Application of triple-helix structural element in RNA (Ribonucleic Acid) drug design

Abstract

The invention discloses application of a triple-helix structural element in RNA (Ribonucleic Acid) drug design, and belongs to the field of biological medicines. According to the application, an ENE element or a mutant of the ENE element is used as a nucleic acid stabilizing element, and the interaction between 3 'UTR and poly (A) is optimized, so that the mRNA stability is enhanced, and the protein translation efficiency is further improved, and therefore, the problem of low protein expression quantity in RNA therapy is effectively solved. The mRNA sequence optimization strategy provided by the invention provides a new method for vaccine design in the field of infectious disease and tumor immunotherapy, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to enhancing the expression of mRNA vaccines by utilizing the ENE element sequence in the non-coding RNA PAN of Kaposi's sarcoma-associated herpesvirus (KSHV) to optimize the design of mRNA vaccine sequences and improve their immunogenicity. Specifically, it involves exploring the insertion position of the ENE element in the 3'UTR of the mRNA vaccine, optimizing mutations based on the triple helix core structure, and applying this element in the field of mRNA vaccine therapy for infectious diseases. Background Technology

[0002] Since the outbreak of the 2019 coronavirus (COVID-19), mRNA vaccines have demonstrated great potential in infectious disease control due to their advantages of rapid development, programmability, and scalability (Qin et al., 2022; Sahin et al., 2014). This progress has driven the rapid development of mRNA-based therapies and vaccines, with mRNA platforms providing a versatile framework for the rapid development of interventions against various infectious diseases, cancer immunotherapies, and protein replacement therapies (Pardiand Krammer, 2024; G. Zhang et al., 2023). However, existing RNA therapies suffer from low cytoplasmic stability and low protein expression levels, resulting in the need for higher doses to achieve the desired effects. This not only increases the risk of toxicity but also raises production and usage costs (Metkar et al., 2023; Weng et al., 2020). Therefore, there is an urgent need to improve the stability and protein expression efficiency of mRNA through sequence optimization and structural modification to further promote its clinical application.

[0003] mRNA molecules typically consist of key components such as a 5' cap, a 5' untranslated region (5'UTR), a coding sequence (CDS), a 3' untranslated region (3'UTR), and a poly(A) tail (Metkar et al., 2023). Optimizing these components is a core strategy for achieving stable and efficient mRNA expression. The 3'UTR plays a crucial role in regulating mRNA dynamics, influencing its subcellular localization, stability, and translation initiation (Matoulkova et al., 2012; Rasekhian et al., 2021). This region often contains regulatory elements, such as microRNA (miRNA) binding sites and RNA-binding protein (RBP) recognition motifs, affecting mRNA stability (Chan et al., 2023; Tian and Manley, 2017).

[0004] The poly(A) tail is another key factor determining mRNA stability and translation efficiency (Passmore and Coller, 2022). Traditionally, the poly(A) tail was considered to be just a simple adenosine chain, with limited potential for optimization in improving translation efficiency; related research has largely focused on tail length selection (Xiang and Bartel, 2021). However, existing research has shown that the structure and composition of the poly(A) tail have a significant impact on mRNA stability and translational activity. Studies have shown that cytidine-intercalated poly(A) tails can protect mRNA from CCR4-NOT complex-mediated deadenylation, thereby significantly prolonging translation duration and increasing protein yield (Li et al., 2022). Similarly, rationally designed multi-tailed poly(A) structures have been shown to significantly improve translation ability without affecting translation initiation (Chen et al., 2024). Furthermore, chimeric DNA-poly(A) tails have also been shown to enhance the immunogenicity of mRNA vaccines. This demonstrates that the poly(A) tail not only plays a role in maintaining mRNA stability, but its structural diversity also provides new possibilities for mRNA optimization.

[0005] Existing studies have attempted to enhance mRNA stability by introducing single or tandem highly stable 3'UTRs derived from genes such as HBA1 or HBB. However, these methods have limitations in improving translation efficiency due to their reliance on a single type of UTR (Holtkamp et al., 2006; Peixeiro et al., 2011). Other strategies include constructing 3'UTR libraries to screen for highly stable variants, but this method is experimentally complex and time-consuming, and the stability of the screened UTRs often exhibits cell type specificity (Niessen et al., 2019). Furthermore, studies have shown that inserting AU-rich elements into 3'UTRs to recruit HuR proteins can enhance mRNA stability and protein expression (Ma et al., 2025). However, the effectiveness of this method in vivo is limited by HuR expression levels. In recent years, deep learning algorithms have provided new tools for mRNA sequence design, often enabling the design of combinatorial optimized sequences to be completed within minutes (Castillo-Hair and Seelig, 2022; H. Zhang et al., 2023). However, relying solely on codon fitness index and minimum free energy parameter still falls short of predicting complex intracellular translation mechanisms (Ma et al., 2025). Therefore, insufficient understanding remains at the molecular level of sequence optimization, limiting further applications.

