridine; even more preferably, natural uridine can be completely replaced with 1-methyl-pseudouridine.

[0027] In the third aspect of the present invention, a vector is provided. The vector includes the above template DNA molecule.

[0028] Preferably, the above vector can be circular DNA or linear DNA.

[0029] More preferably, the above-mentioned circular DNA includes a replication sequence (ori), a selection marker (e.g., kanamycin, ampicillin, chloramphenicol or other antibiotic resistance genes), a promoter for transcribing mRNA, and a DNA sequence encoding the above-mentioned mRNA molecule. The promoter can be a T3 promoter, a T7 promoter, an SP6 promoter, etc.; specifically, it can be a T7 promoter.

[0030] More preferably, the above-mentioned linear DNA can be obtained by digesting the above-mentioned circular DNA with a restriction endonuclease, or can be obtained by PCR amplification using the above-mentioned DNA molecule as a template.

[0031] In the fourth aspect of the present invention, there is provided a method for preparing the above-mentioned mRNA molecule, and the preparation method includes transcribing using the above-mentioned template DNA molecule.

[0032] Preferably, the preparation method further includes a capping step to cap the mRNA. More preferably, the capping step includes a co-transcriptional capping or an enzymatic capping step.

[0033] The co-transcriptional capping reaction system (20 μl) is as follows: 0.1 - 2 μg of the above-mentioned DNA molecule, 2 - 4 μl of T7 EnzymeMix, 2 - 4 μl of 5×Reaction buffer, 1.6 μl each of nucleotides (ATP, CTP, GTP, PseudoUTP) (each individual concentration is 100 mM), 1.6 μl of a cap structure (100 mM), and nuclease-free water is added to make up to 20 μl. After mixing, incubate at 37 °C for 0.5 - 6 hours.

[0034] The enzymatic capping reaction is carried out in two steps. The first step is in vitro transcription, and the second step is in vitro capping.

[0035] Specifically, the enzymatic capping system (20 μl) is as follows: 0.1 - 2 μg of the above DNA molecule, 2 μl of mScript T7 Enzyme Solution, 2 μl of 10×mScript T7 Transcription Buffer, 2 μl of DTT (100 mM), 0.5 μl of ScriptGuard RNase Inhibitor, 7.2 μl of NTP solution, and made up to 20 μl with RNase-free water. After mixing, incubate at 37°C for 0.5 - 4 hours. Then, add 1 μl of RNase-Free DNaseI, mix well, and incubate at 37°C for 0.5 - 2 hours. After the reaction is completed, purify the mRNA by ammonium acetate or magnetic beads, dissolve the purified mRNA in 72 μl of RNase-Free water, incubate at 65°C for 5 - 15 minutes, and then transfer to ice for standby. The second-step capping system (100 μl) is as follows: 72 μl of purified mRNA, 2 μl of 10×Scriptcap Capping Buffer, 5 μl of GTP (20 mM), 2.5 μl of SAM (20 mM), 2.5 μl of ScriptGuard RNase Inhibitor, 4 μl of Scriptcap 2’-O-Methyltransferase (100 U / μl), 4 μl of Scriptcap Capping Enzyme (10 U / μl). After mixing, incubate at 37°C for 0.5 - 2 hours.

[0036] In the fifth aspect of the present invention, there is provided a use of the above template DNA molecule, mRNA molecule or vector in expressing a target polypeptide or protein, and the target polypeptide or protein is derived from any organism.

[0037] Preferably, it is derived from a virus, archaea, prokaryote, eukaryote. More preferably, the organism is derived from a virus, such as varicella-zoster virus (VZV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rabies virus, respiratory syncytial virus, influenza virus. Alternatively, the target polypeptide or protein is derived from an antigen related to an animal or human disease, such as an allergen protein, autoantigen, tumor-specific antigen.

[0038] In a specific embodiment, the target polypeptide or protein includes the gE protein or its mutant of varicella-zoster virus, the S protein or its mutant of severe acute respiratory syndrome coronavirus 2, the G protein (RVG) or its variant of rabies virus CTN-1 strain, the F protein or its mutant of respiratory syncytial virus, the HA protein or its mutant of influenza virus, an allergen protein, an autoantigen, a tumor-specific antigen, etc.

[0039] More preferably, the tumor-specific antigen includes specific antigens from any tumor, OX40L, KRAS, TP53, and the like.

[0040] In the sixth aspect of the present invention, there is provided a use of the above template DNA molecule, mRNA molecule or vector in the preparation of a vaccine.

