A method for rapid mRNA construction and its application

Abstract

The present invention discloses a method for rapid mRNA construction and its application. The method utilizes a pre-prepared double-stranded DNA fragment library, a full-length amplification primer, and a chemically synthesized oligonucleotide (target protein primer) to achieve rapid assembly and synthesis in a sterile environment under the catalytic synthesis of a high-fidelity DNA polymerase. The synthesized linear double-stranded DNA can be used for in vitro transcription reaction to synthesize the target mRNA after simple purification. This rapid mRNA construction method can be used to rapidly synthesize tumor mRNA vaccines encoding single tumor antigen epitopes, and can further be mixed to form an mRNA vaccine library for tumor treatment, greatly shortening the development cycle and cost of tumor vaccines. In addition, the present invention has also developed a rapid identification method for new antigens in tumor patients based on this rapid mRNA construction method. Compared with the use of antigen polypeptide libraries to identify antigen immunogenicity, this method can greatly reduce costs and shorten identification time.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a method for constructing an mRNA drug and its application. Background Art

[0002] Tumor immunotherapy has become a hot area in the development of tumor drugs in recent years due to its significant therapeutic effects. Among them, cancer mRNA vaccines, as an emerging immunotherapy method, have shown broad application prospects in precision treatment and personalized medication for patients. The latest studies have shown that mRNA vaccines based on personalized tumor neoantigens have shown excellent efficacy in multiple clinical trials [1, 2]. However, despite the huge potential of this technology, its widespread application still faces multiple technical bottlenecks: first, the traditional process of manufacturing mRNA vaccines is long, usually taking several weeks, and is costly; second, because personalized cancer mRNA vaccines are designed for a single patient, their flexibility and economy are severely restricted; finally, there is currently a lack of rapid and low-cost experimental methods to efficiently screen tumor neoantigens, which further limits the large-scale development of its application.

[0003] mRNA vaccines have been widely used, and their excellent efficacy and good safety have attracted great attention. They are regarded as an important tool for humans to fight diseases in the future[3]. The design of mRNA drugs simulates the structure of endogenous mRNA in eukaryotic cells, including a 5' cap, a 3' polyadenylic acid tail (PolyA tail), an untranslated region, and a gene coding sequence[4]. At present, the mainstream mRNA manufacturing technology relies on the in vitro transcription reaction of RNA polymerase (such as T7 phage RNA polymerase), in which RNA polymerase recognizes the promoter sequence on the template DNA and transcribes the genetic information encoded by the template DNA into RNA. The traditional mRNA drug manufacturing process includes several key steps such as the construction and production of template plasmid DNA, plasmid linearization and purification, in vitro transcription, and mRNA capping[5]. The traditional mRNA manufacturing process relies on the construction of template plasmids and bacterial culture, resulting in high production costs and long development cycles. At the same time, the bacterial culture tanks take up a lot of space, making it difficult to meet the needs of low-cost and rapid synthesis of multiple different sequence mRNAs.

[0004] Currently, personalized tumor neoantigen mRNA vaccines typically use a design that combines up to 20 antigens in series, allowing a single mRNA to encode as many candidate neoantigen sequences as possible, significantly increasing the likelihood of inducing a positive immune response and achieving clinical efficacy [6]. However, due to the high diversity of genetic mutations in tumor patients, such mRNA vaccines can usually only be designed for individual patients and are difficult to use across patients. In addition, the traditional mRNA manufacturing process is expensive and time-consuming, making the widespread application of such personalized mRNA vaccines in large patient populations a huge challenge.

[0005] On the other hand, the screening of tumor neoantigens and the evaluation of immune responses induced by tumor vaccines usually rely on flow cytometry and ELISPOT technology[7]. These technologies require the use of chemically synthesized peptide libraries to stimulate T cells and thus detect the immune response of T cells to specific antigenic peptides. However, the peptide synthesis cycle is long and the cost of synthesizing peptide libraries is high, which to some extent limits the application efficiency and popularity of these technologies in large-scale tumor neoantigen screening.

[0006] In summary, in order to enable the widespread application of cancer mRNA vaccines, the following three issues need to be optimized: mRNA manufacturing process, tumor personalized mRNA vaccine design, and experimental screening methods for tumor neoantigens. Summary of the Invention

[0007] In response to the above technical problems, the present invention has established a method for synthesizing single antigen epitope mRNA under the catalysis of DNA polymerase using a pre-synthesized double-stranded DNA fragment library, full-length amplification primers and chemically synthesized oligonucleotides (target protein primers), as well as a rapid identification method for neoantigens in tumor patients developed based on this rapid mRNA construction method.

[0008] The first aspect of the present invention provides a rapid mRNA construction method, which comprises the following steps: 1) pre-synthesizing a DNA fragment library and full-length amplification primers; 2) designing target protein primers; 3) a first round of bridge amplification; 4) a second round of in vitro transcription template amplification and optional template purification; and 5) in vitro transcription synthesis of target protein mRNA.

[0009] In certain embodiments, the DNA fragment library in step 1) is selected from a fusion or combination of one or more of 5'UTR, 3'UTR, signal peptide coding sequence, and transmembrane domain coding sequence.

[0010] In certain embodiments, the full-length amplification primer in step 1) is selected from a promoter-containing forward primer and / or a polyT-containing reverse primer.

[0011] In certain embodiments, the promoter is a T7 promoter.

[0012] In certain embodiments, the signal peptide is a signal peptide of a human or mouse major histocompatibility antigen class I protein (MHC-I SP).

[0013] In certain embodiments, the transmembrane domain is the transmembrane domain MITD of a human or mouse major histocompatibility antigen class I protein.

[0014] In certain embodiments, the 5'UTR is fused to the signal peptide coding sequence, and the transmembrane domain coding sequence is fused to the 3'UTR.

