Use of trna in promoting protein-coding ability of mRNA

Abstract

The present invention relates to the use of tRNA in promoting the protein-coding ability of mRNA. The expression level of a target protein is improved by means of overexpressing tRNA, and a codon corresponding to the tRNA can promote or improve the stability of the mRNA. Further provided is a new tRNA+mRNA immunopotentiating vaccine. By means of introducing one or more tRNA molecules, the antigen protein encoding ability of an mRNA vaccine is enhanced, thereby eliciting stronger humoral and cellular immune responses in vivo. Further provided is a recombinant cell for producing an antibody. The recombinant cell overexpresses tRNA capable of increasing the expression level of the antibody, and the tRNA comprises a tRNA isodecoder family. Further provided is a recombinant cell for producing or packaging recombinant AAV, wherein the recombinant cell overexpresses tRNA capable of improving the AAV packaging efficiency.

Description

The role of tRNA in enhancing the protein-coding ability of mRNA Technical Field

[0001] This invention belongs to the field of nucleic acid vaccine technology, specifically relating to the use of tRNA in promoting mRNA protein coding ability and improving mRNA expression, and particularly to an enhanced tRNA+mRNA vaccine in which lipid nanoparticles (LNPs) co-encapsulate tRNA and mRNA. Background Technology

[0002] Nucleic acid vaccines involve directly introducing a foreign gene (DNA or RNA) encoding a specific antigen protein into animal somatic cells. The host cell's expression system then synthesizes the antigen protein, inducing an immune response against it, thereby preventing and treating diseases. Although research on nucleic acid vaccines began in the 1980s and 1990s, no nucleic acid vaccine had been approved for market release before 2020. SARS-CoV-2 infection significantly accelerated the development of nucleic acid vaccines. With the widespread use of SARS-CoV-2 mRNA vaccines, many adverse reactions related to mRNA and lipid nanoparticles (LNPs) have been reported, such as stroke, myocardial infarction, and pulmonary embolism, highlighting the urgent need to enhance the encoding capacity of mRNA and optimize LNP components.

[0003] Protein synthesis is one of the most energy-intensive processes in cells, and efficient translation is crucial for cellular adaptation and function. In recent years, researchers have modified mRNA to achieve higher protein yields, including screening for novel cap-like structures with higher initiation probabilities and modified nucleotides with lower immunogenicity and higher decoding capabilities; optimizing codon distribution and composition; optimizing the secondary structure, length, and sequence of the 5'UTR and 3'UTR to achieve stronger ribosome binding activity, higher stability, and accurate subcellular localization; and designing polyadenylated tails with branched chemical modifications to enhance mRNA stability and translational capacity. Furthermore, circular RNA (circRNA) and self-amplifying RNA (saRNA) or trans-amplifying RNA represent next-generation RNA-based therapies. However, the methodologies for the design, synthesis, purification, delivery, and safety assessment of these RNAs still require further investigation. In summary, we aim to develop a translation accelerator to produce more target proteins with the same amount of mRNA input.

[0004] Translation is the fundamental process of decoding the genetic information of mRNA into proteins. As the translation instruction, the abundance, secondary structure, codon distribution, and composition of mRNA directly determine the efficiency of protein synthesis. tRNA is the primary decoder of the genetic code and a central and dynamic component of translation. The tRNA pool in metazoan cells is dynamically changing; tRNA accessibility (abundance and modification level) affects mRNA and ribosomes in a translation-dependent manner, and is associated with cell differentiation or proliferation, responses to cellular stress, and tumor metastasis. Upregulating tRNA accessibility can increase the optimality of corresponding codons, improving the translation rate and stability of codon-rich mRNAs. tRNA is considered an accelerator of translation, capable of endowing mRNA with stronger protein-coding capabilities. This suggests that the combination of mRNA and tRNA can be a feasible strategy for achieving efficient protein production, known as the "tRNA+" strategy. The "tRNA+" strategy can be used in all translation-dependent RNA therapies or vaccines, enabling the production of more target proteins with the same amount of RNA input, thereby increasing the therapeutic efficacy of RNA therapies and the immune response of RNA vaccines. Based on this, we propose a novel tRNA+mRNA immune-enhancing vaccine. Summary of the Invention

[0005] To address the technical problems of poor encoding capacity, low expression level, and unsatisfactory immunization effect of existing mRNA vaccines, the purpose of this invention is to apply the translation element tRNA to improve the expression level of proteins.