[0006] Studies have found that introducing a poly(U) sequence into the 3'UTR can interact with the poly(A) tail, thereby improving mRNA stability (Geisberg et al., 2014). Furthermore, mechanisms utilizing poly(A) interactions to stabilize RNA also exist in viral native RNA. The most classic example is the expression and nuclear retention element (ENE) in the non-coding PAN RNA of Kaposi's sarcoma-associated herpesvirus, which can extend RNA lifetime by forming a triple helix structure with the poly(A) tail (Mitton-Fry et al., 2010). These findings suggest that the interaction between the 3'UTR and the poly(A) tail may be an important direction for improving mRNA stability and expression levels, but related optimization strategies have not yet been systematically studied and fully utilized. Summary of the Invention

[0007] The purpose of this invention is to provide the application of triple helix structural elements in RNA drug design, specifically a 3'UTR insertion element that enhances the expression efficiency of mRNA proteins, in order to solve the problems of low cell stability and low protein production efficiency of current mRNA vaccines, thereby improving the bioavailability of the protein encoded by the mRNA vaccine.

[0008] In this invention, the term ENE element refers to the ENE sequence from the non-coding RNA PAN of the KSHV virus. The ENE element is a 79-nt sequence with a U-rich inner loop. Crystal structure analysis shows that the ENE element forms a triple helix structure with the poly(A) sequence, preventing the initiation of RNA degradation and extending the half-life of the PAN RNA. This invention inserts the ENE element into the 3'UTR sequence of mRNA and designs ENE-related mutants based on sequence optimization of the triple helix core structure. Protein expression validation revealed that the insertion of the ENE element and its mutant elements into the 3'UTR of this invention can stabilize the target RNA and increase protein expression levels by up to 11-fold, which is more efficient than the 3'UTR of human β-globin mRNA. Furthermore, through sequence optimization design based on the triple helix core structure, an optimization strategy based on the structure of the mRNA itself to enhance mRNA stability and thus improve mRNA translation efficiency is proposed, providing a new reference for mRNA sequence optimization design.

[0009] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a nucleic acid stabilizing element that enhances mRNA expression. The nucleic acid stabilizing element is the ENE sequence in the PAN RNA of KSHV.

[0010] The mRNA molecule comprises a 5' m7G-PPPNm structure, a 5' UTR, a CDS, a 3' UTR, and a poly(A) tail. The 3' UTR sequence is a β-globin 3' UTR or an α-globin 3' UTR, preferably a human β-globin 3' UTR sequence, which is a commonly used 3' UTR sequence for stabilizing mRNA in current mRNA vaccines. A control group with a natural 3' UTR without any inserted sequence is used; the sequence is SEQ ID NO.1.

[0011] This invention starts from the polyadenylation site (AAUAAA) on the β-globin 3'UTR and inserts ENE sequences at 0, 30, 60, and 90 nt upstream of it and at five different positions before the β-globin 3'UTR sequence. The five constructs with different insertion positions are represented by ENE-R, ENE-R1, ENE-M, ENE-L1, and ENE-L. The sequences are SEQ ID NO.3-7.

[0012] Preferably, the mRNA of the present invention is modified with pseudouridine to improve its stability and translation efficiency, and reduce immunogenicity, thereby avoiding a strong inflammatory response.

[0013] Preferably, the target protein expression gene is a fusion protein encoding two genes: the SARS-CoV-2 RBD antigen and the NanoLuc luciferase reporter gene, connected by a linker sequence. The NanoLuc luciferase characterizes the expression of the RBD antigen. The inclusion of the RBD antigen helps to evaluate the application potential of ENE and its mutant elements in the field of mRNA vaccines, especially in the prevention and treatment of infectious diseases (such as novel coronavirus infection), thereby verifying its expression efficiency and immunogenicity as an enhancing element in vaccines.

[0014] Preferably, the pharmaceutical carrier for mRNA molecules is lipid nanoparticles (LNPs), in which mRNA is encapsulated for molecular delivery. Studies have shown that LNPs can encapsulate and protect mRNA molecules, preventing their rapid degradation in vivo, while also facilitating mRNA entry into cells for translation.

[0015] The application manifests itself as follows: (1) Insertion of ENE elements into the 3'UTR sequence can effectively increase protein expression, and the effect of ENE insertion on enhancing protein expression gradually increases over time.

[0016] (2) The insertion position of the ENE element in the 3'UTR affects its ability to enhance protein expression. Insertion at the position of ENE-R did not show a significant enhancement of protein expression, while insertion at other positions except ENE-R enhanced protein expression.

[0017] (3) After replacing β-globin 3'UTR with α-globin 3'UTR, the results showed that ENE-L could still stably increase protein expression levels by about 2-fold in various cell lines.

[0018] (4) Experiments on different cell lines and mouse in vivo imaging experiments showed that when ENE-L, the nucleic acid stabilizing element, is inserted between the CDS and 3' UTR of mRNA, it can stably enhance the protein expression of mRNA.

[0019] Secondly, the present invention provides a core sequence for enhancing the expression of ENE elements.

[0020] Studies have shown that the U-rich inner ring of the ENE element is crucial for its activity. This invention performs site-directed mutagenesis on a portion of the element sequence to identify the core region necessary to maintain its enhancing effect. This invention first mutates the U-rich ring of the ENE, performing mutations such as 16+3U (adding three U bases to the left U ring), 14-5U (deleting the left U ring), U16C (mutating the 16th U base to a C base), U62C (mutating the 62nd U base to a C base), 60-5U (deleting the right U ring), and A65U (mutating the 65th A base to a U base), etc. Figure 12 As shown, the sequence is SEQ ID NO.12-17.