[0015] In certain embodiments, the 5'UTR is fused to the signal peptide coding sequence of major histocompatibility antigen class I protein (5'UTR+MHC-I SP).

[0016] In certain embodiments, the MITD coding sequence is fused to the 3'UTR (MITD+3'UTR).

[0017] In certain embodiments, a forward primer containing a promoter is designed based on the sequence of the 5'UTR; and a reverse primer containing polyT is designed based on the sequence of the 3'UTR.

[0018] In certain embodiments, the 3' end of the promoter-containing forward primer overlaps with the 5' end of the 5' UTR sense strand, and the 3' end of the polyT-containing reverse primer complements the 3' end of the 3' UTR sense strand.

[0019] In certain embodiments, the number of bases of the overlapping or complementary pairing is greater than 12.

[0020] In certain embodiments, the number of bases of the overlapping or complementary pairing is 12-30.

[0021] In certain embodiments, the target protein in step 2) is an antigen, antibody, cytokine or other therapeutic protein.

[0022] In certain embodiments, the antigen is a viral antigen, a tumor antigen, or a bacterial antigen.

[0023] In certain embodiments, the antigen is 20-120 amino acids in length.

[0024] In certain embodiments, the antigen is 20-30 amino acids in length.

[0025] In certain embodiments, the antigen is about 25 amino acids in length.

[0026] In certain embodiments, different numbers of primers are used to assemble inserts of different lengths; preferably, the number of primers is 2-8; preferably, the length of each primer is 42-78 nt, such as about 60 nt.

[0027] In certain embodiments, the antigen primer in step 2) has a sequence of 12 to 30 nt that is complementary to the front segment and the back segment; preferably, the number of bases of the complementary pairing sequence is 15; preferably, the front segment is the primer connected to the front segment, 5'UTR or signal peptide coding sequence; preferably, the back segment is the primer connected to the back segment, 3'UTR or transmembrane domain coding sequence.

[0028] In certain embodiments, the MITD comprises the amino acid sequence shown in SEQ ID NO: 26 (IVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSYSQAACSDSAQGS DVSLTA).

[0029] In certain embodiments, the signal peptide of the human major histocompatibility antigen class I protein (MHC-ISP) comprises the amino acid sequence shown in SEQ ID NO: 27 (MLVMAPRTVLLLLSAALALTETWAGS).

[0030] In certain embodiments, the 5'UTR+SP comprises the nucleotide sequence shown in SEQ ID NO: 1 or 9, and / or the MITD+3'UTR comprises the nucleotide sequence shown in SEQ ID NO: 2 or 10, and / or the MHC-I SP comprises the nucleotide sequence shown in SEQ ID NO: 3, and / or the MITD comprises the nucleotide sequence shown in SEQ ID NO: 4, and / or the 5'UTR comprises the nucleotide sequence shown in SEQ ID NO: 5, and / or the 3'UTR comprises the nucleotide sequence shown in SEQ ID NO: 6, and / or the promoter-containing forward primer comprises the nucleotide sequence shown in SEQ ID NO: 7, and / or the polyT-containing reverse primer comprises the nucleotide sequence shown in SEQ ID NO: 8.

[0031] In certain embodiments, the first-round bridging amplification reaction system in step 3) comprises antigen primers, 5'UTR+MHC-I SP, MITD+3'UTR, and PCR premix.

[0032] In certain embodiments, the amplification program is (1) 98°C for 30s; (2) 98°C for 10s, 60°C for 10s, 72°C for 15s, for 7 cycles; and (3) 72°C for 30s, 4°C∞.

[0033] In certain embodiments, the second round of in vitro transcription template amplification in step 4) comprises adding a forward primer containing a promoter and a reverse primer containing polyT to the reaction in step 3).

[0034] In certain embodiments, the amplification program is: (1) 98°C for 30s; (2) 98°C for 10s, 63°C for 10s, 72°C for 15s, 25 cycles; (3) 72°C for 30s, 4°C∞.

[0035] The second aspect of the present invention provides a target protein mRNA prepared by the method according to the first aspect of the present invention.

[0036] In certain embodiments, the mRNA is an antigen mRNA.

[0037] In certain embodiments, the antigen mRNA is a single-epitope antigen mRNA.

[0038] In certain embodiments, the mRNA comprises, in order from 5' to 3', a 5' cap, a 5' UTR, an MHC-ISP, an antigen epitope sequence in the coding region, a MITD, a 3' UTR and a polyA tail.

[0039] The third aspect of the present invention provides a single-epitope antigen mRNA library, wherein the single-epitope antigen mRNA library comprises two or more single-epitope antigen mRNAs according to the second aspect of the present invention.

[0040] The fourth aspect of the present invention provides the use of the single-epitope antigen mRNA library described in the third aspect of the present invention in the rapid verification of tumor neoantigens.

[0041] In certain embodiments, a mixed library of single-epitope antigen mRNA is used to transfect patient peripheral blood mononuclear cells (PBMC) or B cells, and isolated blood T cells or tumor-infiltrating T cells (Tils) are stimulated. After several days of culture, B cells transfected with a single single-epitope antigen mRNA are used for restimulation, and immunogenic antigen epitopes are screened by detection.

[0042] In certain embodiments, the assay is an Elispot assay.

[0043] The fifth aspect of the present invention provides use of the single-epitope antigen mRNA described in the second aspect of the present invention or the single-epitope antigen mRNA library described in the third aspect of the present invention in the preparation of tumor vaccines or infectious disease vaccines.

[0044] The sixth aspect of the present invention provides a vaccine, which comprises the single-epitope antigen mRNA described in the second aspect of the present invention or the single-epitope antigen mRNA library described in the third aspect of the present invention.

[0045] In certain embodiments, the vaccine further comprises an mRNA delivery vehicle.