[0006] Invention Summary

[0007] This invention is the first to propose that the "tRNA+" strategy can increase the protein coding capacity of mRNA, and can be used as a general strategy to increase the yield of target proteins.

[0008] This invention is the first to develop a novel tRNA+mRNA immune-enhancing vaccine. The enhanced vaccine enhances the antigen protein encoding ability of the mRNA vaccine by introducing one or more tRNA molecules, thereby stimulating a stronger humoral and cellular immune response in the body.

[0009] This invention relates to a delivery system for co-encapsulating tRNA and mRNA in lipid nanoparticles (LNPs). The tRNA molecules used in this invention contain single or multiple site-specific modified nucleotides at positions 18, 26, 34, 37, 47d, 46, 55, and 58, exhibiting higher aminoacylation efficiency, lower immunogenicity, and higher decoding capability compared to unmodified tRNA. The mRNA molecules used in this invention contain complete substitutions of the following single modified nucleotides: A (6Me-A, Z), U (Psi, N1Me-Psi, 5OMe-U, 3Me-5OMe-U), C (5Me-C), and G (N7Me-G).

[0010] The "tRNA+" strategy proposed in this invention involves a method for joint analysis of codon usage frequency and stability coefficient. This method evaluates the contribution of each codon to the stability of the target mRNA, where tRNAs corresponding to codons with high scores are considered to increase the translation level of the target mRNA. The tRNAs have been verified through protein expression level testing to increase the expression level of the target protein.

[0011] The novel tRNA+mRNA immune-enhancing vaccine of this invention enhances the antigen-encoding ability of the mRNA vaccine by introducing one or more tRNA molecules, thereby stimulating a stronger humoral and cellular immune response in the body. The tRNA+mRNA vaccine comprises three main components: lipid nanoparticles (LNPs) (delivery carriers), mRNA (the template for antigen encoding), and tRNA (a promoter that enhances antigen protein expression).

[0012] This invention relates to a delivery system for lipid nanoparticles (LNPs) co-encapsulating tRNA and mRNA, characterized in that the mass ratio of tRNA to mRNA is 1:1, the nitrogen-phosphorus molar ratio is 3:1, the particle size is between 80 and 90 nm, and the encapsulation efficiency is greater than 95%.

[0013] This invention relates to mRNA encoding antigen proteins, characterized in that the mRNA comprises all substitutions of the following single modified nucleotides, including A (6Me-A, Z), U (Psi, N1Me-Psi, 5OMe-U, 3Me-5OMe-U), C (5Me-C), and G (N7Me-G).

[0014] This invention relates to tRNAs with single or multiple site modifications capable of promoting mRNA translation, characterized in that: the tRNA carries a leucine residue and contains single or multiple site-modified nucleotides as follows: position 18 is replaced with Gm (Gm18); position 26 is replaced with m2,2G (m2,2G26); position 34 is replaced with m5C (m5C34); position 34 is replaced with Cm (Cm34); position 37 is replaced with m1G (m1G37); position 47d is replaced with m3C (m3C47d); position 46 is replaced with m7G (…). m7G46); the 55th bit is replaced with Ψ (Ψ55); the 58th bit is replaced with m1A (m1A58); multiple replacements of Gm18 and Ψ55; multiple replacements of m5C34 and m1G37; multiple replacements of Cm34 and m1G37; multiple replacements of m5C34, m1G37 and m1A58; multiple replacements of Cm34, m1G37 and m1A58; multiple replacements of Gm18, m5C34, m1G37, Ψ55 and m1A58; multiple replacements of Gm18, Cm34, m1G37, Ψ55 and m1A58.