[0021] Preferably, the present invention further verifies the functional role of the core sequence in improving mRNA stability by treating the ENE insertion element and mutant at 30°C for different times to verify protein expression.

[0022] The application manifests itself as follows: (1) The U inner loop of the ENE element is crucial for its ability to enhance protein expression. The mutant 16+3U (SEQ ID NO.12), which increases the U base in the U inner loop, further enhanced protein translation, increasing protein expression levels by up to 3-fold. Other mutants that disrupt the U inner loop, 14-5U (SEQ ID NO.13), U16C (SEQ ID NO.14), 60-5U (SEQ ID NO.15), and U62C (SEQ ID NO.16), all caused the original effect of enhancing protein expression to disappear, showing no significant difference in protein expression compared to the WT group. Furthermore, the A65U (SEQ ID NO.17) mutant had a depressant effect on protein expression.

[0023] (2) ENE elements can enhance the solution stability of mRNA vaccines, among which 16+3U (SEQ ID NO.12) shows a better enhancement effect on the stability of target mRNA than the original ENE element.

[0024] Thirdly, the present invention provides an ENE-modified element in the 3'UTR that enhances mRNA expression.

[0025] Studies have shown that ENE elements extend the half-life of RNA by forming a triple helix structure with the poly(A) sequence. Therefore, this variant, based on the triple helix structure formed by the element and the poly(A) tail, is optimized through site-specific mutations. Compared to the natural element, this modified variant further enhances mRNA stability and protein expression levels. The applications are as follows: (1) Increasing the number of U bases on both sides of the U loop of ENE can enhance the efficiency of ENE in improving mRNA translation to a certain extent. However, simply increasing the number of U bases is not directly proportional to the mRNA translation efficiency, and once the number reaches a certain level, it does not continue to enhance protein translation.

[0026] (2) The addition of 3 Us in the left U loop and the addition of 2 Us in the right U loop showed stable translation enhancement in both cell lines. The mutation of adding 3 Us in the left U loop increased protein expression by up to 4-fold, while the mutation of adding 2 Us in the right U loop could increase protein expression by up to 6-fold.

[0027] (3) Adding different numbers of U base mutations to the right U ring on the basis of adding 3 U to the left U ring will not continue to enhance the translation enhancement effect of the 16+3U (SEQ ID NO.12) mutant, but will instead lead to a weakening of translation.

[0028] (4) Replacing UAU in the triple helix core of ENE with CGC can effectively enhance the protein expression of ENE. Replacing one base pair has a stronger effect than replacing two base pairs. After replacing one CGC base pair in the original triple helix core of ENE, the expression in the DC2.4 cell line reached a maximum of 11-fold.

[0029] (5) Replacing one base pair in the triple helix core of the 16+3U (SEQ ID NO.12) mutant increased protein expression by up to 6-fold. Inserting two 16+3U mutants into the 2xM3 (SEQ ID NO.43) mutant further enhanced translation, increasing protein expression by up to 8-fold.

[0030] (6) The deletion mutations M20 (SEQ ID NO.44) and M21 (SEQ ID NO.45) of ENE can form simpler ENE-like structures according to secondary structure prediction, but do not enhance protein expression levels.

[0031] Based on the results of the second and third aspects, this invention proposes an improved nucleic acid stabilizing element, which is formed by performing at least one of the following mutations on the ENE: deletion or point mutation in the U inner loop of the ENE element; addition of 1-6 U bases in the left or right U loop of the ENE element; addition of 3 U bases in the left U loop of the ENE element, followed by addition of 1-6 U bases in the right U loop; replacement of UAU bases with different numbers of CGC bases in the triple helix core structure of the ENE; deletion or point mutation on the stem loops on both sides of the U inner loop of the ENE element; or a combination of multiple ENE mutant sequences.

[0032] Preferably, when the nucleotide sequence of the nucleic acid stabilizing element is one of SEQ ID NO.12, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.39, or SEQ ID NO.43, it has the ability to enhance mRNA stability and protein expression levels.

[0033] In summary, compared with existing technologies, the beneficial effects of this invention are as follows: This invention provides a component that can enhance mRNA stability and protein translation efficiency, and further research has yielded improved mutants with even better effects. These mutants exhibit excellent effects in stabilizing target nucleic acids and enhancing target protein expression in mRNA vaccines, and are expected to have wider applications in the health and medical fields. Furthermore, this invention's sequence optimization design based on the formation of a triple helix structure from ENE and poly(A) to enhance mRNA stability and thus protein translation provides a new reference and strategy for mRNA vaccine sequence design. Attached Figure Description

[0034] Figure 1 This is a diagram of the carrier template in Example 1.

[0035] Figure 2 This is a schematic diagram of a carrier structure in Example 1 in which the target ENE element is inserted at different positions in the 3'UTR.

[0036] Figure 3The relative expression levels of the ENE element inserted at different positions in the 3'UTR at different times in the 293T cell line in Example 1.