[0046] In certain embodiments, the delivery vehicle is a lipid nanoparticle (LNP).

[0047] The advantages of the present invention over the prior art are:

[0048] 1) The present invention designs a rapid mRNA construction method: it significantly shortens the time to construct the target mRNA - after the sequence is designed, all processes from primer synthesis to mRNA synthesis can be completed within one day at the earliest; it significantly reduces the cost of constructing the target mRNA - this method eliminates the process steps such as bacterial culture, plasmid purification, and enzyme linearization, saving the cost of these processes; it significantly reduces the production space required for mRNA development and production - this method does not require large culture tanks, and a small-volume reactor can be used to develop and prepare mRNA in large quantities; mRNA construction has the advantage of modularity - preparing different DNA fragments can easily replace functional sequences such as the untranslated region and signal peptide in the mRNA.

[0049] 2) The single-epitope antigen mRNA designed in this invention utilizes a rapid construction method, enabling the rapid and cost-effective construction of a single-epitope antigen mRNA library. This library can be flexibly combined into personalized neoantigen hybrid vaccines, avoiding the problem of mutual interference between multiple antigens on the same amino acid chain. This hybrid vaccine not only exhibits excellent tumor-specific immune induction and tumor treatment efficacy in animal models, but also exhibits a highly effective induction effect on human peripheral blood mononuclear cells, achieving a shorter production cycle and more flexible application scenarios than traditional tandem mRNA.

[0050] 3) Rapid validation of tumor neoantigens using a rapidly synthesized single-epitope mRNA antigen library significantly reduces costs and shortens validation cycles compared to traditional peptide library validation methods. Furthermore, this rapid validation method innovatively involves direct mRNA transfection into PBMCs or easily amplified B cells to amplify antigen-recognizing T cells, which are then restimulated with B cells. This method is more convenient and less expensive than traditional DC stimulation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0052] Figure 1A schematic diagram of the rapid mRNA construction method (using a single-epitope tumor antigen mRNA as an example) is shown. A target fragment in the middle of the mRNA coding region is designed and assembled using short oligonucleotide primers. The remaining relatively fixed coding region (CDS) and untranslated region (UTR) sequences are presynthesized. These fragments are bridged through a first round of 7 cycles of PCR amplification. A second round of 25 cycles of PCR amplification is then performed using full-length primers containing a T7 promoter and polythymidine (PolyT). The resulting template DNA is purified and used for in vitro transcription to synthesize the target mRNA.

[0053] Figure 2 Figure 3. Single-epitope antigen mRNA design, rapid construction results, and protein expression verification. (A) Schematic diagram of single-epitope antigen mRNA design: From 5' to 3', the single-epitope mRNA consists of CAP1 or other cap, 5' UTR, SP (human MHC-I signal peptide), Ag (antigen epitope sequence, encoding approximately 25 amino acids), MITD (human MHC-I transmembrane domain), 3' UTR, and polyA (polyadenylic acid, typically 120 A residues). (B) Agarose gel electrophoresis results of the single-epitope antigen DNA template synthesized using the rapid construction method. (C) Agarose gel electrophoresis results of the single-epitope antigen mRNA synthesized using the rapid construction method. (D) Western blot results showing protein expression of the rapidly constructed single-epitope antigen mRNA in HEK293T cells.

[0054] Figure 3 Rapidly constructed single-epitope antigen mRNAs enable rapid, large-scale validation of candidate tumor neoantigens in patients. (A) Schematic diagram of the rapid validation method for tumor neoantigens based on single-epitope antigen mRNAs. After candidate tumor neoantigens are identified, single-epitope mRNA libraries for these antigens are synthesized within a day. PBMCs or B cells are transfected with the mixed single-epitope mRNA library, followed by a first round of T cell stimulation. After overnight cytokine-free culture, T cells are restimulated with B cells transfected with a single single-epitope mRNA, and positive antigens are detected by ELISpot. (B) Immunogenicity validation results of tumor neoantigens from three patients with ovarian cancer using the above method.

[0055] Figure 4Figure 1. Exploration of the conditions for the rapid mRNA construction method. (A) Schematic diagram of the rapid mRNA construction method for constructing Gaussia luciferase (Gluc) mRNA. (B) Agarose gel electrophoresis of template DNA at different temperatures for the first and second rounds of PCR. (C) Schematic diagram of constructing Gluc mRNA using primers of varying complementary sequence lengths. (D) Agarose gel electrophoresis of template DNA constructed using primers of varying complementary sequence lengths. (E) Comparison of Gluc signals for mRNA constructed using primers of varying complementary sequence lengths.

[0056] Figure 5 Exploration of methods for rapid construction of long target fragments. (A) Schematic diagram of inserting a 75-345 nt target fragment using two or more primers. (B) Agarose gel electrophoresis of template DNA constructed with different numbers of inserted primers. (C) Comparison of Glucose signals for mRNA constructed with different numbers of inserted primers.

[0057] Figure 6 Comparison of the immune-inducing abilities of single antigen mix mRNAs (4-antigen mix, 20-antigen mix) synthesized using the rapid construction method with traditional 20-antigen concatenated mRNA. (A) The proportion of CD8 T cells (IFNγ-positive) responding to mRNAs containing a mix of 4 positive antigens, antigens P9, and antigen B3, among all splenic CD8 T cells. (B) The proportion of CD4 T cells (IFNγ-positive) responding to mRNAs containing a mix of 4 positive antigens, antigens P9, antigen P15, and antigen B5, among all splenic CD4 T cells.

[0058] Figure 7 This image shows the immune-inducing effects of mixed and traditional concatenated viral antigen mRNAs synthesized using the rapid construction method on healthy human PBMCs. Twenty mixed viral single antigen mRNAs (MIX) and the corresponding concatenated mRNAs (Concat) were used to stimulate T cells. The results were then analyzed using IFNγ elispot assays. The lower two panels used PBMCs from two different healthy individuals.