[0015] Invention Details

[0016] This invention provides the use of tRNA in enhancing the protein-coding ability of mRNA by overexpressing tRNA to increase the expression level of the target protein. It also provides a novel tRNA+mRNA immunostimulatory vaccine that enhances the antigen-protein encoding ability of the mRNA vaccine by introducing one or more tRNA molecules, thereby stimulating a stronger humoral and cellular immune response in vivo. The tRNA+mRNA vaccine utilizes a lipid nanoparticle (LNP) delivery system; the tRNA molecule contains single or multiple site-modified nucleotides at positions 18, 26, 34, 37, 47d, 46, 55, and 58, exhibiting higher aminoacylation efficiency, lower immunogenicity, and higher decoding ability compared to unmodified tRNA; the mRNA contains complete substitutions of the following single modified nucleotides: A (6Me-A, Z), U (Psi, N1Me-Psi, 5OMe-U, 3Me-5OMe-U), C (5Me-C), and G (N7Me-G). The aforementioned tRNA+mRNA immune-enhancing vaccine can be applied to the preparation of viral mRNA vaccines and tumor mRNA vaccines with stronger immune effects, the development of protein-alternative mRNA therapies with higher protein expression levels, and can also be applied to empower other forms of RNA therapy and vaccines, such as circular RNA and self-replicating RNA. This invention also provides a recombinant cell for antibody production, wherein the recombinant cell overexpresses tRNA capable of increasing antibody expression levels, the tRNA including the tRNA isodecoder family, preferably Val-tRNA-AAC, Val-tRNA-CAC, Ile-tRNA-AAT, and Gln-tRNA-CTG. This invention also provides a recombinant cell for producing or packaging recombinant AAV, characterized in that the recombinant cell overexpresses tRNA capable of improving AAV packaging efficiency, the tRNA including Asp-GTC-2-1, Ile-AAT-1-1, Ile-GAT-1-1, Leu-TAA-1-1, and Arg-TCT-3-2.

[0017] Specifically:

[0018] On the one hand, this application provides the use of tRNA to promote the protein coding ability of mRNA or to improve mRNA expression, characterized in that the codon corresponding to the tRNA can promote or improve the stability of the mRNA.

[0019] Furthermore, the use of the tRNA described in this invention to promote the protein-coding ability of mRNA or to increase mRNA expression is characterized in that the mRNA encodes an antibody, preferably a monoclonal antibody; and the tRNA increases the expression level of the antibody.

[0020] Furthermore, the use of the tRNA described in this invention to promote mRNA protein coding ability or enhance mRNA expression is characterized in that the monoclonal antibody is expressed by recombinant cells, and the tRNA includes the tRNA isodecoder family, preferably Val-tRNA-AAC, Val-tRNA-CAC, Ile-tRNA-AAT, Gln-tRNA-CTG, Ala-tRNA-AGC, Asn-tRNA-GTT, Asp-tRNA-GTC, Gly-tRNA-GCC, Lys-tRNA-CTT, Ser-tRNA-AGA, Ser-tRNA-GCT, Thr-tRNA-AGT.

[0021] Furthermore, the use of the tRNA described in this invention to promote mRNA protein coding ability or improve mRNA expression is characterized in that the mRNA encodes AAV packaging-related proteins, preferably encoding AAV capsid proteins or recombinant exogenous genes; the tRNA improves the packaging efficiency of recombinant AAV viruses.

[0022] Furthermore, the use of the tRNA described in this invention to promote mRNA protein coding ability or improve mRNA expression is characterized in that the AAV is a recombinant AAV produced by packaging using a two-plasmid system or a three-plasmid system, and the tRNA includes Asp-GTC-2-1, Ile-AAT-1-1, Ile-GAT-1-1, Leu-TAA-1-1, and Arg-TCT-3-2.

[0023] Furthermore, the tRNA of the present invention comprises a single or multiple modifying nucleotide selected from the following: positions 18, 26, 34, 37, 47d, 46, 55, and 58 on the tRNA molecule; and the modifying nucleotide is selected from: Gm, m2, 2G, m5C, Cm, m3C, m1G, m7G, Ψ, and m1A.

