Rapid mRNA construction method and use thereof

Abstract

The present invention provides a rapid mRNA construction method and a use thereof. The method uses a pre-prepared double-stranded DNA fragment library and full-length amplification primer and a chemically synthesized oligonucleotide (a target protein primer) to achieve rapid assembly synthesis under the catalytic synthesis of a high-fidelity DNA polymerase, and the synthesized linear double-stranded DNA, after simple purification, can be used for an in vitro transcription reaction for synthesis of target mRNA. The rapid mRNA construction method can be used for rapidly synthesizing a tumor mRNA vaccine encoding a single tumor antigen epitope, and the tumor mRNA vaccine can further be mixed to form a mRNA vaccine library for tumor treatment. In addition, in the present invention, a rapid identification method for neoantigens in tumor patients is further developed on the basis of the rapid mRNA construction method.

Description

A rapid mRNA construction method and its application Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the construction method of mRNA drugs and their applications. Background Technology

[0002] Tumor immunotherapy has become a hot area in cancer drug development in recent years due to its significant efficacy. Among them, cancer mRNA vaccines, as an emerging immunotherapy approach, have shown broad application prospects in precision treatment and personalized medication for patients. Recent studies have shown that mRNA vaccines based on personalized tumor neoantigens have demonstrated excellent efficacy in multiple clinical trials [1, 2]. However, despite the great potential of this technology, its widespread application still faces several technical bottlenecks: First, the traditional process for manufacturing mRNA vaccines is time-consuming, usually requiring several weeks, and is costly; second, because personalized cancer mRNA vaccines are designed specifically for individual patients, their flexibility and cost-effectiveness are severely limited; finally, there is currently a lack of rapid, low-cost experimental methods to efficiently screen tumor neoantigens, which further restricts the large-scale development of its application.

[0003] mRNA vaccines have been widely used during the COVID-19 pandemic. Their excellent efficacy and good safety have attracted great attention and are regarded as an important tool for humans to fight diseases in the future [3]. The design of mRNA drugs mimics the structure of endogenous mRNA in eukaryotic cells, including a 5' cap, a 3' polyadenylate tail, an untranslated region, and a gene coding sequence [4]. Currently, 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, bacterial culture tanks occupy a lot of space, making it difficult to meet the demand for low-cost and rapid synthesis of various different mRNA sequences.

[0004] Currently, personalized tumor neoantigen mRNA vaccines typically employ a design with up to 20 antigen tandems, enabling a single mRNA to encode as many candidate neoantigen sequences as possible, thereby significantly increasing the likelihood of inducing a positive immune response and achieving clinical efficacy [6]. However, due to the high diversity of gene mutations in cancer patients, these mRNA vaccines are usually designed for individual patients and are difficult to use universally among patients. In addition, traditional mRNA manufacturing processes are costly and time-consuming, posing a significant challenge to the widespread application of such personalized mRNA vaccines in large patient populations.

[0005] On the other hand, the screening of tumor neoantigens and the assessment 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, thereby detecting the immune response of T cells to specific antigenic peptides. However, the synthesis cycle of peptides is long and the cost of synthesizing peptide libraries is high, which to some extent limits the efficiency and popularity of these technologies in large-scale tumor neoantigen screening.

[0006] In summary, to enable the widespread application of cancer mRNA vaccines, optimization is needed in the following three areas: mRNA manufacturing process, personalized mRNA vaccine design for tumors, and experimental screening methods for tumor neoantigens. Summary of the Invention

[0007] To address the above technical problems, this invention establishes a method for synthesizing single antigenic epitope mRNA using a pre-synthesized double-stranded DNA fragment library, full-length amplification primers, and chemically synthesized oligonucleotides (target protein primers) under the catalysis of DNA polymerase, 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 this invention provides a rapid method for constructing mRNA, the method comprising the following steps: 1) pre-synthesizing a DNA fragment library and full-length amplification primers; 2) designing target protein primers; 3) first round of bridging amplification; 4) second round of in vitro transcription template amplification and optionally template purification; 5) in vitro transcription synthesis of target protein mRNA.

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

[0010] In some implementations, the full-length amplification primers in step 1) are selected from forward primers containing promoters and / or reverse primers containing polyT.

[0011] In some implementations, the promoter is the T7 promoter.

[0012] In some embodiments, the signal peptide is the signal peptide of a major histocompatibility antigen class I protein (MHC-I SP) in humans or mice.

[0013] In some embodiments, the transmembrane domain is the MITD (Transmembrane Domain of Major Histocompatibility Antigen Class I Proteins of Humans or Mice).