[0059] Figure 8 Figure 1 shows the efficacy of a rapid synthesis method for the treatment of a subcutaneous melanoma mouse model using a mixed mRNA containing eight antigens. (A) and (B) Tumor anatomy, tumor growth curves, and mouse body weight changes on day 17 after treatment of a subcutaneous B16F10 mouse model with a mixed mRNA containing eight single epitopes (B16-8MIX) and a tandem mRNA containing eight antigens (B16-8). (C) and (D) The proportions of CD8 and CD4 T cells in the spleen that respond to neoantigens (IFN-γ positive) are shown. DETAILED DESCRIPTION

[0060] Rapid mRNA construction method

[0061] The present invention provides a modular, rapid mRNA construction method that is independent of bacteria and plasmids. The method primarily uses chemically synthesized oligonucleotides to assemble target gene fragments via DNA polymerase chain reaction (PCR). These fragments are then combined with other fixed-sequence modular fragment libraries to assemble and amplify a complete template for mRNA synthesis.

[0062] The mRNA rapid construction method of the present invention is divided into three main parts: 1. Preparation of DNA fragment library, full-length amplification primers and single-stranded DNA oligonucleotide primers; 2. Assembly, amplification and purification of DNA fragments; 3. In vitro transcription and purification of mRNA (such as Figure 1 ).

[0063] The mRNA rapid construction method of the present invention comprises the following steps: (taking the single epitope antigen mRNA designed by the present invention as an example)

[0064] 1) Fragment preparation: The 5'UTR+SP and MITD+3'UTR plasmids were digested with BSAI endonuclease (enzyme cleavage site: GGTCTC, SEQ ID NO: 11) and recovered from gel.

[0065] Fixed primer preparation: A forward primer (T7-F, SEQ ID NO: 7) containing a T7 phage promoter and a reverse primer (PolyA120-R, SEQ ID NO: 8) containing 120 thymines were designed based on the sequences of the 5'UTR (e.g., SEQ ID NO: 5) and 3'UTR (e.g., SEQ ID NO: 6), respectively. These primers were synthesized in large quantities by Huzhou Hippo Biological in advance.

[0066] 2) Antigen primer design: The principle of primer design in this method is that the primer connected to the fragment has a sequence of 12 nt or more that is complementary to the previous and next fragments (i.e., 5'UTR+SP, SEQ ID NO: 1; MITD+3'UTR, SEQ ID NO: 2), and the primers connected to the previous and next fragments also have a sequence of 12 nt or more that is complementary to each other.

[0067] 3) First round of bridge amplification (50ul amplification system as an example): Use 2×Hieff AdvanceFast PCR Master Mix was mixed with 5 ng of 5'UTR+SP, 5 ng of MITD+3'UTR fragment, and 0.5 pmol / antigen primer. 50 μl of amplification system was prepared by adding appropriate amount of ddH2O. Seven cycles of amplification were performed according to the following protocol: (1) 98°C for 30 s; (2) 98°C for 10 s, 60°C for 10 s, and 72°C for 15 s, seven cycles; (3) 72°C for 30 s, 4°C for ∞.

[0068] 4) Second round of in vitro transcription template amplification: Add 10 pmol of T7-F and 10 pmol of PolyA120-R primer to the reaction in step 3. Amplify for 25 cycles according to the following protocol: (1) 98°C for 30 s; (2) 98°C for 10 s, 63°C for 10 s, and 72°C for 15 s, for 25 cycles; (3) 72°C for 30 s, 4°C for ∞.

[0069] 5) The reaction system in step 4 was run on an agarose gel and the gel was cut for recovery and purification.

[0070] 6) In vitro transcription of single-epitope antigen mRNA: Using Yisheng Bio's T7 High Yield RNA Synthesis Kit for Co-transcription, mix ATP, N1-Me-Pseudo UTP, CTP, GTP, GAG, and 10× Transcription Buffer, add the template DNA purified in step 5 (500 ng for 20 μl) and T7 RNA Polymerase Mix. Add an appropriate amount of RNase-Free Water, mix, and incubate at 37°C for 2 h. Purify the RNA and quality check.

[0071] Single epitope antigen mRNA and mRNA library

[0072] The present invention also provides a single-epitope antigen mRNA prepared according to the above method, which mainly comprises an antigen epitope sequence in the coding region (encoding about 25 amino acids), a signal peptide (MHC-I signal peptide, SP) and a transmembrane sequence (MHC-I transmembrane domain, MITD) of the human major histocompatibility complex (MHC-I) at both ends, as well as a 5'UTR, a 3'UTR, a 5' cap, and a polyA tail.

[0073] In certain embodiments, the single-epitope antigen mRNA comprises, in order from 5' to 3', a 5' cap, a 5' UTR, an MHC-ISP, an antigen epitope sequence in the coding region, a MITD, a 3' UTR and a polyA tail.

[0074] The present invention also provides a single-epitope antigen mRNA library, wherein the mRNA library comprises two or more single-epitope antigen mRNAs.

[0075] In some embodiments, the mRNA library comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 single-epitope antigen mRNAs.

[0076] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0077] Unless otherwise defined, technical or scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0078] The present invention first explored the optimal response profile for a rapid mRNA construction method. The ability of single-epitope antigen mRNA to induce antigen-specific T cells was then validated in mice and in healthy human PBMCs. Finally, a mixed single-epitope antigen mRNA vaccine was designed for mouse B16F10 cells, demonstrating superior immune induction and tumor therapeutic efficacy compared to a tandem antigen mRNA vaccine.