[0041] Preferably, the vaccine is used for preventing or treating diseases related to polypeptides or proteins, such as diseases caused by viruses, archaea, prokaryotes, eukaryotes, etc. More preferably, the diseases are caused by viral infections, such as varicella-zoster virus (VZV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rabies virus, respiratory syncytial virus, influenza virus, and the like. Alternatively, the diseases are allergic diseases, autoimmune diseases or tumors, and the like.

[0042] In the seventh aspect of the present invention, there is provided an mRNA vaccine, which includes the above mRNA molecule.

[0043] Preferably, the mRNA vaccine encodes the gE protein of varicella-zoster virus or a mutant thereof.

[0044] More preferably, the mRNA vaccine is used for preventing varicella-zoster virus infection.

[0045] Preferably, the mRNA vaccine encodes the S protein of severe acute respiratory syndrome coronavirus 2 or a mutant thereof.

[0046] More preferably, the mRNA vaccine is used for preventing severe acute respiratory syndrome coronavirus 2 infection.

[0047] Preferably, the mRNA vaccine further includes a delivery system. Preferably, the delivery system includes cationic lipid nanoparticles, cationic liposomes or cationic polymers, etc. More preferably, the delivery system is cationic lipid nanoparticles (LNP).

[0048] Preferably, the LNP components include protonatable cationic lipids, structural lipids, helper lipids and PEG lipids.

[0049] Preferably, the molar ratio of the protonatable cationic lipid is 50-65%, the molar ratio of the structural lipid is 30-40%, the molar ratio of the helper lipid is 4-10%, and the molar ratio of the PEG lipid is 0.5-2%.

[0050] Preferably, the mass ratio of the protonatable cationic lipid to mRNA is (8-10):1.

[0051] Preferably, the protonatable cationic lipid includes SM-102, ALC-0315, Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA, C12-200, DlinDMA, etc., and preferably SM-102.

[0052] Preferably, the structural lipid includes cholesterol, cholesterol ester, steroid hormone, steroid vitamin, bile acid, etc., and preferably cholesterol.

[0053] Preferably, the helper lipid includes DSPC, DOPE, DOPC, DOPS, etc., and preferably DSPC.

[0054] Preferably, the PEG lipid includes ALC-0159, DMG-PEG2000, PEG-DSPE, etc., and preferably DMG–PEG2000.

[0055] In the eighth aspect of the present invention, a method for preparing the above mRNA vaccine is provided, and the preparation method includes encapsulating the above mRNA molecule in a delivery system.

[0056] The delivery system is as defined above.

[0057] Preferably, the delivery system is LNP, and the preparation method for LNP to encapsulate mRNA includes,

[0058] 1) Dissolve mRNA in a buffer solution, the buffer solution is acetic acid buffer solution or citric acid buffer solution, the pH value is 3-6, preferably acetic acid buffer solution, and the mRNA concentration is 0.05-1 mg / ml.

[0059] 2) Dissolve the protonatable cationic lipid, structural lipid, helper lipid and PEG lipid in an organic solution according to the formula ratio to obtain an organic phase. Preferably, the organic solution is C1-C4 lower carbon alcohol, and preferably ethanol.

[0060] 3) The volume ratio of the aqueous phase to the organic phase is 3:1.

[0061] 4) Use a microfluidic device to mix the aqueous phase and the organic phase, and the flow rate is not less than 3 ml / min.

[0062] 5) After mixing, dilute and ultrafiltrate and concentrate 10-50 times with a dilution buffer solution, and the dilution buffer solution is physiological saline, PBS, Tris buffer solution, etc., and preferably Tris buffer solution.

[0063] More preferably, the mRNA vaccine further includes a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be a carrier, diluent, adjuvant or adjuvant-encoding nucleotide sequence, solubilizer, binder, lubricant, suspending agent, transfection promoter, etc.

[0064] More preferably, the transfection promoter includes, but is not limited to, surfactants such as immunostimulating complexes, Freunds incomplete adjuvant, LPS analogs (such as monophosphoryl lipid A), muramyl peptides, benzoquinone analogs, squalene, hyaluronic acid, lipids, lipids, calcium ions, viral proteins, cations, polycations (such as poly-L-glutamic acid (LGS)), or nanoparticles or other known transfection promoters.

[0065] The nucleotide sequence encoding the adjuvant is a nucleotide sequence encoding at least one of the following adjuvants: GM-CSF, IL-17, IFNg, IL-15, IL-21, anti-PD1 / 2, lactoferrin, protamine, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-α, INF-γ, Lymphotoxin-α, hGH, MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF,, CD40, CD40L, vascular growth factor, fibroblast growth factor, nerve growth factor, vascular endothelial growth factor, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAPK, SAP-1, JNK, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANKLIGAND, Ox40, Ox40LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof.