[0024] Preferably, the tRNA contains single- or multiple-site modifying nucleotides selected from the group consisting of:

[0025] (1) Replace the 18th bit with Gm (Gm18);

[0026] (2) Replace the 26th bit with m2,2G(m2,2G26);

[0027] (3) Replace the 34th bit with m5C (m5C34);

[0028] (4) Replace the 34th bit with Cm (Cm34);

[0029] (5) Replace the 37th bit with m1G (m1G37);

[0030] (6) Replace the 47d position with m3C (m3C47d);

[0031] (7) Replace the 46th bit with m7G (m7G46);

[0032] (8) Replace the 55th position with Ψ(Ψ55);

[0033] (9) Replace the 58th bit with m1A (m1A58);

[0034] (10) Replace the 18th bit with Gm (Gm18) + replace the 55th bit with Ψ (Ψ55);

[0035] (11) Replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37);

[0036] (12) Replace the 34th bit with Cm (Cm34) + replace the 37th bit with m1G (m1G37);

[0037] (13) Replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37) + replace the 58th bit with m1A (m1A58);

[0038] (14) Replace the 34th bit with Cm (Cm34) + replace the 37th bit with m1G (m1G37) + replace the 58th bit with m1A (m1A58);

[0039] (15) Replace the 18th bit with Gm (Gm18) + replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37) + replace the 55th bit with Ψ (Ψ55) + replace the 58th bit with m1A (m1A58);

[0040] (16) Replace the 18th position with Gm (Gm18) + replace the 34th position with Cm (Cm34) + replace the 37th position with m1G (m1G37) + replace the 55th position with Ψ (Ψ55) + replace the 58th position with m1A (m1A58).

[0041] Furthermore, the use of the tRNA of the present invention to promote the protein coding ability of mRNA or improve mRNA expression is characterized in that: the tRNA carries leucine; preferably, it contains one or more modified nucleotides based on the Leu-StRNA-CUA shown in SEQ ID NO:1, or contains one or more modified nucleotides based on the Leu-tRNA-CAG shown in SEQ ID NO:2.

[0042] Furthermore, the use of the tRNA described in this invention to promote mRNA protein coding ability or enhance mRNA expression is characterized in that the mRNA comprises the complete replacement of a single modifying nucleotide, preferably including the following modifications:

[0043] (1) Replace all A's on the mRNA with modified nucleotides selected from 6Me-A and Z;

[0044] (2) Replace all the Us on the mRNA with modified nucleotides selected from Psi, N1Me-Psi, 5OMe-U, 3Me-5OMe-U;

[0045] (3) Replace all Cs on the mRNA with the modified nucleotide 5Me-C;

[0046] (4) Replace all Gs on the mRNA with the modified nucleotide N7Me-G.

[0047] Furthermore, the use of the tRNA described in this invention to promote mRNA protein coding ability or improve mRNA expression is characterized in that the tRNA and the mRNA use the same delivery system, preferably co-encapsulated and delivered by lipid nanoparticles (LNPs).

[0048] The tRNA and mRNA have a mass ratio of 1:1, a nitrogen-phosphorus molar ratio of 3:1, a particle size between 80 and 90 nm, and an encapsulation efficiency greater than 95%.

[0049] Secondly, the present invention provides the use of tRNA in the preparation of mRNA vaccines, wherein the tRNA acts as an expression promoter to increase the expression level of mRNA, and the codon corresponding to the tRNA can promote or improve the stability of the mRNA; preferably, the tRNA further comprises a single or multiple modifying nucleotide selected from the following: positions 18, 26, 34, 37, 47d, 46, 55, and 58 on the tRNA molecule; and the modifying nucleotide is selected from: Gm, m2, 2G, m5C, Cm, m1G, m7G, Ψ, and m1A.

[0050] Furthermore, the use of the tRNA described in this invention in the preparation of mRNA vaccines is characterized in that the tRNA, by increasing the expression level of mRNA, stimulates the body to produce a stronger humoral immune response and cellular immune response.