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

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

[0016] In some implementations, the MITD encoded sequence and the 3'UTR are fused (MITD+3'UTR).

[0017] In some implementations, 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 some embodiments, the 3' end of the promoter-containing forward primer overlaps with the 5' end of the 5'UTR positive strand, and the 3' end of the polyT-containing reverse primer is complementary to the 3' end of the 3'UTR positive strand.

[0019] In some embodiments, the number of overlapping or complementary base pairs is greater than 12.

[0020] In some embodiments, the number of overlapping or complementary base pairs is 12-30.

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

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

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

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

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

[0026] In some embodiments, different numbers of primers are used to assemble insert fragments of different lengths; preferably, the number of primers is 2-8; preferably, each primer is 42-78 nt in length, for example, about 60 nt.

[0027] In some embodiments, the antigen primer in step 2) has a sequence of 12 to 30 nt complementary to the preceding and following fragments; preferably, the number of complementary base pairs is 15; preferably, the preceding fragment is a primer, 5'UTR, or signal peptide coding sequence linked to the preceding fragment; preferably, the following fragment is a primer, 3'UTR, or transmembrane domain coding sequence linked to the following fragment.

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

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

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

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

[0032] In some implementations, the amplification program is (1) 98℃ for 30s; (2) 98℃ for 10s, 60℃ for 10s, 72℃ for 15s, repeated 7 times; (3) 72℃ for 30s, 4℃ for ∞.

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

[0034] In some embodiments, the amplification program is: (1) 98℃ for 30s; (2) 98℃ for 10s, 63℃ for 10s, 72℃ for 15s, repeated 25 times; (3) 72℃ for 30s, 4℃ for ∞.

[0035] The second aspect of the present invention provides a method for preparing target protein mRNA according to the method described in the first aspect of the present invention.

[0036] In some embodiments, the mRNA is antigen mRNA.

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

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

[0039] A third aspect of the present invention provides a monoepisode antigen mRNA library, wherein the monoepisode antigen mRNA library contains two or more monoepisode antigen mRNAs as described in the second aspect of the present invention.

[0040] The fourth aspect of this invention provides the application of the single epitope antigen mRNA library described in the third aspect of this invention in the rapid validation of tumor neoantigens.

[0041] In some implementations, peripheral blood mononuclear cells (PBMCs) or B cells of patients are transfected with a mixed library of single epitope antigen mRNAs to stimulate isolated blood T cells or tumor-infiltrating T cells (Tils). After culturing for several days, B cells transfected with a single single epitope antigen mRNA are used for re-stimulation, and antigenic epitopes with immunogenicity are screened by detection.

[0042] In some implementations, the detection is an Elispot detection.

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

[0044] The sixth aspect of the present invention provides a vaccine comprising 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 some embodiments, the vaccine further includes an mRNA delivery vector.

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

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

[0048] 1) This invention designs a rapid mRNA construction method: significantly shortens the time for constructing the target mRNA—after designing the sequence, all processes from primer synthesis to mRNA synthesis can be completed in as little as one day; significantly reduces the cost of constructing the target mRNA—this method eliminates the processes of culturing bacteria, purifying plasmids, and linearizing with enzyme digestion, saving the cost of these processes; significantly reduces the production space required for mRNA development and production—this method does not require large culture tanks, and mRNA can be developed and prepared in large quantities using small-volume reactors; mRNA construction has the advantage of modularity—preparing different DNA fragments can easily replace functional sequences such as untranslated regions and signal peptides in mRNA.

[0049] 2) The single-epitope antigen mRNA designed in this invention employs a rapid construction method, enabling the rapid and low-cost construction of a single-epitope antigen mRNA library. This library can be flexibly combined to create personalized neoantigen mixed vaccines for tumors, avoiding the problem of interference between multiple antigens on the same amino acid chain. This mixed vaccine not only exhibits excellent tumor-specific immune induction and tumor therapeutic effects in animal models but also demonstrates very good induction effects on human peripheral blood mononuclear cells, achieving a shorter production cycle and more flexible application scenarios compared to traditional tandem mRNA.

[0050] 3) The method of rapidly validating tumor neoantigens using a rapidly synthesized single-epitope mRNA antigen library significantly reduces costs and shortens the validation cycle compared to the traditional method using peptide libraries. Furthermore, this rapid validation method innovatively transfects mRNA directly into PBMCs or easily amplified B cells to amplify antigen-recognizing T cells, and then restimulates T cells with B cells, making it more convenient and lower cost-effective than the traditional DC stimulation method. Attached Figure Description

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

[0052] Figure 1 shows a schematic diagram of a rapid mRNA construction method (taking tumor single epitope antigen mRNA as an example). After designing the target fragment in the middle of the mRNA coding region, short oligonucleotide primers are synthesized and assembled. The remaining relatively fixed coding region (CDS) and uncoding region (UTR) sequences are pre-synthesized. This fragment is bridged by a first round of 7 cycles of PCR amplification. Then, a second round of 25 cycles of PCR amplification is performed using full-length primers containing the T7 promoter and polythymine (PolyT), respectively. The resulting template DNA is purified and can be used for in vitro transcription to synthesize the target mRNA.