[0066] In a ninth aspect of the present invention, a method for treating or preventing a disease is provided, the method comprising administering the mRNA molecule or mRNA vaccine to an individual.

[0067] Preferably, the diseases include diseases related to the target polypeptide or protein, such as diseases caused by viruses, archaea, prokaryotes, eukaryotes, etc. More preferably, the diseases are infections caused by viruses, such as varicella-zoster virus (VZV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rabies virus, respiratory syncytial virus, influenza virus, and so on. Alternatively, the diseases are allergic diseases, autoimmune diseases, tumors, and so on.

[0068] The beneficial effects of the present invention are as follows:

[0069] 1. For the improved template DNA molecule, by screening the 5'UTR and 3'UTR according to certain principles, such as defining the length and sequence structure of the UTR, the expression efficiency of mRNA can be improved, and various target polypeptides or proteins can be efficiently expressed, including, for example, target polypeptides or proteins derived from viruses, eukaryotes, etc.

[0070] 2. With a polyA not exceeding 100A paired with the UTR backbone, a polyA as short as 40A can effectively express the target protein sequence, thus avoiding the adverse effect of easy recombination and loss of polyA during the amplification process;

[0071] 3. The template DNA molecule, mRNA molecule, or vector of the present invention can be used to prepare DNA vaccines or mRNA vaccines, which can efficiently express antigens and produce high-titer antibodies, indicating that the vaccines of the present invention can effectively produce humoral and cellular immune protection.

[0072] 4. The template DNA molecule, mRNA molecule, or vector of the present invention can be used to prepare or express drugs for target polypeptides or proteins.

[0073] The above only summarizes some aspects of the present invention and should not be considered as limiting the present invention in any aspect.

[0074] All patents and publications mentioned in this specification are incorporated into the present invention by reference as a whole. Those skilled in the art should recognize that certain changes can be made to the present invention without departing from the concept or scope of the present invention.

[0075] The following examples further illustrate the present invention in detail and should not be considered as limiting the present invention or the scope of the specific methods described in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings, wherein:

[0077] Figure 1 Shown is a schematic diagram of the design of the mRNA vaccine transcription template;

[0078] Figure 2 The expression results of different 5’UTR and 3’UTR combinations are shown;

[0079] Figure 3 The expression results with the 5’UTR and 3’UTR of beta globin as a control are shown;

[0080] Figure 4 The fluorescence rate percentages of cells after transfection for 24 h with different 5’UTR replacements are shown;

[0081] Figure 5 The fluorescence rate percentages of cells after transfection for 24 h with different 3’UTR replacements are shown;

[0082] Figure 6 The expression results of different lengths of polyA are shown;

[0083] Figure 7 The gE-specific antibody results 28 days after immunization with the varicella-zoster gE protein mRNA vaccine are shown;

[0084] Figure 8 The activation levels of IFN-γ and IL-2 28 days after immunization with the varicella-zoster gE protein mRNA vaccine are shown;

[0085] Figure 9 The S-protein specific antibody results 28 days after immunization with the SARS-CoV-2 S protein mRNA vaccine are shown. Specific implementation manners

[0086] [[ID=3…]]The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions fall within the protection scope of the present invention.

[0087] In each of the following embodiments, the main equipment and materials are obtained from the several companies indicated below:

[0088] Table 1: Main equipment

[0089]

[0090] Table 2: Main materials

[0091] [[ID=5…]]

[0092] Example 1: mRNA preparation template construction strategy

[0093] In the solution of the present invention, the DNA molecule used as the template for mRNA preparation sequentially includes a promoter, a 5'UTR sequence, a Kozak sequence, an antigen coding region (CDS) sequence, a 3'UTR sequence, and a polyadenylic acid (polyA) sequence; under the drive of the promoter, the 5'UTR sequence, the Kozak sequence, the antigen coding region sequence, the 3'UTR sequence, and the polyadenylic acid (polyA) sequence can be co-transcribed to generate a co-transcript. As Figure 1 shown.

[0094] The specific construction and mRNA preparation methods include:

[0095] I. In vitro transcription

[0096] The nucleic acid sequence composed of the promoter, 5'UTR sequence, Kozak sequence, antigen coding region (CDS) sequence, 3'UTR sequence, and polyadenylic acid (polyA) sequence in series is synthesized onto the pUC57-kana vector (Nanjing GenScript Biotechnology Co., Ltd.), and the pUC57-kana vector is linearized by EcoRI and BspQI. The EcoRI and BapQI restriction enzyme sequences are not retained during synthesis, and the BapQI restriction enzyme sequence is contained downstream of the polyA sequence of the synthesized sequence. The synthesized plasmid is amplified by Escherichia coli. When preparing the in vitro transcription template, the in vitro transcription template can be generated by digesting the plasmid with BspQI, or can be obtained by PCR amplification using the synthesized plasmid as the template. In the present invention, the in vitro transcription template is prepared by digesting the plasmid with BspQI.