[0037] Figure 4 The protein expression levels in the 293T and DC2.4 cell lines were determined by the insertion of the ENE element at different positions after altering the 3'UTR in Example 1.

[0038] Figure 5 The protein expression levels of the two insertion sites, ENE-R1 and ENE-L, in the HeLa cell line in Example 1 are shown.

[0039] Figure 6 This was to verify the effect of the ENE element on mRNA protein expression levels at the in vivo level in Example 1.

[0040] Figure 7 The value represents the protein expression level of ENE in the 293T cell line after the U inner loop mutation in Example 2.

[0041] Figure 8 The value represents the protein expression level of ENE in the DC2.4 cell line after the U inner loop mutation in Example 2.

[0042] Figure 9 The ELISA test in Example 2 was used to verify the protein expression level of the mutant ENE-enhanced mRNA vaccine.

[0043] Figure 10 This describes the effect of the ENE element and the optimized element 16+3U on the stability level of mRNA in solution in Example 2.

[0044] Figure 11 The cytotoxicity of ENE and the optimized element 16+3U at different concentrations in the 293T cell line was determined in Example 2.

[0045] Figure 12 This is a schematic diagram of the ENE element optimized based on the triple helix core structure in Example 3.

[0046] Figure 13 The protein expression levels of the ENE element and optimized elements M1-M14 in the DC2.4 cell line in Example 3 are shown.

[0047] Figure 14 The protein expression levels of the ENE element in Example 3 and the optimized element obtained by further increasing the number of U bases on M3 in the DC2.4 cell line.

[0048] Figure 15The protein expression levels of the ENE element in Example 3, the optimized element obtained by replacing UAU bases with different numbers of CGC bases on the ENE, and the multiple sequence combinations and deletion mutations, are shown in the DC2.4 cell line. Detailed Implementation

[0049] The present invention will be further described below with reference to specific embodiments. The listed embodiments are only used to help understand the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various adjustments, improvements or equivalent substitutions to the embodiments without departing from the spirit and substance of the present invention, and these should all be considered to fall within the scope of protection of the present invention.

[0050] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods known in the art; the materials, reagents, or equipment used, unless otherwise specified, can be obtained through conventional commercial channels; the sequences provided are DNA sequences corresponding to specific fragments in mRNA. The description of the following embodiments can provide reference for those skilled in the art, but should not limit the understanding and application of the present invention.

[0051] Example 1: Filtering the position of ENE element insertion in 3'UTR This study used the NanoLuc luciferase gene as a reporter gene to investigate the location where ENE element insertion into the 3'UTR enhances mRNA expression. The regulatory effects on NanoLuc protein expression in 293T cells were investigated, including the control group with β-globin 3'UTR and experimental groups with ENEs inserted at different positions in the β-globin 3'UTR. Furthermore, the regulatory effects on NanoLuc mRNA expression in the control and experimental groups were investigated in 293T and DC2.4 cell lines after replacing the β-globin 3'UTR with the α-globin 3'UTR. In addition, the effect of ENE insertion on protein expression enhancement was verified in vivo. The specific process is as follows: (1) Carrier construction.

[0052] (1.1) The nucleotide sequences required for constructing the control group vector were obtained through NCBI, including the RBD sequence, NanoLuc sequence, β-globin 3'UTR sequence, and α-globin 3'UTR sequence of SARS-CoV-2. Using the linearized template vector CloningKit for mRNA Template (Takara, Cat:6143) as a template, the gene was synthesized by Beijing Qingke Biotechnology Co., Ltd. This synthesized plasmid served as the control group vector, representing a vector without the insertion of the ENE element. The nucleotide sequences of the β-globin 3'UTR control group and the α-globin 3'UTR control group as transcription templates are shown in SEQ ID NO. 1-2, respectively, containing the T7 promoter, 5'UTR, CDS, 3'UTR, and poly(A) portion. The plasmid map of the β-globin 3'UTR control group is shown below. Figure 1 As shown.

[0053] (1.2) The nucleotide sequence of KSHV PAN-ENE, 79 bp in length, was obtained by NCBI and synthesized by Beijing Qingke Biotechnology Co., Ltd. The synthesized PAN-ENE sequence was inserted into different positions in the 3'UTR of the control vector via homologous recombination. After transformation into T1 competent cells, correct single colonies were picked to obtain the experimental plasmid, which was verified to be correct by sequencing. The nucleotide sequences of ENE inserted into different positions of the β-globin 3'UTR are shown in SEQ ID NO. 3-7, respectively, corresponding to... Figure 2 The nucleotide sequences of ENE-R, ENE-R1, ENE-M, ENE-L1, and ENE-L, which are inserted at different positions in the α-globin 3'UTR, are shown in SEQ ID NO.8-11.

[0054] (2) mRNA in vitro transcription, modification and purification.

[0055] (2.1) Obtaining linearized templates.

[0056] The obtained recombinant plasmid was digested with the restriction endonuclease HindIII overnight at 37°C. The linear fragment was separated by 1.5% gel electrophoresis and purified by gel extraction using the FastPure Gel DNA Extraction Mini Kit. Nanodrop quantification was used to detect the DNA fragments, and the purity was confirmed by 1.5% agarose gel electrophoresis.