[0051] Furthermore, the use of the tRNA in the preparation of mRNA vaccines according to the present invention is characterized in that: the tRNA contains single or multiple site-modifying nucleotides selected from the following group:

[0052] (1) Replace the 18th bit with Gm (Gm18);

[0053] (2) Replace the 26th bit with m2,2G(m2,2G26);

[0054] (3) Replace the 34th bit with m5C (m5C34);

[0055] (4) Replace the 34th bit with Cm (Cm34);

[0056] (5) Replace the 37th bit with m1G (m1G37);

[0057] (6) Replace the 47d position with m3C (m3C47d);

[0058] (7) Replace the 46th bit with m7G (m7G46);

[0059] (8) Replace the 55th position with Ψ(Ψ55);

[0060] (9) Replace the 58th bit with m1A (m1A58);

[0061] (10) Replace the 18th bit with Gm (Gm18) + replace the 55th bit with Ψ (Ψ55);

[0062] (11) Replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37);

[0063] (12) Replace the 34th bit with Cm (Cm34) + replace the 37th bit with m1G (m1G37);

[0064] (13) Replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37) + replace the 58th bit with m1A (m1A58);

[0065] (14) Replace the 34th bit with Cm (Cm34) + replace the 37th bit with m1G (m1G37) + replace the 58th bit with m1A (m1A58);

[0066] (15) Replace the 18th bit with Gm (Gm18) + replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37) + replace the 55th bit with Ψ (Ψ55) + replace the 58th bit with m1A (m1A58);

[0067] (16) Replace the 18th position with Gm (Gm18) + replace the 34th position with Cm (Cm34) + replace the 37th position with m1G (m1G37) + replace the 55th position with Ψ (Ψ55) + replace the 58th position with m1A (m1A58).

[0068] Furthermore, the use of the tRNA of the present invention in the preparation of mRNA vaccines is characterized in that: the tRNA carries leucine; preferably, it contains one or more modified nucleotides based on the Leu-StRNA-CUA shown in SEQ ID NO:1, or contains one or more modified nucleotides based on the Leu-tRNA-CAG shown in SEQ ID NO:2.

[0069] Furthermore, the use of the tRNA in the preparation of mRNA vaccines according to the present invention is characterized in that the mRNA comprises the complete substitution of a single modified nucleotide, preferably including the following modifications:

[0070] (1) Replace all A's on the mRNA with modified nucleotides selected from 6Me-A and Z;

[0071] (2) Replace all the Us on the mRNA with modified nucleotides selected from Psi, N1Me-Psi, 5OMe-U, 3Me-5OMe-U;

[0072] (3) Replace all Cs on the mRNA with the modified nucleotide 5Me-C;

[0073] (4) Replace all Gs on the mRNA with the modified nucleotide N7Me-G.

[0074] Furthermore, the use of the tRNA in the preparation of mRNA vaccines according to the present invention is characterized in that the tRNA and the mRNA use the same delivery system, preferably lipid nanoparticles (LNPs) for co-encapsulation and delivery;

[0075] The tRNA and mRNA have a mass ratio of 1:1, a nitrogen-phosphorus molar ratio of 3:1, a particle size between 80 and 90 nm, and an encapsulation efficiency greater than 95%.

[0076] Thirdly, the present invention provides a vaccine composition comprising:

[0077] (1) mRNA, wherein the mRNA encodes one or more antigenic epitopes;

[0078] (2) tRNA, wherein the codon corresponding to the tRNA can promote or improve the stability of the mRNA, thereby increasing the expression level of the mRNA; preferably, the tRNA also contains single or multiple site-modifying nucleotides.

[0079] And, optional

[0080] (3) Delivery system.

[0081] Furthermore, the vaccine composition of the present invention is characterized in that: the tRNA comprises single or multiple site-modifying nucleotides selected from the group consisting of:

[0082] (1) Replace the 18th bit with Gm (Gm18);

[0083] (2) Replace the 26th bit with m2,2G(m2,2G26);

[0084] (3) Replace the 34th bit with m5C (m5C34);

[0085] (4) Replace the 34th bit with Cm (Cm34);

[0086] (5) Replace the 37th bit with m1G (m1G37);

[0087] (6) Replace the 47d position with m3C (m3C47d);

[0088] (7) Replace the 46th bit with m7G (m7G46);