[0097] II. In vitro transcription

[0098] After the preparation of the in vitro transcription template is completed, the co-transcription capping method is used for mRNA preparation.

[0099] The method for preparing mRNA by co-transcription capping is as follows:

[0100] The co-transcription capping reaction system (20 μl) is: 0.1 - 2 μg of the above DNA molecule, 2 - 4 μl of T7 EnzymeMix, 2 - 4 μl of 5×Reaction buffer, 1.6 μl each of nucleotides (ATP, CTP, GTP, PseudoUTP) (each single concentration is 100 mM), 1.6 μl of the cap structure (100 mM), and RNase-free water is added to make up to 20 μl. After mixing, it is incubated at 37°C for 0.5 - 6 hours.

[0101] Capping can also be carried out by enzymatic capping reaction.

[0102] The enzymatic capping reaction is carried out in two steps, the first step is in vitro transcription, and the second step is in vitro capping.

[0103] The specific enzymatic capping system (20 μl) is as follows:

[0104] In vitro transcription:

[0105] 0.1 - 2 μg of the above DNA molecule, 2 μl of mScript T7 Enzyme Slution, 2 μl of 10×mScriptT7Transcription Buffer, 2 μl of DTT (100 mM), 0.5 μl of ScriptGuard RNase Inhibitor, 7.2 μl of NTP solution, and make up to 20 μl with RNase-free water. After mixing, incubate at 37 °C for 0.5 - 4 hours. Then, add 1 μl of RNase-Free DNaseI, mix well and incubate at 37 °C for 0.5 - 2 hours. After the reaction is completed, purify the mRNA by ammonium acetate or magnetic beads, dissolve the purified mRNA in 72 μl of RNase-Free water, incubate at 65 °C for 5 - 15 minutes and then transfer to ice for standby.

[0106] In vitro capping system (100 μl): 72 μl of purified mRNA, 2 μl of 10×Scriptcap Capping Buffer, 5 μl of GTP (20 mM), 2.5 μl of SAM (20 mM), 2.5 μl of ScriptGuard RNase Inhibitor, 4 μl of Scriptcap2’-O-Methyltransferase (100 U / μl), 4 μl of Scriptcap Capping Enzyme (10 U / μl).

[0107] III. Detection of mRNA expression

[0108] After mRNA expression, it is detected by fluorescence observation or immunological methods. The detection methods are as follows:

[0109] If the coding region includes a reporter gene, the expression of the target polypeptide or protein can be detected by detecting the protein encoded by the reporter gene. For example, when the protein encoded by the reporter gene is enhanced green fluorescent protein (EGFP), the expression of green fluorescent protein can be observed by a fluorescence microscope, and the proportion of cells expressing green fluorescent protein can be analyzed by a flow cytometer;

[0110] If the coding region does not include a reporter gene, the expression of the polypeptide or protein can be detected by ELISA method.

[0111] The specific method is as follows:

[0112] 1. Seed HEK293 cells into a 12-well plate, and control the cell number at 200,000 cells / well.

[0113] 2. Observe the 12-well plate after culturing in a cell incubator at 37 °C for 12 - 16 hours. When the cell density reaches 60 - 80% confluence, mRNA transfection can be carried out.

[0114] 3. Transfect the mRNA into HEK293 cells using Lipofectamine MessengerMAX transfection reagent. The ratio of the volume of the transfection reagent to the mass of the mRNA is 3 μl:1 μg. The specific operation is carried out according to the transfection reagent instruction manual. Culture at 37 °C in a cell incubator for 24 hours.

[0115] 4. After 24 hours of transfection, directly observe the green fluorescence expression of the cells in the 12-well plate under a fluorescence microscope and take pictures.

[0116] 5. When analyzing by flow cytometry, discard the cell culture medium, wash the cells once with PBS, add 100 μl of trypsin to digest the cells, and then neutralize the trypsin digestion with 500 μl of complete medium. Transfer the cell resuspension to a 1.5 ml centrifuge tube, centrifuge at 1000 g for 2 minutes in a centrifuge, discard the supernatant, resuspend the cells with 500 μl of PBS, and then the cells can be loaded onto the flow cytometer for detection. Set the cells without transfected mRNA as the negative control group. Detect the negative control cells using a flow cytometer and circle the target cell signal on the dot plot. Read the FITC fluorescence signal using a histogram, and adjust the detector sensitivity and voltage to determine the range of the green fluorescence positive cell fluorescence signal on the histogram, and gate accordingly. Detect each sample in turn and record the percentage of green fluorescence positive cells. Read 10,000 signals for each sample.