[0057] (2.2) The obtained linearized plasmid template was transcribed in vitro using the EasyCap T7 Co-transcription Kit with CAG Trimer (Vazyme, Cat:DD4203-01).

[0058] (2.2.1) On ice, add the following reagents to 200 μL microcentrifuge tubes according to Table 1.

[0059] Table 1: In vitro transcription system

[0060] (2.2.2) After thorough mixing and centrifugation, the reaction tubes were incubated at 37°C for 4 hours. Then, 1.5 μL LDNase I was added to remove the linearized template, and the reaction was continued at 37°C for 30 minutes. The transcribed mRNA was purified using the RNA Purification Kit (TransGenBiotech, Cat:ER701-01). The concentration and purity of the purified mRNA product were determined using Nanodrop, and its quality was assessed by 1.5% agarose gel electrophoresis.

[0061] (3) Prepare mRNA-LNP mixture.

[0062] SM102 (Cat:O02010), distearate phosphatidylcholine DSPC (Cat:S01005), high-purity cholesterol CHO-HP (Cat:57-88-5), and DMG-PEG2000 (Cat:O02005), purchased from Aivit (Shanghai) Pharmaceutical Technology Co., Ltd., were dissolved in anhydrous ethanol and prepared into a lipid ethanol solution at a molar ratio of 50:10:38.5:1.5. The purified mRNA was diluted with citrate-sodium citrate buffer (pH=4.5), and the lipid ethanol solution was thoroughly mixed with the mRNA dilution solution and repeatedly pipetted to obtain an mRNA-LNP mixture.

[0063] (4) NanoLuc detection.

[0064] Cell lines: including at least 293T, DC2.4, and HeLa cell lines. (4.1) Take 293T cells in logarithmic growth phase (culture medium: 89% DMEM + 10% FBS + 1% Penicillin-Streptomycin Solution), DC2.4 cells (culture medium: 89% RPMI + 10% FBS + 1% Penicillin-Streptomycin Solution), and HeLa cells (culture medium: 89% DMEM + 10% FBS + 1% Penicillin-Streptomycin Solution), and distribute them at 4 x 10⁻⁶ cells per well. 4 Seeds were seeded per well into 48-well plates and cultured overnight. 2 μL of the prepared mRNA-LNP was added to each well of the 48-well plate, with 4 μL of Fluc-containing mRNA-LNP added to each well as a control. Fluorescence intensity was measured 24 h after transfection.

[0065] (4.2) Use The kit was used to detect NanoLuc fluorescence intensity. The reagents were allowed to thaw at room temperature. After 24 hours of transfection, the cell plate was removed, and the cells were gently pipetted off. 20 μL of the corresponding sample cell suspension was added to each well of an opaque, white 96-well plate, and an equal volume of ONE-Glo was added to each well. TM Incubate the cells with EX Reagent at room temperature for at least 5 minutes to allow for complete cell lysis, then use a microplate reader to measure fluorescence intensity. After detection, add an equal volume of NanoDLR to each well as the original culture medium. TM Stop & Glo® Reagent, place it in a microplate reader to detect the fluorescence intensity of NanoLuc.

[0066] The results showed that insertion of the ENE element into the 3'UTR effectively increased mRNA protein expression levels. Figure 3 As shown, for the β-globin 3'UTR, in the 293T cell line, ENE-R1, ENE-M, ENE-L1, and ENE-L (SEQ ID NO: 4-7) increased mRNA protein expression levels by 2-fold at 72 h. Changing the 3'UTR to the α-globin 3'UTR resulted in... Figure 4 As shown, ENE-R1 (SEQ ID NO. 9) and ENE-L (SEQ ID NO. 11) significantly enhanced protein expression in the 293T and DC2.4 cell lines, respectively. ENE-R1 (SEQ ID NO. 9) increased protein expression by up to 1.8-fold and 2.8-fold in the 293T and DC2.4 cell lines, respectively, while ENE-L (SEQ ID NO. 11) increased protein expression by up to 2.4-fold and 2.1-fold in the 293T and DC2.4 cell lines, respectively. Figure 5 As shown, in the HeLa cell line, ENE-R1 (SEQ ID NO.9) increased protein expression by a maximum of only 1.4-fold, while ENE-L (SEQ ID NO.11) increased it by a maximum of 2.5-fold. Therefore, it was determined that directly inserting ENE into the 3'UTR front end has the ability to enhance mRNA protein expression in different cell lines.

[0067] (5) In vivo imaging.

[0068] (5.1) To obtain mRNA with the NanoLuc sequence removed and only the RBD remaining in the coding region, the vector construction, template preparation, mRNA preparation, and lipid nanoparticle encapsulation were performed sequentially according to the steps described in Example 1. The obtained mRNA-LNP was placed in a Slide-A-Lyzer™ mini dialysis cup (10 KMWCO, 0.1 mL), and the filter membrane was soaked with PBS solution in a centrifuge tube. The mixture was then dialyzed overnight on a shaker at 4°C.