[0079] Example 1 mRNA Rapid Construction Method

[0080] In order to verify the feasibility of the rapid mRNA construction method, the mRNA of the Gaussia luciferase (GLUC) reporter gene was selected to facilitate the detection of the activity of the mRNA product. By enzyme digestion of plasmid DNA, a fragment containing 5'UTR and GLUC 1-147bp (SEQ ID NO: 12) and a fragment of GLUC 223-558bp and 3'UTR (SEQ ID NO: 13) were obtained. At the same time, two 60nt oligonucleotide primers (SEQ ID NO: 14-15 Suzhou Jinweizhi synthesis) were designed to assemble the 75 bases of GLUC 148-222bp and complement the two ends. Two rounds of PCR reactions were designed. The first round of reaction had 7 amplification cycles to achieve the bridge between the primer and the fragment. In the second round of reaction, two primers (SEQ ID NO: 16-17) containing T7 promoter and 120 thymines (reverse primer, the template chain after synthesis was 120 adenines) were added and amplified for 25 cycles to amplify the DNA template that can be used for in vitro transcription ( Figure 4A). The PCR reaction was performed using a common three-step amplification method, including denaturation, annealing, and extension (using 2×Hieff CanaceAdvanceFast PCR Master Mix, Yisheng Biotechnology). The inventors first explored the use of a range of annealing temperatures for two rounds of PCR reactions ( Figure 4 B), it was found that different annealing temperatures in the first round of PCR reaction had little effect on the synthesis efficiency, while the annealing temperature of the second round of PCR reaction had the best amplification efficiency when it was between 63°C and 65°C. In order to find the most suitable primer length and the length of the complementary sequence between the primer and the fragment, a GLUC construction method with a primer inserted in the middle was designed ( Figure 4 C), the fragment of 5'UTR+"GLUC 1-147bp" is shown in SEQ ID NO: 12, and the fragment of "GLUC 178-558bp"+3'UTR is shown in SEQ ID NO: 25. The reaction conditions of this method are the same as those of Figure 2 The same as in A, but different lengths of intermediate primers (42nt, 48nt, 54nt, 60nt, 66nt, 72nt, 78nt, SEQ ID NO: 18-24, respectively) were used. The corresponding primers and the complementary sequence lengths of the front and back fragments were 6nt, 9nt, 12nt, 15nt, 18nt, 21nt, and 24nt. The results showed that the target DNA could be amplified only when the complementary sequence length was 12nt, while the optimal amplification yield and purity were achieved when the complementary sequence length was 15nt or longer ( Figure 4 D). GLUC mRNA was synthesized using the template obtained from the reaction and transfected into HEK293T cells to detect mRNA activity. It was found that there was no significant difference in the activity of GLUC mRNA constructed with complementary sequence 15nt-24nt primers, and there was no significant difference in the activity of these mRNAs and the control GLUC mRNA synthesized with a linearized plasmid template (with the same UTR sequence and polyA length) ( Figure 4 E). In summary, the method designed by the present invention successfully constructed highly active mRNA.

[0081] Similarly, the GLUC mRNA construction method was further used to test the insertion of gene fragments of different lengths to explore how long the target gene fragment can be inserted. The inventors tried to use different numbers (2-8) of 60nt primers to assemble insert fragments of different lengths (75nt-345nt) to synthesize the complete GLUC mRNA ( Figure 5A), the first to eighth primers are shown in SEQ ID NOs: 14-15 and SEQ ID NOs: 28-33, respectively. The agarose gel results of the synthesized template DNA showed that when the number of primers increased from 2 to 8, that is, when the length of the inserted sequence increased to 345 nt, the method of the present invention still successfully synthesized high-purity template DNA ( Figure 5 B). GLUC mRNA was further synthesized and its activity was tested. It was found that when the number of primers was 2-4, the mRNA activity did not decrease significantly, but when the number of primers was 6-8, the mRNA activity decreased ( Figure 5 C) This may be because too many primers with different sequences increase the probability of incorrect complementary pairing, resulting in more mutant GLUC mRNA.

[0082] The antigen sequence recognized by T cells is usually only 9-25 amino acids. Based on this, the present invention designs a short single antigen epitope mRNA, which carries the necessary 5'UTR, 3'UTR, signal peptide and transmembrane sequence of human major histocompatibility antigen class I protein, and also carries about 25 amino acid sequences of the target antigen ( Figure 1 After analyzing the antigen epitope, constructing such a single epitope mRNA only requires replacing the target antigen sequence in the middle, which can be easily constructed using the rapid construction method of the present invention. According to this method ( Figure 1 ), synthesized 10 pairs of 60nt primers, and successfully constructed DNA templates and mRNAs for ten different antigens ( Figure 2 A, Figure 2 B), as shown in Table 1 below. Western blot results further showed that these mRNAs successfully expressed the target protein in HEK293T cells ( Figure 2 C) In summary, the rapid construction method of the present invention can be used for the rapid synthesis of single antigen epitope mRNA.

[0083] Table 1 10 antigen primers and DNA full-length information

[0084]

[0085]

[0086] Note: The primers in Table 1 are DNA sequences.