[0053] Figure 2 shows the design of monoepisode antigen mRNA, its rapid construction results, and protein expression validation. (A) Schematic diagram of monoepisode antigen mRNA design: The monoepisode mRNA consists of CAP1 or other types of caps from 5' to 3', 5' UTR, SP (human MHC-I signal peptide), Ag (antigenic epitope sequence, encoding approximately 25 amino acids), MITD (human MHC-I transmembrane domain), 3' UTR, and polyA (polyadenylate, typically 120 A's). (B) Agarose gel electrophoresis results of the monoepisode antigen DNA template synthesized using the rapid construction method. (C) Agarose gel electrophoresis results of the monoepisode antigen mRNA synthesized using the rapid construction method. (D) Western blot results showing the protein expression of the rapidly constructed monoepisode antigen mRNA in HEK293T cells.

[0054] Figure 3 shows the rapid large-scale validation of candidate tumor neoantigens in patients using rapidly constructed single-episode antigen mRNA. (A) Schematic diagram of the rapid validation method for tumor neoantigens based on single-episode antigen mRNA. After analyzing the candidate tumor neoantigens, a single-episode mRNA library of these antigens was synthesized within one day. PBMCs or B cells were transfected with the mixed single-episode mRNA library, followed by the first round of T cell stimulation. After T cells were cultured overnight without cytokines, they were restimulated with B cells transfected with a single single-episode mRNA, and finally, positive antigens were detected by elispot assay. (B) Immunogenicity results of tumor neoantigens from three ovarian cancer patients were validated using the above method.

[0055] Figure 4 shows the conditions for exploring the rapid mRNA construction method. (A) Schematic diagram of Gaussian luciferase (GLUC) mRNA construction using the rapid mRNA construction method. (B) Agarose gel electrophoresis results of template DNA at different temperatures during the first and second rounds of PCR. (C) Schematic diagram of GLUC mRNA construction using primers with complementary sequences of different lengths. (D) Agarose gel electrophoresis results of template DNA constructed using primers with complementary sequences of different lengths. (E) Comparison of GLUC signals of mRNA constructed using primers with complementary sequences of different lengths.

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

[0057] Figure 6 shows the immunogenicity of single antigen-mixed mRNAs (4-positive and 20-antigen mixtures) synthesized using a rapid construction method compared to conventional 20-antigen tandem mRNAs. (A) The proportion of CD8 T cells (IFNγ-positive) responding to the mixed 4-positive antigen, antigen P9, and B3 antigen mRNAs out of all splenic CD8 T cells. (B) The proportion of CD4 T cells (IFNγ-positive) responding to the mixed 4-positive antigen, antigen P9, P15, and B5 antigen mRNAs out of all splenic CD4 T cells.

[0058] Figure 7 shows the effect of mixed viral antigen mRNA synthesized using a rapid construction method on the immune induction of healthy human PBMCs. Twenty mixed viral single antigen mRNAs (MIX) and their corresponding tandem mRNAs (Concat) were used to stimulate T cells, followed by re-stimulation of T cells with single antigen mRNAs, and the IFNγ elispot detection results were obtained. The lower two halves of the figure use PBMCs from two different healthy individuals.

[0059] Figure 8 shows the treatment of a mouse subcutaneous melanoma model with 8-antigen mixed mRNA synthesized using a rapid construction method. (A) and (B) Tumor anatomy photographs, tumor growth curves, and mouse body weight changes on day 17 of the mouse subcutaneous B16F10 tumor model treated with 8 mixed single epitope antigen mRNA (B16-8MIX) and 8 antigen tandem mRNA (B16-8). (C) and (D) Proportion of neoantigen-responsive (IFN-γ positive) CD8 and CD4 T cells in the mouse spleen. Detailed Implementation

[0060] Rapid mRNA Construction Method

[0061] This invention provides a modular, rapid mRNA construction method that is independent of bacteria and plasmids. The method primarily uses chemically synthesized oligonucleotides to assemble the target gene fragment via DNA polymerase chain reaction (PCR), and simultaneously assembles and amplifies a complete template for mRNA synthesis using other modular fragment libraries with fixed sequences.