[0069] (5.2) Six-week-old female C57BL / 6 mice were purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd. Dialyzed mRNA-LNP was administered to the mice via intramuscular injection (n=5). Fluorescence detection was performed on the mice at 6h, 12h, 24h and 48h after administration.

[0070] (5.3) Furimazine (Fz) is a substrate for NanoLuc luciferase. First, Furimazine was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mg / mL stock solution. Then, the stock solution was diluted with PBS to prepare a 0.25 mg / mL working solution. The appropriate amount was injected according to the ratio of 1.25 mg / kg Fz to mouse body weight. 100 μL of the working solution was administered to mice via intraperitoneal injection, and bioluminescence imaging was performed on the mice using a small animal in vivo imaging system.

[0071] The results showed that, Figure 6 As shown, ENE-L (SEQ ID NO.7) can also increase mRNA protein expression levels by about 2-fold in vivo. From 6h to 48h, the fluorescence intensity of luciferase after protein translation of injected mRNA-LNP gradually decreased, while ENE-L (SEQ ID NO.7) still maintained a protein expression level of about 2-fold compared to the control group at 6h, 12h, and 24h.

[0072] Example 2: Mutation to determine the core sequence of ENE-enhanced mRNA expression (1) NanoLuc detection.

[0073] Following the steps described in Example 1, vector construction, template preparation, mRNA preparation, and LNP encapsulation were performed sequentially to obtain vectors that mutate the U inner loop of ENE. The sequences were 16+3U (M3, SEQ ID NO. 12), 14-5U (M7, SEQ ID NO. 13), U16C (M8, SEQ ID NO. 14), 60-5U (M15, SEQ ID NO. 15), U62C (M16, SEQ ID NO. 16), and A65U (M17, SEQ ID NO. 17), and the corresponding mRNA-LNP compositions were obtained. The sequences were inserted at the front of the 3' UTR, and the 3' UTR was a β-globin 3' UTR. The mRNA was encapsulated in 4x10 wells. 4 293T and DC2.4 cells were seeded into 48-well plates and cultured overnight. 6 μL of prepared mRNA-LNP was added to each well of the 48-well plate, with 6 μL of Fluc-containing mRNA-LNP added to each well as a control. Fluorescence intensity was measured 24 h after transfection following the NanoLuc assay procedure in Example 1.

[0074] The results showed that the inner U loop of ENE is crucial for its ability to enhance mRNA protein expression levels. For example... Figure 7 and Figure 8 As shown, in both the 293T and DC2.4 cell lines, deletion or point mutation of the inner U loop of ENE resulted in the loss or even reduction of ENE's ability to enhance protein expression. However, the mutation 16+3U (SEQ ID NO.12) which adds 3 U bases to the left U loop of ENE can enhance ENE's ability to enhance mRNA protein expression, increasing protein expression by up to 2.1-fold and 3.3-fold in the 293T and DC2.4 cell lines, respectively.

[0075] (2) ELISA detection of RBD antigen expression.

[0076] Cell lines: including at least the 293T cell line (2.1) Take 293T cells in the logarithmic growth phase and divide them into 2 x 10 cells per well. 5 Cells were seeded per well in a 6-well plate and cultured overnight. Prepared mRNA-LNP was added at a rate of 2 μg / well to the 6-well plate, and cell proteins were extracted 48 h after transfection.

[0077] (2.2) SARS-CoV-2(2019-nCoV)Spike RBD ELISA Kit.

[0078] Remove the kit from 4°C and allow it to equilibrate at room temperature for at least half an hour. Prepare washing buffer, standard, and universal sample diluent. Dilute the standard and sample with the sample diluent. After dilution, add 100 μL of standard and sample to each well and incubate at room temperature for 2 hours. Remove the liquid from the plate, add washing buffer to each well, and squeeze to remove the liquid. Repeat this washing process three times. Add 100 μL of antibody working solution to each well and incubate at room temperature for 1 hour. Remove the liquid from the plate and wash three times. Add 100 μL of substrate solution to each well and incubate at room temperature in the dark for 20 minutes. Stop the reaction by adding 100 μL of stop solution to each well. Measure the OD450 and OD630 absorbance using a microplate reader.

[0079] The results showed that, in addition to the dual-luciferase reporter gene system, ELISA experiments also demonstrated an increase in the expression capacity of the 16+3U group (SEQ ID NO. 12) compared to the original ENE-enhancing protein. Figure 9 As shown, compared with the control group, the 16+3U group (SEQ ID NO.12) increased protein expression level by nearly 2 times.

[0080] (3) Solution stability (3.1) Dilute 25 μg of mRNA in 1350 μL of citrate-sodium citrate buffer (pH=4.5) and treat at 30 °C. Take samples at 2 h, 4 h, 8 h, 16 h, 24 h and 48 h, with each sample being about 200 μL.

[0081] (3.2) The mRNA samples taken at different time points were encapsulated according to the steps for preparing the mRNA-LNP mixture in Example 1. Each well contained 4 x 10 mRNA molecules. 4 293T cells were seeded into 48-well plates and cultured overnight. 6 μL of prepared mRNA-LNP was added to each well of the 48-well plate, with 6 μL of Fluc-containing mRNA-LNP added to each well as a control. Fluorescence intensity was measured 24 h after transfection using the NanoLuc assay procedure described in Example 1.