[0117] 6. When detecting antigen expression by ELISA method, discard the cell culture medium, wash the cells once with PBS, add 100 μl of trypsin to digest the cells, and then neutralize the trypsin digestion with 500 μl of complete medium. Transfer the cell resuspension to a 1.5 ml centrifuge tube, centrifuge at 1000 g for 2 minutes in a centrifuge, discard the supernatant, add 150 μL of NP - 40 cell lysis buffer to resuspend the cells, and lyse on ice for 10 - 30 minutes. Centrifuge at 12,000 rpm at 4 °C for 3 min, take the supernatant, and perform the subsequent ELISA experiment. Add the cell lysate supernatant to the enzyme - labeled plate pre - coated with the primary antibody, and incubate at room temperature for 2 hours. After incubation, wash the plate 3 times with 0.05% PBST washing solution, 300 μL / well, and pat dry with a clean paper. Add the HRP - labeled secondary antibody and incubate at room temperature for 1 hour. After incubation, wash the plate 4 times with 0.05% PBST washing solution, 300 μL / well, and pat dry with a clean paper. Add the ELISA chromogenic solution for color development, 100 μL / well, and incubate at room temperature for 10 min. After color development, add 50 μL / well of the termination solution, detect at a wavelength of 450 nm and a reference wavelength of 630 nm, and record the OD value.

[0118] Example 2: Screening of different 5'UTR and 3'UTR sequences

[0119] Design different 5'UTR and 3'UTR to screen for suitable sequences to express the target antigen CDS. Other references are made to Example 1.

[0120] Screen for suitable UTR combinations from the human genome, as shown below, where

[0121] 1. ZF2101

[0122] 5'UTR (HLA-A): SEQ ID NO:1, 3'UTR (HLA-A): SEQ ID NO:2.

[0123] 2. ZF2102

[0124] 5'UTR (HLA-DMA): SEQ ID NO:3, 3'UTR (HLA-DMA): SEQ ID NO:4.

[0125] 3. ZF2103

[0126] 5'UTR (HLA-A): SEQ ID NO:1, 3'UTR (HLA-DMA): SEQ ID NO:4.

[0127] 4. ZF2104

[0128] 5'UTR (HLA-F): SEQ ID NO:5, 3'UTR (HLA-F): SEQ ID NO:6.

[0129] 5. ZF2105

[0130] 5'UTR (HLA-DMA): SEQ ID NO:3, 3'UTR (HLA-F): SEQ ID NO:6.

[0131] Use the 5'UTR and 3'UTR of beta globulin as a control,

[0132] 5'UTR (beta globulin): SEQ ID NO:7, 3'UTR (beta globulin): SEQ ID NO:8.

[0133] The CDS selects the reporter gene dEGFP, and this sequence contains a degradation signal, reducing the half-life of EGFP from 17 hours to 2 hours (X Li, X Zhao, Y Fang, et al., 1998; S Holtkamp, S Kreiter, A Selmi, et al., 2006.).

[0134] The length of polyA is 17 bp.

[0135] The immunofluorescence results are as Figure 2 and 3 shown, where Figure 2 the immunofluorescence results of ZF2101, ZF2102, ZF2103, ZF2104, and ZF2105 are respectively, Figure 3 and that of the control. The results show that the fluorescence expression efficiency of ZF2105 is close to that of the control group, and the expression effect is the best.

[0136] Using ZF2105 as the backbone, the polyA sequence was replaced with A40+GTGAGTCTTC+A60, named ZF2105.1. Then, based on ZF2105.1, the 5'UTR sequence and 3'UTR sequence were replaced respectively to screen other available 5'UTR sequences and 3'UTR sequences.

[0137] Table 3: Replaced 5'UTR

[0138] 5'UTR Name Sequence Number Sequence (5ˊ-3ˊ) [[ID=—]]CLCN6 5UTR1 SEQ ID NO:9 NADK 5UTR2 SEQ ID NO:10 DFFA 5UTR3 SEQ ID NO:11 EXOSC10 5UTR4 SEQ ID NO:12 CCDC27 5UTR5 SEQ ID NO:13 DFFA 5UTR6 SEQ ID NO:14

[0139] Replace the 5'UTR in ZF2105.1 with the 5'UTR in Table 3 to obtain multiple backbones, express dEGFP in them, and detect after transfection for 24 hours according to the above method.