[0062] The rapid mRNA construction method of the present invention consists of 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 (as shown in Figure 1).

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

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

[0065] Primer preparation: Based on the sequences of the 5'UTR (e.g., SEQ ID NO: 5) and 3'UTR (e.g., SEQ ID NO: 6), forward primers (T7-F, SEQ ID NO: 7) containing the T7 phage promoter and reverse primers (PolyA120-R, SEQ ID NO: 8) containing 120 thymines were designed and were prepared in advance by Huzhou Hippo Biotechnology Co., Ltd.

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

[0067] 3) First round of bridging amplification (taking a 50ul amplification system as an example): using Yisheng Bio's 2×Hieff The AdvanceFast PCR Master Mix was mixed with 5 ng of 5'UTR+SP, 5 ng of MITD+3'UTR fragments and 0.5 pmol / each of antigen primers, and an appropriate amount of ddH2O was added to prepare a 50 μL amplification system. The amplification was performed according to the following program for 7 cycles: (1) 98℃ for 30 s; (2) 98℃ for 10 s, 60℃ for 10 s, 72℃ for 15 s, for 7 cycles; (3) 72℃ for 30 s, 4℃ for ∞.

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

[0069] 5) Run the reaction system from step 4 on an agarose gel and then cut the gel for purification.

[0070] 6) In vitro transcription synthesis of single epitope antigen mRNA: Using the T7 High Yield RNA Synthesis Kit for Co-transcription from Yisheng Biotechnology, ATP, N1-Me-Pseudo UTP, CTP, GTP, GAG, and 10×Transcription Buffer were mixed sequentially. The template DNA purified in step 5 (500 ng in a 20 μL system) and T7 RNA Polymerase Mix were added, along with an appropriate amount of RNase-free Wattite. The mixture was incubated at 37°C for 2 hours. The RNA was purified and quality tested.

[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 includes an antigenic epitope sequence in the coding region (encoding about 25 amino acids), a signal peptide (MHC-I signal peptide, SP) and a 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 some embodiments, the single epitope antigen mRNA comprises, from 5' to 3', a 5' cap, a 5' UTR, an MHC-I SP, an antigenic epitope sequence in the coding region, MITD, a 3' UTR, and a polyA tail.

[0074] The present invention also provides a monoepisode antigen mRNA library, wherein the mRNA library contains two or more monoepisode antigen mRNAs.

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

[0076] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

[0078] The embodiments of this invention first explored the optimal reaction strips for a rapid mRNA construction method. Then, the antigen-specific T cell induction ability of single-episode antigen mRNA was verified in mice and healthy human PBMCs. Finally, a hybrid single-episode antigen mRNA vaccine for mouse B16F10 cells was designed, and its immune induction and tumor therapeutic effects were found to be superior to tandem antigen mRNA vaccines.

[0079] Example 1: Rapid mRNA Construction Method

[0080] To verify the feasibility of the rapid mRNA construction method, the mRNA of the Gaussian luciferase (GLUC) reporter gene was constructed to facilitate the detection of mRNA product activity. Fragments containing the 5' UTR and GLUC 1-147 bp (SEQ ID NO: 12) and GLUC 223-558 bp and 3' UTR (SEQ ID NO: 13) were obtained by digesting plasmid DNA. Two 60 nt oligonucleotide primers (SEQ ID NO: 14-15, synthesized by Suzhou Genewiz) were designed to assemble 75 bases of GLUC 148-222 bp and complement the ends. Two rounds of PCR were designed. In the first round, seven amplification cycles were performed to bridge the primers and fragments. In the second round, two primers (SEQ ID NO: 16-17) containing the T7 promoter and 120 thymine (reverse primers, resulting in a template strand of 120 adenine) were added, and 25 amplification cycles were performed to amplify a DNA template suitable for in vitro transcription (Figure 4A). The PCR reaction employed a common three-step amplification method, including denaturation, annealing, and extension (using 2×Hieff Canace AdvanceFast PCR Master Mix, Yisheng Biotechnology). The inventors first explored two rounds of PCR using a series of annealing temperatures (Figure 4B), finding that different annealing temperatures had little effect on synthesis efficiency in the first round, while the second round achieved the best amplification efficiency at 63℃ to 65℃. To find the optimal primer length and the length of complementary sequences between the primers and fragments, a GLUC construction method with an intermediate primer was designed (Figure 4C). The fragment with 5'UTR + "GLUC 1-147bp" is shown in SEQ ID NO: 12, and the fragment with "GLUC 178-558bp" + 3'UTR is shown in SEQ ID NO: 25. The reaction conditions for this method are the same as in Figure 2A, but different lengths of intermediate primers (42nt, 48nt, 54nt, 60nt, 66nt, 72nt, and 78nt, SEQ ID NO: 18-24, respectively) were used, with corresponding complementary sequence lengths of 6nt, 9nt, 12nt, 15nt, 18nt, 21nt, and 24nt. The results showed that the target DNA could only be amplified 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 4D). GLUC mRNA was synthesized using the template obtained from the reaction and transfected into HEK293T cells to detect mRNA activity. It was found that the activity of GLUC mRNA constructed using primers with complementary sequences of 15nt-24nt showed no significant difference, and the activity of these mRNAs was also not significantly different from that of control GLUC mRNA synthesized using a linearized plasmid template (with the same UTR sequence and polyA length) (Figure 4E).In summary, the method designed in this invention successfully constructed highly active mRNA.