[0082] The results showed that inserting an ENE enhanced the stability of mRNA in solution, and the 16+3U group (SEQ ID NO.12) further improved the ability of the ENE to stabilize mRNA in solution. Figure 10 As shown, mRNA began to degrade gradually after 16 hours of treatment at 30°C, while the protein expression level of the 16+3U group remained 2-3 times that of the WT group after 24 and 48 hours of treatment at 30°C.

[0083] (4) Cytotoxicity test.

[0084] 293T cells in logarithmic growth phase were harvested and distributed at a density of 1.5 x 10⁻⁶ cells per well. 4Seeds were seeded per well into 96-well plates and incubated overnight. Prepared mRNA-LNP was added to the 96-well plates at concentrations of 10, 50, 100, 250, 500, and 1000 ng / mL. 24 h after transfection, 10 μL of CCK8 solution was added to each well, and the plates were incubated for another hour before measuring the absorbance at 450 nm.

[0085] The results showed that inserting or mutating ENE elements did not cause additional cytotoxicity to the mRNA vaccine. Figure 11 As shown, within a certain concentration range, mRNA vaccines containing ENE elements do not cause cytotoxicity after in vitro transfection of cells.

[0086] Example 3: Optimizing the ENE sequence can significantly enhance mRNA expression. (1) NanoLuc detection.

[0087] Cell line: DC2.4 cell line Following the steps described in Example 1, vector construction, template preparation, mRNA preparation, and LNP encapsulation were performed sequentially to obtain a vector for sequence optimization of ENE, such as... Figure 12The diagram shows all the designed mutants, including mutations that add different numbers of U bases to the left and right U rings, mutations that add different numbers of U bases to the right U ring based on the 16+3U mutant, mutations that change the triple helix core structure UAU to CGC, and deletion mutations. The sequences are M1 (SEQ ID NO.18), M2 (SEQ ID NO.19), M3 (SEQ ID NO.12), M4 (SEQ ID NO.20), M5 (SEQ ID NO.21), M6 (SEQ ID NO.22), M9 (SEQ ID NO.23), M10 (SEQ ID NO.24), and M11 (SEQ ID NO.24). NO.25), M12 (SEQ ID NO.26), M13 (SEQ ID NO.27), M14 (SEQ ID NO.28), M3+M9 (SEQ ID NO.29), M3+M10 (SEQ ID NO.30), M3+M11 (SEQ ID NO.31), M3+M12 (SEQ ID NO.32), M3+M13 (SEQ ID NO.33), M3+M14 (SEQ ID NO.34), M(a) (SEQ ID Sequences M(a+b) (SEQ ID NO. 35), M(a+b) (SEQ ID NO. 36), M(a+b+c) (SEQ ID NO. 37), M3+M(a) (SEQ ID NO. 38), M3+M(a+b) (SEQ ID NO. 39), M3+M(a+b+c) (SEQ ID NO. 40), M3+M18 (SEQ ID NO. 41), M3+M19 (SEQ ID NO. 42), 2xM3 (SEQ ID NO. 43), M20 (SEQ ID NO. 44), and M21 (SEQ ID NO. 45) were used to obtain the corresponding mRNA-LNP compositions. The sequences were inserted into the 3' UTR front end, where the 3' UTR was a β-globin 3' UTR, and the same applies below. The mRNA-LNP compositions were prepared according to a 4x10 well ratio. 4 293T and DC2.4 cells were seeded into 48-well plates and cultured overnight. 2 μL of prepared mRNA-LNP was added to each well of the 48-well plate, with 4 μL of Fluc-containing mRNA-LNP added to each well as a control. Fluorescence intensity was measured 24 h after transfection, following the NanoLuc detection procedure in Example 1.

[0088] The results showed that sequence mutations designed based on the ENE triple-helix core structure significantly enhanced translation performance. For example... Figure 13As shown, M1-M6 (SEQ ID NO.12, SEQ ID NO.18-22) are mutants with an additional U base in the left U ring, and M9-M14 (SEQ ID NO.23-28) are mutants with an additional U base in the right U ring. Adding a U base solely to one side of the inner U ring effectively enhances the ability of ENE to boost mRNA translation, but the number of additional U bases is not positively correlated with the enhanced translation ability. Among the mutants with additional U bases on both sides of the U ring, M3 (SEQ ID NO.12) and M10 (SEQ ID NO.24) showed the most significant enhancement. The M3 (SEQ ID NO.12) mutant with 3 additional U bases in the left U ring showed the highest enhancement of protein expression level by 4.3-fold in the DC2.4 cell line; the M10 (SEQ ID NO.24) mutant with 2 additional U bases in the right U ring showed the highest enhancement of protein expression level by 6.2-fold in the DC2.4 cell line.

[0089] like Figure 14 As shown, mutants M3+M9 (SEQ ID NO.29), M3+M10 (SEQ ID NO.30), M3+M11 (SEQ ID NO.31), M3+M12 (SEQ ID NO.32), M3+M13 (SEQ ID NO.33), and M3+M14 (SEQ ID NO.34), which further increase the number of U bases on the right U ring of the M3 (SEQ ID NO.12) mutant, do not further enhance the translation level. Furthermore, mutants M3+M18 (SEQ ID NO.41) and M3+M19 (SEQ ID NO.42), which mutate the UG bases on both sides of the U ring to CG bases on the M3 (SEQ ID NO.12) mutant, also did not show enhanced protein translation.