[0140] The results are as Figure 4 and shown in Table 4. The fluorescence cell ratio of each of the above backbones is comparable to that of ZF2105.1, and there is no significant difference statistically (p>0.001), and they can be used to express polypeptides or proteins.

[0141] Table 4: Percentage of cell fluorescence rate after 5'UTR replacement

[0142] 5'UTR Name Sequence Number Repeat 1 Repeat 2 Repeat 3 Average p value Positive Control ZF2105.1 61.18％ 61.35％ 59.29％ 60.61％ CLCN6 5UTR1 56.64％ 58.59％ 56.10％ 57.11％ 0.0253 NADK 5UTR2 60.48％ 59.57％ 61.75％ 60.60％ 0.9945 DFFA 5UTR3 55.08％ 52.87％ 53.79％ 53.91％ 0.0019 EXOSC10 5UTR4 54.71％ 58.77％ 57.80％ 57.09％ 0.0649 CCDC27 5UTR5 55.55％ 53.46％ 50.57％ 53.19％ 0.0095 DFFA 5UTR6 53.14％ 53.91％ 55.63％ 54.23％ 0.003

[0143] Table 5: Replaced 3'UTR

[0144] 3'UTR Name Sequence Number Sequence (5ˊ-3ˊ) SAMD11 3UTR1 SEQ ID NO:15 HES4 3UTR2 SEQ ID NO:16 ISG15 3UTR3 SEQ ID NO:17 TTLL10 3UTR4 SEQ ID NO:18 AURKAIP1 3UTR5 SEQ ID NO:19 MIB2 3UTR6 SEQ ID NO:20 MMP23B 3UTR7 SEQ ID NO:21 CDK11B 3UTR8 SEQ ID NO:22 TMEM52 3UTR9 SEQ ID NO:23

[0145] Replace the 3'UTR in ZF2105.1 with the 3'UTR in Table 5 to obtain multiple backbones, express dEGFP in them, and detect after transfection for 24 hours according to the above method.

[0146] The results are as Figure 5 and shown in Table 6. The fluorescence cell ratio of each of the above backbones is close to or significantly higher than that of ZF2105.1, and they can be used to express polypeptides or proteins.

[0147] Table 6: Percentage of cell fluorescence rate after 3'UTR replacement

[0148] 3'UTR Name Sequence Number Repeat 1 Repeat 2 Repeat 3 Average p value Positive Control ZF2105.1 61.18％ 61.35％ 59.29％ 60.61％ SAMD11 3UTR1 67.02％ 64.13％ 65.03％ 65.39％ 0.0114 HES4 3UTR2 61.99％ 60.34％ 58.21％ 60.18％ 0.7552 ISG15 3UTR3 57.32％ 60.10％ 61.95％ 59.79％ 0.6148 TTLL10 3UTR4 69.79％ 61.20％ 70.35％ 67.11％ 0.0986 AURKAIP1 3UTR5 79.23％ 74.99％ 74.71％ 76.31％ 0.0006 MIB2 3UTR6 67.15％ 67.70％ 63.62％ 66.16％ 0.0182 MMP23B 3UTR7 58.10％ 58.76％ 59.66％ 58.84％ 0.0918 CDK11B 3UTR8 76.46％ 65.81％ 73.81％ 72.03％ 0.025 TMEM52 3UTR9 66.61％ 64.28％ 63.71％ 64.87％ 0.0183

[0149] Example 3: Screening of polyA with different lengths

[0150] Based on the UTR combination of ZF2105, the length of polyA was screened.

[0151] Three groups of polyA with different lengths were selected for screening, 40 consecutive A (A40), 60 consecutive A (A60), 100 A (A100), where A100 is 40 consecutive A and 60 consecutive A in series with an interval of 10 bases in the middle, and the structure of the 10 bases of the interval is GTGAGTCTTC (SEQ ID NO: 24). The control used untransfected cells.

[0152] Others are as in Example 2.

[0153] The results are as Figure 6 shown, indicating that there is no significant difference in the fluorescence ratio of the three experimental groups. When using the ZF2105 UTR backbone, a shorter polyA length can be used, such as A40, and the percentage of fluorescent cells can reach more than 80%, while the percentage of fluorescent cells with A60 can reach more than 90%, and basically the expression effect of A100 can be achieved.

[0154] After 25 passages in Escherichia coli, the plasmid remained stable, and the performance of the mRNA prepared by in vitro transcription of the prepared template was stable, and the percentage of fluorescent cells could reach more than 80%.

[0155] Example 4: Immunogenicity of varicella-zoster gE protein mRNA vaccine

[0156] The immunological activity of the varicella-zoster gE protein mRNA vaccine (ZF006) was detected by using the ZF2105 UTR backbone in combination with A60, where the coding region of the gE protein was optimized based on the original sequence (Gene ID: 1487709), and the sequence was as SEQ ID NO: 25.