[0081] Similarly, the GLUC mRNA construction method was further tested to insert gene fragments of different lengths to explore the maximum length of the target gene fragment that this method can insert. The inventors attempted to assemble insert fragments of different lengths (75nt-345nt) using different numbers (2-8) of 60nt primers to synthesize complete GLUC mRNA (Figure 5A). Primers 1 to 8 are shown in SEQ ID NO: 14-15 and SEQ ID NO: 28-33, respectively. Agarose gel electrophoresis of the synthesized template DNA showed that when the number of primers increased from 2 to 8, i.e., the insert sequence length increased to 345nt, the method of this invention still successfully synthesized high-purity template DNA (Figure 5B). Further synthesis of GLUC mRNA and detection of its activity revealed 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 5C). This may be because too many different sequence primers increase the probability of incorrect complementary pairing, leading to the generation of more mutated GLUC mRNA.

[0082] T cell-recognized antigen sequences typically consist of only 9-25 amino acids. Based on this, this invention designs a short, single-epitope mRNA containing the essential 5'UTR, 3'UTR, signal peptide of the major human histocompatibility antigen class I protein, and a transmembrane sequence, as well as approximately 25 amino acid sequences of the target antigen (Figure 1). After identifying the epitope, constructing such a single-epitope mRNA simply requires replacing the intermediate target antigen sequence, making it easily constructable using the rapid construction method of this invention. According to this method (Figure 1), 10 pairs of 60nt primers were synthesized, successfully constructing DNA templates and mRNAs for ten different antigens (Figure 2A, Figure 2B), as shown in Table 1 below. Western blot results further showed that these mRNAs successfully expressed the target protein in HEK293T cells (Figure 2C). In summary, the rapid construction method of this invention can be used for the rapid synthesis of single-epitope mRNAs.

[0083] Table 1. Information on primers and full-length DNA for 10 antigens

[0084] Note: The primers in Table 1 are DNA sequences.

[0085] Example 2: Immunoinduction effect of mixed single-episode tumor antigen mRNA in mice

[0086] Traditional personalized tumor neoantigen mRNA vaccines typically use up to 30 neoantigens tandemly to form a single mRNA vaccine. However, the rapid mRNA construction method and single-epitope mRNA designed in this invention can synthesize dozens of single-epitope mRNA vaccines in a very short time. These single-epitope mRNAs can be easily combined, offering greater flexibility in later clinical applications compared to multi-epitope tandem mRNAs, and saving time in plasmid construction. To evaluate the immunogenicity of the mixed single-epitope antigen mRNAs, this invention selected 20 mouse-derived mutant tumor neoantigens containing 4 positive epitopes and 16 negative epitopes, constructing them all into single-epitope mRNA vaccines. Simultaneously, tandem mRNAs of these 20 epitopes were constructed using conventional methods for comparison. The mRNA sequences of the 20 antigenic epitopes are shown in SEQ ID NO: 64-83, and the sequence of the tandem mRNA is shown in SEQ ID NO: 84. Mice were immunized with a mixed antigen vaccine containing 4 positive antigens (4MIX), a mixed antigen vaccine containing all 20 antigens (20MIX), and a tandem 20 antigen mRNA vaccine. In this invention, mice were injected with two doses of vaccine, one week apart, 5 μg / mouse each time. After the second dose, splenic lymphocytes were isolated and transfected with a mixture of four positive epitope mRNAs or four positive epitope mRNAs separately to detect the proportion of IFNγ-positive T cells. The results showed that when splenic lymphocytes were transfected with mixed positive mRNAs, the group immunized with a mixture of four positive antigens (4MIX) had the highest proportion of CD8 and CD4 T cells secreted by IFNγ, followed by the group immunized with a mixture of 20 antigens (20MIX), and the group immunized with tandem mRNAs (20C) had the lowest proportion (Figure 6A). This result indicates that both mixed antigen vaccines had good immune induction effects. Analyzing the proportion of T cells responding to each of the four positive antigens (IFNγ-positive), this invention found that splenic lymphocytes in both the 4MIX and 20MIX groups responded well to all four positive antigens (Figure 6B). While group 20C had the highest proportion of B3-specific CD8 T cells, its response to the other three antigens was poor, with almost no P9-specific CD8 T cells detected. This phenomenon may suggest that some epitopes in tandem mRNAs may be ineffective, while single-episode mRNAs, because each epitope is translated separately, may avoid the ineffectiveness of positive epitopes. These results indicate that vaccines containing a mixture of multiple single-episode antigen mRNAs have excellent immunogenicity, while the immunogenicity of tandem epitope mRNA vaccines varies greatly depending on the epitope, and induction failure can even occur.