[0087] Example 2 Immune Induction Effect of Mixed Single-Epitope Tumor Antigen mRNA in Mice

[0088] Traditional personalized tumor neoantigen mRNA vaccines usually take the form of up to 30 neoantigens concatenated into a single mRNA vaccine. The mRNA rapid construction method and single antigen epitope mRNA designed by the present invention can synthesize dozens of single antigen epitope mRNA vaccines in a very short time. These single antigen epitope mRNAs can be conveniently combined, which is more flexible in later clinical applications than multi-epitope concatenated mRNAs, and saves the time of constructing plasmids. In order to evaluate the immune induction effect of mixed single epitope antigen mRNA, the present invention selected 20 mouse-derived mutant tumor neoantigens containing 4 positive epitopes and 16 negative epitopes, all of which were constructed into single epitope mRNA vaccines. At the same time, the concatenated mRNA of these 20 epitopes was also constructed using traditional methods for comparison. The mRNA sequences of the 20 antigen epitopes are shown in SEQ ID NO: 64-83, respectively, and the sequence of the concatenated mRNA is shown in SEQ ID NO: 84. The present invention immunized mice with a mixed antigen vaccine (4MIX) of 4 positive antigens, a mixed antigen vaccine (20MIX) of all 20 antigens, and a concatenated 20 antigen mRNA vaccine. The present invention injected two doses of vaccine, one week apart, each time with 5ug / mouse. After the second dose of vaccine, the mouse spleen lymphocytes were isolated and transfected with mixed 4 positive epitope mRNA or transfected with 4 positive epitope mRNAs separately to detect the proportion of IFNγ-positive T cells. The results showed that when the mixed positive mRNA was transfected into spleen lymphocytes, the proportion of CD8 and CD4 T cells secreting IFNγ in the group immunized with 4 positive antigens mixed (4MIX) was the highest, followed by the group immunized with 20 antigens mixed (20MIX), and the group immunized with tandem mRNA (20C) had the lowest proportion ( Figure 6 A). This result shows that the immune induction effect of the two mixed antigen vaccines is very good. The proportion of T cells responding to the four positive antigens (IFNγ positive) was analyzed separately. The present invention found that the spleen lymphocytes of the 4MIX and 20MIX groups had a good response to the four positive antigens ( Figure 6 B). While group 20C had the highest proportion of B3-specific CD8 T cells, responses to the other three antigens were relatively poor, with almost no P9-specific CD8 T cells detected. This phenomenon may suggest that certain epitopes in tandem mRNA may be ineffective, while single-epitope mRNA, because each epitope is translated separately, may avoid the ineffectiveness of positive epitopes. These results suggest that vaccines containing a mixture of multiple single-epitope antigen mRNAs have a strong immune-inducing effect, while tandem epitope mRNA vaccines have a wide range of immune-inducing effects among different epitopes, and may even fail to induce immunity.

[0089] Table 2. mRNA sequences of 20 mouse-derived mutant tumor neoantigens

[0090] Antigen name mRNA sequences Snapc4 SEQ ID NO: 64 Kif18b SEQ ID NO: 65 Pcnx SEQ ID NO: 66 Obsl1 SEQ ID NO: 67 Zfp938 SEQ ID NO: 68 Arfgef1 SEQ ID NO: 69 Cant1 SEQ ID NO: 70 Clasp1 SEQ ID NO: 71 Dpy19l1 SEQ ID NO: 72 Dtx3 SEQ ID NO: 73 Fbf1 SEQ ID NO: 74 Klhl7 SEQ ID NO: 75 Nfat5 SEQ ID NO: 76 Pdpr SEQ ID NO: 77 Prrc2b-I SEQ ID NO: 78 Tubb3 SEQ ID NO: 79 Rab11fip5 SEQ ID NO: 80 Slc16a13 SEQ ID NO: 81 Vps13b SEQ ID NO: 82 Tep1 SEQ ID NO: 83

[0091] Note: SEQ ID NO: 64-84 are full-length mRNA sequences

[0092] Example 3 Ability of mixed single-epitope viral antigen mRNA to induce human immune cell responses

[0093] In order to test the ability of the single-epitope mRNA synthesized by the rapid construction method to induce human immune cell responses, the inventors synthesized single epitopes and tandem mRNAs of 20 viral antigens. The mRNA sequences of the 20 viral antigens are shown in SEQ ID NO: 85-104, respectively, and the sequence of the tandem mRNA is shown in SEQ ID NO: 105 (Table 3). First, mixed single-epitope mRNA or tandem mRNA was transfected into healthy human PBMCs and amplified for 7 days. These PBMCs were then stimulated with B cells transfected with a single single-epitope mRNA, and the number of IFN-γ-positive T cells was detected using elispot. The response results of the 20 viral antigens showed that the mixed single-epitope mRNA was more effective in inducing IFN-γ-positive T cells than the tandem mRNA ( Figure 7 ).

[0094] Table 3 Sequence information of 20 viral antigens

[0095] antigen mRNA sequences Viral-II-1 SEQ ID NO: 85 Viral-II-2 SEQ ID NO: 86 Viral-II-3 SEQ ID NO: 87 Viral-II-4 SEQ ID NO: 88 Viral-II-5 SEQ ID NO: 89 Viral-II-6 SEQ ID NO: 90 Viral-II-7 SEQ ID NO: 91 Viral-II-8 SEQ ID NO: 92 Viral-II-9 SEQ ID NO: 93 Viral-II-10 SEQ ID NO: 94 Viral-I-1 SEQ ID NO: 95 Viral-I-2 SEQ ID NO: 96 Viral-I-3 SEQ ID NO: 97 Viral-I-4 SEQ ID NO: 98 Viral-I-5 SEQ ID NO: 99 Viral-I-6 SEQ ID NO: 100 Viral-I-7 SEQ ID NO: 101 Viral-I-8 SEQ ID NO: 102 Viral-I-9 SEQ ID NO: 103 CMV SEQ ID NO: 104

[0096] Note: SEQ ID NO: 85-104 are full-length mRNA sequences

[0097] Example 4 Verification of the Tumor Therapeutic Effect of Mixed Single-Epitope mRNA Vaccines in Animal Tumor Models