[0157] mRNA was encapsulated with LNP, and the preparation method included,[[]]

[0158] 1) Dissolve the mRNA in a buffer solution, which is acetic acid buffer solution or citric acid buffer solution, with a pH value of 3-6, preferably acetic acid buffer solution, and the mRNA concentration is 0.05-1 mg / ml.

[0159] 2) Dissolve the protonatable cationic lipid, structural lipid, co-lipid, and PEG lipid in an organic solution according to the formulation ratio to obtain an organic phase. Preferably, the organic solution is a C1-C4 lower alcohol, preferably ethanol.

[0160] 3) The volume ratio of the aqueous phase to the organic phase is 3:1.

[0161] 4) Mix the aqueous phase and the organic phase using a microfluidic device with a flow rate not less than 3 ml / min.

[0162] 5) After mixing, dilute and ultrafiltrate and concentrate 10 - 50 times with a dilution buffer, which is physiological saline, PBS, Tris buffer, etc., preferably Tris buffer.

[0163] BALB / C mice at 6 - 8 weeks of age were subcutaneously immunized with varicella virus one month in advance to establish a model, and one month later, were intramuscularly injected with the herpes zoster gE protein mRNA vaccine at an immunization dose of 5 μg / 100 μl / mouse, with a 28-day interval between two injections. 28 days after the two injections in the mice, gE-specific antibodies were detected in the serum by ELISA method.

[0164] The ELISA detection method is as described in Example 1. The enzyme-linked immunosorbent assay plate was coated with gE protein, then the serum of the immunized mice was added and incubated, followed by incubation with HRP-labeled secondary antibody and addition of substrate for color development.

[0165] The results are as Figure 7 shown. The gE-specific antibody titer of the vaccine of the present invention can reach 1.77×10 6 , obtaining a high level of gE-specific antibodies. The vaccine of the present invention does not contain an adjuvant to assist the body's activation, but the antibody level produced is comparable to that of the commercially available GSK herpes zoster vaccine containing an adjuvant.

[0166] Meanwhile, the activation levels of IFN-γ and IL-2 secreted by CD4+ T cells 28 days after the second immunization were detected. Among them, the injection of physiological saline was used as the control group (NEG).

[0167] The results are as Figure 8 shown. In terms of cellular immunity, the activation levels of IFN-γ and IL-2 are significantly higher than those of the control commercially available GSK herpes zoster vaccine, reaching 18975 and 20677 respectively.

[0168] The above results indicate that using the ZF2105 UTR backbone in combination with A60 as the mRNA vaccine backbone to express the gE antigen can effectively activate humoral immunity and cellular immunity.

[0169] Example 5: Immunogenicity of the mRNA Vaccine against the Spike Protein of the Omicron Strain of SARS-CoV-2

[0170] The immunological activity of the novel coronavirus S protein mRNA vaccine (ZF008) was detected using the ZF2105 UTR backbone in combination with A100. Among them, the coding region of the novel coronavirus S protein was optimized based on the original sequence (Gene ID: 43740568), and the sequence is as shown in SEQ ID NO: 26.

[0171] The vaccine preparation method refers to Example 4.

[0172] BALB / C mice aged 4 - 6 weeks were immunized at a dose of 3 μg / 100 μl per mouse, and the two injections were spaced 28 days apart. 28 days after the two - injection immunization of the mice, S - protein - specific antibodies were detected in the serum by ELISA as described above.

[0173] The results are as Figure 9 shown. The titer of the S - protein - specific antibody of the vaccine of the present invention can reach 6.4×10 6 , and a high level of S - protein - specific antibody was obtained (see Figure 9 the left figure); the results of the pseudovirus neutralization experiment showed that the ID50 value of the post - immunization serum against the Omicron - strain pseudovirus was greater than 65610 (see Figure 9 the right figure).

[0174] In terms of cellular immunity, there was also good activation, and the activation levels of IFN - γ and IL - 2 could reach 14169 and 20187 respectively.

[0175] The above results indicate that using the ZF2105 UTR backbone in combination with A100 as the mRNA vaccine backbone to express the novel coronavirus S protein antigen can effectively activate humoral immunity and cellular immunity.

[0176] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above - mentioned embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0177] In addition, it should be noted that, among the various specific technical features described in the above - mentioned specific embodiments, they can be combined in any suitable way without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0178] Furthermore, any combination can be made between different embodiments of the present invention as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A template DNA molecule, characterized in that, the template DNA molecule comprises a 5'-untranslated region (5'UTR), a coding region for a target polypeptide or protein, a 3'-untranslated region (3'UTR), and a polyadenylate (polyA) region. The UTR sequences are derived from the UTRs with translation function in the human genome. The 5'UTR sequence is as shown in any one of SEQ ID NO: 3, 9-14, and the 3'UTR sequence is as shown in any one of SEQ ID NO: 6, 15-23.