[0087] Table 2. mRNA sequences of 20 mouse-derived mutant tumor neoantigens

[0088] Note: SEQ ID NO: 64-84 is the full-length mRNA sequence.

[0089] Example 3: The ability of mixed single-epitope viral antigen mRNA to induce human immune cell responses

[0090] To test the ability of monoepisode mRNAs synthesized using a rapid construction method to induce responses in human immune cells, the inventors synthesized monoepisodes and tandem mRNAs of 20 viral antigens. The mRNA sequences of the 20 viral antigens are shown in SEQ ID NO:85-104, and the sequences of the tandem mRNAs are shown in SEQ ID NO:105 (Table 3). First, mixed monoepisode mRNAs or tandem mRNAs were transfected into PBMCs of healthy individuals and amplified for 7 days. Then, these PBMCs were stimulated with B cells transfected with a single monoepisode mRNA, and the number of IFN-γ-positive T cells was detected using elispot assay. The response results for the 20 viral antigens showed that the mixed monoepisode mRNAs induced IFN-γ-positive T cells more effectively than the tandem mRNAs (Figure 7).

[0091] Table 3. Sequence information of 20 viral antigens

[0092] Note: SEQ ID NO: 85-104 is the full-length mRNA sequence.

[0093] Example 4: Validating the tumor therapeutic effect of the mixed single-episode mRNA vaccine in an animal tumor model.

[0094] The ultimate goal of developing tumor vaccines is to efficiently induce tumor antigen-specific T cells that recognize and kill tumor cells. The results above indicate that the mixed single-episode mRNA vaccine has a good induction effect on T cell immunity in both mice and humans. This prompted this invention to verify the tumor therapeutic effect of the mixed single-episode mRNA vaccine in animal tumor models. This invention synthesized single-episode mRNA and tandem mRNA of eight neoantigens of B16F10 tumor cells. After packaging with SM102 LNP, these were injected subcutaneously into mice with B16F10 tumors. The mRNA sequences of the eight tumor neoantigens 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, tumor growth was significantly inhibited in both the mixed single-episode mRNA vaccine (B16-8MIX) and the tandem mRNA vaccine (B16-8) groups (Figures 8A and 8B), with the B16-8MIX group showing the smallest tumor volume on day 17. Splenic lymphocytes were isolated on day 17 after tumor inoculation, and neoantigen-specific T cells were detected by flow cytometry. The B16-8MIX group showed the highest proportions of neoantigen-specific CD8 and CD4 T cells. These results indicate that the mixed single-epitope mRNA vaccine induces higher levels of tumor neoantigen-specific immunity and better tumor treatment efficacy.

[0095] Table 4. Neoantigen sequence information of 8 B16F10 tumor cells

[0096] Note: SEQ ID NO: 116-114 is the full-length mRNA sequence.

[0097] Example 5: Rapid Validation Method for Tumor Neoantigens Based on Single Epitope Antigen mRNA Library

[0098] Peripheral blood mononuclear cells (PBMCs) or B cells from patients were transfected with a mixed library of single-epitope antigen mRNAs (SEQ ID NO: 115-173, with 120 nt polyA added). This stimulated isolated blood T cells or tumor-infiltrating T cells (Tils). After 7 days of culture, B cells transfected with a single single-epitope antigen mRNA were used for restimulation. Finally, Elispot assays were performed to identify immunogenic antigenic epitopes (Figure 3A). Using this rapid validation method, antigenic epitopes that could be recognized by the T cells of three ovarian cancer patients were detected (Figure 3B). The specific rapid validation method is as follows:

[0099] 1) Analyze and synthesize a patient monoepithelial antigen mRNA library.

[0100] 2) Thaw frozen PBMCs.