[0090] like Figure 15As shown, among the mutants M(a) (SEQ ID NO. 35, whose sequence includes the mutant ENE sequence, 3' UTR, and mutant poly(A) tail, and the same as SEQ ID NO. 36-40), M(a+b) (SEQ ID NO. 36), and M(a+b+c) (SEQ ID NO. 37) with UAU bases replaced by different numbers of CGC bases in the original ENE, only M(a) (SEQ ID NO. 35) and M(a+b) (SEQ ID NO. 36) had different effects on mRNA translation levels. M(a) (SEQ ID NO. 35) and M(a+b) (SEQ ID NO. 36) both further enhanced protein translation, increasing protein expression levels by up to 11.2 and 5.1 times, respectively, while M(a+b+c) (SEQ ID NO. 37) did not increase protein expression levels. The mutants M3+M(a)(SEQ ID NO.38), M3+M(a+b)(SEQ ID NO.39), and M3+M(a+b+c)(SEQ ID NO.40), which replace the UAU bases with different numbers of CGC bases based on the M3(SEQ ID NO.12) mutant, also had different effects on protein translation. Among them, M3+M(a)(SEQ ID NO.38) further enhanced protein translation, increasing the protein expression level by up to 6.7 times; M3+M(a+b)(SEQ ID NO.39) did not further enhance the protein expression level based on M3, only increasing the protein expression level by about 3 times compared to the control group; M3+M(a+b+c)(SEQ ID NO.40) did not enhance protein expression. At the same time, 2xM3(SEQ ID NO.43), which inserts two M3(SEQ ID NO.12) mutants, also further enhanced protein translation, increasing the protein expression level by up to 8.2 times. Furthermore, the M20 (SEQ ID NO.44) and M21 (SEQ ID NO.45) mutants with deletion mutations in ENE did not show a trend toward protein enhancement.

[0091] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, any equivalent substitutions or improvements made without departing from the technical concept disclosed in the present invention should be considered to fall within the scope of protection of the present invention. The scope of protection of the present invention should be determined by the appended claims.

Claims

1. A nucleic acid stabilizing element for enhancing mRNA expression, characterized in that, The element comprises an ENE element based on the triple helix structure of Kaposi's sarcoma-associated herpesvirus or a mutant thereof.

2. The nucleic acid stabilizing element according to claim 1, characterized in that, The mutant includes at least one of the following mutations: Deletion or point mutation is performed within the U-loop of the ENE element; Add 1-6 U bases to the left or right U ring of the ENE element, respectively; Based on adding 3 U bases to the left U ring of the ENE element, 1-6 U bases are added to the right U ring; In the ENE triple helix core structure, the UAU bases are replaced with different numbers of CGC bases; Deletion or point mutation is performed on the stem loops on both sides of the U inner loop of the ENE element; A combination of multiple ENE mutant sequences.

3. The nucleic acid stabilizing element according to claim 1 or 2, characterized in that, The nucleotide sequence of the mutant is one of SEQ ID NO.12-45.

4. The nucleic acid stabilizing element according to claim 3, characterized in that, The mutant is one of SEQ ID NO.12, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.39, and SEQ ID NO.

43.

5. A method for enhancing mRNA stability and / or protein translation efficiency, characterized in that, The nucleic acid stabilizing element described in claim 1 is inserted between the CDS and 3' UTR of the mRNA to form a triple helix structure with poly(A).

6. The method according to claim 5, characterized in that, The mRNA contains a 5' cap, a 5' UTR, a CDS, a 3' UTR, and a poly(A) tail, wherein the 3' UTR is a β-globin 3' UTR or an α-globin 3' UTR.

7. An mRNA vaccine, characterized in that, It comprises the mRNA prepared by the method of claim 5 and a pharmaceutical carrier, for use in enhancing immune and therapeutic effects.

8. The mRNA vaccine as described in claim 7, characterized in that, The CDS region of the mRNA encodes the RBD antigen of SARS-CoV-2; the pharmaceutical carrier is a lipid nanoparticle, and the mRNA molecule is encapsulated in the lipid nanoparticle.

9. A method for preparing the mRNA vaccine according to claim 7, characterized in that, Includes the following steps: 1) Construct a recombinant plasmid comprising a T7 promoter, a 5' UTR, a coding region, a 3' UTR, and a poly(A), wherein the nucleic acid stabilizing element of claim 1 is inserted into the 3' UTR; 2) Linearize the recombinant plasmid using a restriction endonuclease to obtain a linearized template; 3) Transcribe the linearized template using an in vitro transcription kit to obtain mRNA; 4) Mix the mRNA with lipid nanoparticles to prepare an mRNA-lipid nanoparticle composition.

10. Use of a nucleic acid stabilizing element according to any one of claims 1-4 or an mRNA vaccine according to claim 7 in the preparation of a medicament for the prevention and / or treatment of infectious diseases and / or tumors.

Images