2. The template DNA molecule according to any one of claim 1, characterized in that, the polyA does not exceed 100 bp.

3. The template DNA molecule according to any one of claims 1-2, characterized in that, The polyA includes A40, A60 or A100, wherein A100 is 40 consecutive As and 60 consecutive As in series with an interval of 10 bases in the middle, and the structure of the 10 intervening bases is as shown in GTGAGTCTTC.

4. The template DNA molecule according to any one of claims 1-2, characterized in that, the template DNA molecule further comprises a 5' cap structure.

5. The template DNA molecule according to claim 4, wherein The 5' cap structure sequence is a standard cap structure or a modified cap structure.

6. The template DNA molecule according to any one of claims 1-2, characterized in that, the target polypeptide or protein is derived from any organism.

7. The template DNA molecule according to any one of claims 1-2, wherein the target polypeptide or protein is derived from a virus, a prokaryote, or a eukaryote.

8. The template DNA molecule according to claim 6, wherein the organism is derived from varicella-zoster virus (VZV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rabies virus, respiratory syncytial virus, influenza virus.

9. The template DNA molecule according to claim 6, wherein the target polypeptide or protein comprises the gE protein or its mutant of varicella-zoster virus, the S protein or its mutant of severe acute respiratory syndrome coronavirus 2, the G protein or its variant of rabies virus CTN-1 strain, the F protein or its mutant of respiratory syncytial virus, the HA protein or its mutant of influenza virus.

10. An mRNA molecule, characterized in that, the mRNA molecule is transcribed from the template DNA molecule according to any one of claims 1-9.

11. The mRNA molecule according to claim 10, characterized in that, the mRNA molecule is natural or modified RNA.

12. A vector, characterized in that, the vector comprises the template DNA molecule according to any one of claims 1-9.

13. A method for preparing the mRNA molecule according to any one of claims 10-11, characterized in that, the preparation method comprises transcribing the template DNA molecule according to any one of claims 1-9.

14. The preparation method according to claim 13, characterized in that, the preparation method further comprises a capping step of capping the mRNA.

15. The preparation method according to claim 14, characterized in that, The capping step comprises a co-transcriptional capping or an enzymatic capping step.

16. Use of the template DNA molecule according to any one of claims 1-9, the mRNA molecule according to any one of claims 10-11, or the vector according to claim 12 in the expression of a target polypeptide or protein.

17. The use according to claim 16, wherein the target polypeptide or protein is derived from any organism.

18. The use according to any one of claims 16-17, wherein the target polypeptide or protein is derived from a virus, a prokaryote, or a eukaryote.

19. The use according to claim 17, wherein the organism is derived from varicella-zoster virus (VZV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rabies virus, respiratory syncytial virus, or influenza virus.

20. The use according to any one of claims 16-17, wherein the target polypeptide or protein comprises the gE protein or a mutant thereof of varicella-zoster virus, the S protein or a mutant thereof of severe acute respiratory syndrome coronavirus 2, the G protein or a variant thereof of rabies virus strain CTN-1, the F protein or a mutant thereof of respiratory syncytial virus, or the HA protein or a mutant thereof of influenza virus.

21. Use of the template DNA molecule according to any one of claims 1-9, the mRNA molecule according to any one of claims 10-11, or the vector according to claim 12 in the preparation of a vaccine.

22. The use according to claim 21, wherein the vaccine is used for preventing or treating a disease related to the target polypeptide or protein.

23. An mRNA vaccine, characterized in that the mRNA vaccine comprises the mRNA molecule according to any one of claims 10-11.

24. The mRNA vaccine according to claim 23, wherein the mRNA encodes the gE protein or a mutant thereof of varicella-zoster virus, the S protein or a mutant thereof of severe acute respiratory syndrome coronavirus 2, the G protein or a variant thereof of rabies virus strain CTN-1, the F protein or a mutant thereof of respiratory syncytial virus, or the HA protein or a mutant thereof of influenza virus.

25. The mRNA vaccine according to any one of claims 23-24, characterized in that the mRNA vaccine further comprises a delivery system.

26. The mRNA vaccine according to claim 25, wherein the delivery system comprises a cationic liposome or a cationic polymer.

27. A method for preparing the mRNA vaccine according to any one of claims 23-26, characterized in that the preparation method comprises encapsulating the mRNA molecule in a delivery system.

Images