[0101] 3) Using CALNP TMmRNA in vitro transfection reagent (Dona Medical), 500 ng of mixed patient single epitope antigen mRNA per well.

[0102] 4) DC cell maturation needs to be promoted 8 hours after transfection: Do not change the medium after 8 hours, add 100ul DC medium containing GM-CSF (1ul / mL, PeproTech) and IL4 (5ul / mL, PeproTech), and add twice the amount of LPS (2×, 20ul / ml, Sigma) and IFN-γ (2×, 2ul / ml, PeproTech). Promote maturation for 16 hours.

[0103] 5) Carefully replace half of the culture medium and culture with T cell culture medium, while adding PeproTech IL-2 (0.5ul / ml, PeproTech), IL-7 (2ul / ml, PeproTech), and IL-15 (2ul / ml, PeproTech). From day two to day seven, replace half of the medium every 2-3 days: discard half of the supernatant, add 2× or more cytokines to T median, and continue culturing.

[0104] 6) On day 8, merge cells from the same group, count them, resuspend them at 100 w / ml, and add IL2, IL7, and IL5 to 24 wells. Treat PBMCs: centrifuge at 400g for 5 min, discard the supernatant. Culture overnight with T median (without cytokines).

[0105] 7) At the same time, prepare the B cells and B medium to be used, and add IL-4 (2ul / ml, Peprotech), IL21 (2ul / ml, ACROBiosystems), CD40L (3ul / ml, ACROBiosystems), and BME (1ul / ml, Sigma-Aldrich) for culture.

[0106] 8) Co-incubation of B cells and T cells: All B cells were aspirated, centrifuged at 400g for 5min, the supernatant was discarded, and the cells were resuspended in 2ml of empty culture. The cells were counted and replenished to 100w / ml. T cells were aspirated from 24-well plates, counted directly, centrifuged at 400g for 5min, the supernatant was discarded, and the cells were resuspended in empty culture to 200w / ml.

[0107] 9) Use GenNano-W0021 (Maianna) to prepare a single single epitope antigen mRNA transfection complex and add it to each well of a 96-well plate. Add 50 μL of B cells (5w), 50 μL of T cells (10w), and 50 μL of empty culture to each well of the RNA transfection complex.

[0108] After thoroughly mixing the B-cell and T-cell co-incubation system, carefully add it to the Elispot wells (each tube corresponds to each well), incubate at 37°C in a 5% CO2 incubator for 40 hours, and then perform the detection.

[0109] As shown in Figure 3B, the SUPV3L1 antigen in patient OC41, VIRMA, KIAA1217, AP1G2 in patient OC44, and GART antigen epitope in patient OC50 activated patient T cells, demonstrating immunogenicity and clinical application value.

[0110] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the scope of the invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0111] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This description is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0112] References

[0113] 1.Rojas LA, Sethna Z, Soares KC, et al.Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer.Nature.2023;618(7963):144-150.doi:10.1038 / s41586-023-06063-y

[0114] 2.Weber JS,Carlino MS,Khattak A,et al.Individualised neoantigen therapy mRNA-4157(V940)plus pembrolizumab versus pembrolizumab monotherapy in resected melanoma(KEYNOTE-942):a randomised,phase 2b study.Lancet.2024;403(10427):632-644.doi:10.1016 / S0140-6736(23)02268-7

[0115] 3.Baden LR,El Sahly HM,Essink B,et al.Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine.N Engl J Med.2021;384(5):403-416.doi:10.1056 / NEJMoa2035389

[0116] 4.Rohner E,Yang R,Foo KS,Goedel A,Chien KR.Unlocking the promise of mRNA therapeutics.Nat Biotechnol.2022;40(11):1586-1600.doi:10.1038 / s41587-022-01491-z

[0117] 5.Bancel S,ISSA WJ,AUNINS JG,Chakraborty T.Manufacturing methods for production of rna transcripts.Published online September 25,2014.Accessed December 28,2024.https: / / patents.google.com / patent / WO2014152027A1 / en

[0118] 6.Cafri G,Gartner JJ,Zaks T,et al.mRNA vaccine-induced neoantigen-specific T cell immunity in patients with gastrointestinal cancer.J Clin Invest.2020;130(11):5976-5988.doi:10.1172 / JCI134915

[0119] 7.Lybaert L,Lefever S,Fant B,et al.Challenges in neoantigen-directed therapeutics.Cancer Cell.2023;41(1):15-40.doi:10.1016 / j.ccell.2022.10.013

Claims

1. A rapid method for constructing mRNA, characterized in that, The process includes the following steps: 1) pre-synthesizing a DNA fragment library and full-length amplification primers; 2) designing target protein primers; 3) first-round bridging amplification; 4) second-round in vitro transcription template amplification and optional template purification; 5) in vitro transcription synthesis of target protein mRNA.