[0098] The ultimate goal of developing tumor vaccines is to be able to efficiently induce tumor antigen-specific T cells that recognize and kill tumor cells. The above results show that the mixed single-epitope mRNA vaccine has a good induction effect on T cell immunity in both mice and humans. This prompted the present invention to verify the tumor treatment effect of the mixed single-epitope mRNA vaccine in an animal tumor model. The present invention synthesized 8 single-epitope mRNAs and tandem mRNAs of new antigens of B16F10 tumor cells, packaged them with SM102 LNP, and injected them into mice subcutaneously inoculated with B16F10 tumors. The mRNA sequences of the 8 tumor new antigens are shown in SEQ ID NO: 106-113, and the sequence of the tandem mRNA is shown in SEQ ID NO: 114 (Table 4). The results showed that compared with the control group, the tumor growth of the mixed single-epitope mRNA vaccine (B16-8MIX) and tandem mRNA vaccine (B16-8) groups were significantly inhibited ( Figure 8(A, 8B) The B16-8MIX group had the smallest tumor volume on day 17. Splenic lymphocytes were isolated 17 days after tumor inoculation, and neoantigen-specific T cells were analyzed by flow cytometry. The B16-8MIX group had the highest proportions of both neoantigen-specific CD8 and CD4 T cells. These results demonstrate that mixed single-epitope mRNA vaccines induce higher levels of tumor neoantigen-specific immunity and better tumor treatment efficacy.

[0099] Table 4 Neoantigen sequence information of 8 B16F10 tumor cells

[0100] Antigen name mRNA sequences Ppp1r7 SEQ ID NO: 106 Sema3b SEQ ID NO: 107 Obsl1 SEQ ID NO: 108 Actn4 SEQ ID NO: 109 Kif18b SEQ ID NO: 110 Tubb3 SEQ ID NO: 111 Def8 SEQ ID NO: 112 Tm9sf3 SEQ ID NO: 113

[0101] Note: SEQ ID NO: 116-114 are full-length mRNA sequences

[0102] Example 5 Rapid Validation Method for Tumor Neoantigens Based on Single-Epitope Antigen mRNA Library

[0103] The patient's peripheral blood mononuclear cells (PBMCs) or B cells were transfected with a mixed library of single-epitope antigen mRNAs (SEQ ID NOs: 115-173, with a 120-nt poly A added), and isolated blood T cells or tumor-infiltrating T cells (Tils) were stimulated. After 7 days of culture, B cells transfected with a single single-epitope antigen mRNA were re-stimulated, and finally, Elispot assay was performed to obtain the immunogenic antigen epitopes (such as Figure 3 A). This rapid validation method was used to detect antigen epitopes that can be recognized by the patients' T cells in three ovarian cancer patients (such as Figure 3 B). The specific quick verification method is as follows:

[0104] 1) Analyze and synthesize patient single epitope antigen mRNA libraries.

[0105] 2) Thawing of cryopreserved PBMCs.

[0106] 3) Use CALNP TM mRNA in vitro transfection reagent (Dona Pharmaceuticals) was used to transfect 500 ng of mixed patient single-epitope antigen mRNA per well.

[0107] 4) 8 hours after transfection, DC cell maturation needs to be promoted: 8 hours later, do not change the medium, add 100ul DC culture medium containing

[0108] GM-CSF (1 ul / mL, PeproTech) and IL4 (5 ul / mL, PeproTech) were added, along with twice the amount of LPS (2×, 20 ul / ml, Sigma) and IFN-γ (2×, 2 ul / ml, PeproTech) to promote maturation for 16 h.

[0109] 5) Carefully replace half of the culture medium and culture with T cell culture medium, while adding PeproTech's IL-2

[0110] (0.5ul / ml, PeproTech), IL-7 (2ul / ml, PeproTech) and IL-15 (2ul / ml,

[0111] PeproTech). From the second to the seventh day, change half of the medium every 2-3 days: discard half of the supernatant, add 2× T median

[0112] The above cytokines were expressed and cultured continuously.

[0113] 6) On day 8, cells from the same group were pooled, counted, and resuspended at 100 μg / ml. IL-2, IL-7, and IL-5 were added and plated in 24-well plates. PBMCs were treated by centrifugation at 400 g for 5 minutes and the supernatant discarded. Culture was continued overnight in T media (without cytokines).

[0114] 7) Prepare the B cells and B medium to be used, and add IL-4 (2ul / ml, Peprotech)

[0115] IL21 (2ul / ml, ACROBiosystems), CD40L (3ul / ml, ACROBiosystems), BME (1ul / ml, Sigma-Aldrich) were cultured.

[0116] 8) B cell and T cell co-incubation: All B cells were aspirated, centrifuged at 400g for 5 minutes, the supernatant was discarded, the suspension was resuspended in 2ml of empty culture medium, counted, and supplemented to 100w / ml; T cells were aspirated from the 24-well plate, 2ml was directly counted,

[0117] Centrifuge at 400g for 5 minutes, discard the supernatant, and resuspend the culture medium to 200w / ml.

[0118] 9) Use GenNano-W0021 (Miana) to prepare a single single epitope antigen mRNA transfection complex into each well of a 96-well plate. Add 50ul (5w) of B cells, 50ul (10w) of T cells, and 50ul of empty culture medium to each well of the RNA transfection complex.

[0119] The B cell and T cell co-incubation system was mixed evenly and then carefully added into Elispot (Dakoway) wells (each tube corresponds to each well), and cultured in a 37° C., 5% CO 2 incubator for 40 h before detection.

[0120] Depend on Figure 3As shown in B, the verified SUPV3L1 antigen of patient OC41, VIRMA, KIAA1217, and AP1G2 antigens of patient OC44, and GART antigen epitopes in the antigen of patient OC50 activated the patient's T cells, were immunogenic, and had clinical application value.

[0121] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, from all points of view, the embodiments should be regarded as illustrative and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and range of equivalents of the claims are included in the present invention. Any reference signs in the claims should not be construed as limiting the claim to which they relate.