[0089] (8) Replace the 55th position with Ψ(Ψ55);

[0090] (9) Replace the 58th bit with m1A (m1A58);

[0091] (10) Replace the 18th bit with Gm (Gm18) + replace the 55th bit with Ψ (Ψ55);

[0092] (11) Replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37);

[0093] (12) Replace the 34th bit with Cm (Cm34) + replace the 37th bit with m1G (m1G37);

[0094] (13) Replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37) + replace the 58th bit with m1A (m1A58);

[0095] (14) Replace the 34th bit with Cm (Cm34) + replace the 37th bit with m1G (m1G37) + replace the 58th bit with m1A (m1A58);

[0096] (15) Replace the 18th bit with Gm (Gm18) + replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37) + replace the 55th bit with Ψ (Ψ55) + replace the 58th bit with m1A (m1A58);

[0097] (16) Replace the 18th position with Gm (Gm18) + replace the 34th position with Cm (Cm34) + replace the 37th position with m1G (m1G37) + replace the 55th position with Ψ (Ψ55) + replace the 58th position with m1A (m1A58).

[0098] Furthermore, the vaccine composition of the present invention is characterized in that: the tRNA carries leucine; preferably, it contains one or more modified nucleotides based on the Leu-StRNA-CUA shown in SEQ ID NO:1, or contains one or more modified nucleotides based on the Leu-tRNA-CAG shown in SEQ ID NO:2.

[0099] Furthermore, the vaccine composition of the present invention is characterized in that the mRNA comprises the complete replacement of a single modified nucleotide, preferably including the following modifications:

[0100] (1) Replace all A's on the mRNA with modified nucleotides selected from 6Me-A and Z;

[0101] (2) Replace all the Us on the mRNA with modified nucleotides selected from Psi, N1Me-Psi, 5OMe-U, 3Me-5OMe-U;

[0102] (3) Replace all Cs on the mRNA with the modified nucleotide 5Me-C;

[0103] (4) Replace all Gs on the mRNA with the modified nucleotide N7Me-G.

[0104] Furthermore, the vaccine composition of the present invention is characterized in that the tRNA and the mRNA use the same delivery system, preferably lipid nanoparticles (LNPs) for co-encapsulation and delivery;

[0105] The tRNA and mRNA have a mass ratio of 1:1, a nitrogen-phosphorus molar ratio of 3:1, a particle size between 80 and 90 nm, and an encapsulation efficiency greater than 95%.

[0106] Fourthly, the present invention provides a method for screening enhanced tRNAs, comprising the following steps:

[0107] (1) Analyze the frequency of codon usage for the target mRNA;

[0108] (2) Calculate the R-value using the Pearson correlation between the frequency of codons on transcripts and the half-life of the target mRNA, which is the correlation coefficient between the frequency of codon use and the stability of the target mRNA.

[0109] (3) Evaluate the contribution of each codon to the stability of the target mRNA, select the codons that contribute highly to the stability of the target mRNA or promote the stability of the target mRNA, and use their corresponding tRNAs as enhanced tRNAs.

[0110] Furthermore, the method for screening enhanced tRNAs according to the present invention is characterized in that the enhanced tRNA is a tRNA that can promote the protein coding ability of the target mRNA or increase the expression of the target mRNA.

[0111] Fifthly, the present invention provides a recombinant cell for antibody production, characterized in that the recombinant cell overexpresses tRNA capable of increasing antibody expression, wherein the tRNA includes the tRNA isodecoder family, preferably Val-tRNA-AAC, Val-tRNA-CAC, Ile-tRNA-AAT, Gln-tRNA-CTG, Ala-tRNA-AGC, Asn-tRNA-GTT, Asp-tRNA-GTC, Gly-tRNA-GCC, Lys-tRNA-CTT, Ser-tRNA-AGA, Ser-tRNA-GCT, Thr-tRNA-AGT.