2. The method according to claim 1, wherein the DNA fragment library in step 1) is selected from one or more fusions or combinations of 5'UTR, 3'UTR, signal peptide coding sequence, and transmembrane domain coding sequence; the full-length amplification primers are selected from forward primers containing a promoter and / or reverse primers containing polyT; preferably, the promoter is the T7 promoter; preferably, the signal peptide is the signal peptide (MHC-I SP) of a major histocompatibility antigen class I protein of humans or mice; preferably, the transmembrane domain is the transmembrane domain MITD of a major histocompatibility antigen class I protein of humans or mice.

3. The method according to claim 2, wherein the 5'UTR and the signal peptide coding sequence are fused, and the transmembrane domain coding sequence is fused with the 3'UTR; preferably, the 5'UTR and the signal peptide coding sequence of a major histocompatibility antigen class I protein are fused (5'UTR+MHC-I SP); preferably, the MITD coding sequence is fused with the 3'UTR (MITD+3'UTR).

4. The method according to claim 2, wherein the 3' end of the forward primer containing the promoter overlaps with the 5' end of the 5' UTR positive strand, and the 3' end of the reverse primer containing polyT is complementary to the 3' end of the 3' UTR positive strand; preferably, the number of overlapping or complementary bases is greater than 12; preferably, the number of overlapping or complementary bases is 12-30.

5. The method according to any one of claims 1-4, wherein the target protein in step 2) is an antigen, antibody, cytokine, or other therapeutic protein; preferably, the antigen is a viral antigen, tumor antigen, or bacterial antigen; preferably, the length of the antigen is 20-120 amino acids; more preferably, the length of the antigen is 20-30 amino acids; even more preferably, the length of the antigen is about 25 amino acids.

6. The method according to any one of claims 1-5, wherein different numbers of primers are used to assemble insert fragments of different lengths; preferably, the number of primers is 2-8; preferably, the length of each primer is 42-78 nt, for example, about 60 nt.

7. The method according to any one of claims 1-6, wherein the antigen primer in step 2) has a sequence of 12 to 30 nt complementary to the preceding and following fragments; preferably, the number of complementary base pairs is 15; preferably, the preceding fragment is a primer, 5'UTR, or signal peptide coding sequence connected to the preceding fragment; preferably, the following fragment is a primer, 3'UTR, or transmembrane domain coding sequence connected to the following fragment.

8. The method according to claims 1-7, wherein 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.

9. The method according to any one of claims 1-8, wherein the first round of bridging amplification reaction system in step 3) comprises antigen primers, 5'UTR+MHC-I SP, MITD+3'UTR and PCR premix; preferably, the amplification program is (1) 98℃ for 30s; (2) 98℃ for 10s, 60℃ for 10s, 72℃ for 15s, for 7 cycles; (3) 72℃ for 30s, 4℃ for ∞.

10. The method according to any one of claims 1-9, wherein step 4) second round of in vitro transcription template amplification includes adding a forward primer containing a promoter and a reverse primer containing polyT to the reaction of step 3); preferably, the amplification program is: (1) 98℃ for 30s; (2) 98℃ for 10s, 63℃ for 10s, 72℃ for 15s, for 25 cycles; (3) 72℃ for 30s, 4℃ for ∞.

11. The target protein mRNA prepared by the method according to any one of claims 1-10; preferably, the mRNA is an antigen mRNA; more preferably, the antigen mRNA is a single epitope antigen mRNA; even more preferably, the single epitope antigen mRNA contains, in sequence from 5' to 3', a 5' cap, a 5' UTR, an MHC-ISP, an antigenic epitope sequence of the coding region, MITD, a 3' UTR, and a polyA tail.

12. A single epitope antigen mRNA library, characterized in that, It contains two or more single epitope antigen mRNAs as described in claim 11.

13. The application of the single epitope antigen mRNA library according to claim 14 in rapid validation of tumor neoantigens; preferably, the application includes transfecting patient peripheral blood mononuclear cells (PBMCs) or B cells with a mixed library of single epitope antigen mRNAs, stimulating isolated blood T cells or tumor-infiltrating T cells (Tils), culturing for several days, and then restimulating with B cells transfected with a single single epitope antigen mRNA, and screening for immunogenic antigen epitopes by detection; preferably, the detection is the Elispot assay.

14. The use of the single epitope antigen mRNA of claim 11 or the single epitope antigen mRNA library of claim 12 in the preparation of tumor vaccines or infectious disease vaccines.

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