[0122] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0123] References

[0124] 1.Rojas LA, Sethna Z, Soares KC, et al.Personalized RNA neoantigenvaccines stimulate T cells in pancreatic cancer.Nature.2023;618(7963):144-150.doi:10.1038 / s41586-023-06063-y

[0125] 2.Weber JS,Carlino MS,Khattak A,et al.Individualised neoantigentherapy mRNA-4157(V940)plus pembrolizumab versus pembrolizumab monotherapy inresected melanoma(KEYNOTE-942):a randomised,phase 2bstudy.Lancet.2024;403(10427):632-644.doi:10.1016 / S0140-6736(23)02268-7

[0126] 3.Baden LR,El Sahly HM,Essink B,et al.Efficacy and Safety of themRNA-1273SARS-CoV-2Vaccine.N Engl J Med.2021;384(5):403-416.doi:10.1056 / NEJMoa2035389

[0127] 4.Rohner E,Yang R,Foo KS,Goedel A,Chien KR.Unlocking the promise ofmRNA therapeutics.Nat Biotechnol.2022;40(11):1586-1600.doi:10.1038 / s41587-022-01491-z

[0128] 5.Bancel S,ISSA WJ,AUNINS JG,Chakraborty T.Manufacturing methods forproduction of rna transcripts.Published online September 25,2014.AccessedDecember 28,2024.https: / / patents.google.com / patent / WO2014152027A1 / en

[0129] 6.Cafri G,Gartner JJ,Zaks T,et al.mRNA vaccine-induced neoantigen-specific T cell immunity in patients with gastrointestinal cancer.J ClinInvest.2020;130(11):5976-5988.doi:10.1172 / JCI134915

[0130] 7.Lybaert L,Lefever S,Fant B,et al.Challenges in neoantigen-directedtherapeutics.Cancer Cell.2023;41(1):15-40.doi:10.1016 / j.ccell.2022.10.013。

Claims

1. A rapid method for constructing single-epitope antigen mRNA, characterized in that: The process includes the following steps: 1) pre-synthesis of DNA fragment library and full-length amplification primers; 2) design of target protein primers; 3) first round of bridge amplification; 4) second round of in vitro transcription template amplification; 5) in vitro transcription synthesis of target protein mRNA; wherein the DNA fragment library in step 1) comprises a 5'UTR, a 3'UTR, a signal peptide coding sequence, and a transmembrane domain coding sequence; the signal peptide is the signal peptide MHC-I SP of a human or mouse major histocompatibility antigen class I protein; the transmembrane domain is the transmembrane domain MITD of a human or mouse major histocompatibility antigen class I protein; the 5'UTR is fused to the signal peptide coding sequence of the major histocompatibility antigen class I protein, i.e., 5'UTR+MHC-I SP; the MITD coding sequence is fused to the 3'UTR, i.e., MITD+3'UTR; the full-length amplification primer is selected from a forward primer containing a T7 promoter and a reverse primer containing polyT; the 3' end of the forward primer containing the T7 promoter overlaps with the 5' end of the 5'UTR sense strand, and the 3' end of the reverse primer containing polyT is complementary to the 3' end of the 3'UTR sense strand; the number of bases of the overlapping or complementary pairing is 12-30; The target protein in step 2) is an antigen, antibody, cytokine or other therapeutic protein; the antigen is a viral antigen, tumor antigen or bacterial antigen; the length of the antigen is 20-30 amino acids; Different numbers of primers are used to assemble inserts of different lengths; the number of primers is 2-8; and the length of each primer is 42-78 nt; Wherein, the antigen primer in step 2) has a sequence of 12 to 30 nt that is complementary to the front segment and the back segment; the front segment is the primer, 5'UTR or signal peptide coding sequence connected to the front segment; the back segment is the primer, 3'UTR or transmembrane domain coding sequence connected to the back segment; The first round of bridge amplification reaction system in step 3) includes antigen primers, 5'UTR+MHC-I SP, MITD+3'UTR and PCR premix; the amplification program is (1) 98°C for 30s; (2) 98°C for 10s, 60°C for 10s, 72°C for 15s, 7 cycles; (3) 72°C for 30s, 4°C ∞; The second round of in vitro transcription template amplification in step 4) includes adding a forward primer containing a promoter and a reverse primer containing polyT to the reaction in step 3); the amplification program is: (1) 98°C for 30s; (2) 98°C for 10s, 63°C for 10s, 72°C for 15s, 25 cycles; (3) 72°C for 30s, 4°C ∞.

2. The method according to claim 1, wherein the 5'UTR+SP is the nucleotide sequence shown in SEQ ID NO: 1 or 9, the MITD+3'UTR is the nucleotide sequence shown in SEQ ID NO: 2 or 10, the MHC-I SP is the nucleotide sequence shown in SEQ ID NO: 3, the MITD is the nucleotide sequence shown in SEQ ID NO: 4, the 5'UTR is the nucleotide sequence shown in SEQ ID NO: 5, the 3'UTR is the nucleotide sequence shown in SEQ ID NO: 6, the promoter-containing forward primer is the nucleotide sequence shown in SEQ ID NO: 7, and the polyT-containing reverse primer is the nucleotide sequence shown in SEQ ID NO:

8.

3. The single epitope antigen mRNA prepared according to the method of claim 1 or 2. 4 . The single-epitope antigen mRNA according to claim 3 , further comprising, from 5′ to 3′, a 5′ cap, a 5′ UTR, an MHC-I SP, an antigen epitope sequence in the coding region, a MITD, a 3′ UTR, and a polyA tail.

5. A single epitope antigen mRNA library, characterized in that: Comprising two or more single-epitope antigen mRNAs according to claim 3 or 4.

6. Use of the method for constructing single epitope antigen mRNA according to claim 1 or 2 in the preparation of tumor vaccines or infectious disease vaccines.

Images