[0112] In a sixth aspect, the present invention provides recombinant cells for producing or packaging recombinant AAV, characterized in that the recombinant cells overexpress tRNAs capable of improving AAV packaging efficiency, wherein the tRNAs include Asp-tRNA-GTC, Ile-tRNA-AAT, Ile-tRNA-GAT, Leu-tRNA-TAA, Arg-tRNA-TCT, Phe-tRNA-GAA, Leu-tRNA-CAG, Leu-tRNA-GAG, Val-tRNA-CAC, Ala-tRNA-AGC, Tyr-tRNA-GTA, Thr-tRNA-AGT, Gly-tRNA-GCC, Pro-tRNA-GGG, Asn-tRNA-ATT, Lys-tRNA-TTT, and Glu-tRNA-TTC.

[0113] Advantages and beneficial effects of the present invention:

[0114] This invention is based on the principles of mRNA translation in eukaryotic cells, where tRNA accessibility is a crucial factor influencing protein production efficiency. A "tRNA+" strategy is proposed, the core idea of ​​which is that increased accessibility of a certain tRNA promotes the translation of homologous codon-rich mRNAs, thereby increasing the expression level of the target protein. Through combined analysis of codon usage frequency and stability coefficients, the contribution of each codon to the stability of the target mRNA was evaluated, and protein expression function experiments verified the screening of synergistic tRNAs that promote target protein expression. Secondly, by introducing modifying nucleotides into mRNA through in vitro transcription, beneficial modifying nucleotide types that enhance the protein-coding ability of mRNA were identified. Furthermore, drawing on the modification paradigm of natural tRNA, the impact of specific modifying nucleotides at specific sites on tRNA decoding ability was evaluated, and beneficial modification sites and nucleotide types were screened. Finally, by analyzing the synergistic effects between nucleotides at different sites on tRNA, combinations of modifying nucleotides at different sites were screened, verifying the identification of more functional modification sites and nucleotide combination paradigms. Finally, a tRNA+mRNA vaccine formulation was obtained by co-encapsulating site-modified tRNA and SARS-CoV-2 Spike (B.1.1.529) mRNA onto lipid nanoparticles, and its humoral and cellular immune response efficacy was evaluated in mice. Compared with the prior art, the technical solution of the present invention has at least the following beneficial effects:

[0115] First, the "tRNA+" strategy proposed in this invention has been experimentally verified to show that overexpression of tRNA can significantly promote the expression level of the target protein. This invention provides a novel tRNA+mRNA immune-enhancing vaccine, which enhances the antigen-protein encoding ability of the mRNA vaccine by introducing one or more tRNA molecules, thereby stimulating a stronger humoral and cellular immune response in vivo, indicating that tRNA+mRNA is an immune-enhancing vaccine.

[0116] Second, this invention discovered that different modified nucleotides at different sites have varying effects on the decoding ability of tRNA. The tRNA molecules used in this invention contain single or multiple site-specific modified nucleotides at positions 18, 26, 34, 37, 47d, 46, 55, and 58, exhibiting higher aminoacylation efficiency, lower immunogenicity, and higher decoding ability compared to unmodified tRNA. Among all site-specific modified Leu-stRNA-CUAs, Cm34 significantly enhances tRNA reading efficiency, while Cm34+m1G37, Cm34+m1G37+m1A58, and Gm18+Cm34+m1G37+Ψ55+m1A58 further enhance it. In all site-specific modified Leu-stRNA-CUAs, tRNAs containing Cm34 modification exhibit superior reading ability compared to tRNAs modified with m5C34. Among all site-modified Leu-tRNA-CAGs, the m1G37-modified tRNA exhibited the highest decoding efficiency among single-site modified tRNAs. The following modifiers further enhanced this decoding efficiency: m5C34+m1G37, Cm34+m1G37, m5C34+m1G37+m1A58, Cm34+m1G37+m1A58, Gm18+m5C34+m1G37+Ψ55+m1A58, and Gm18+Cm34+m1G37+Ψ55+m1A58. In all site-modified Leu-tRNA-CAGs, tRNAs containing m5C34 modification consistently demonstrated superior decoding capabilities compared to Cm34-modified tRNAs.

[0117] Third, this invention has discovered that different modified nucleotides have different effects on the protein-coding ability of mRNA. The mRNA molecules used in this invention contain full substitutions of a single modified nucleotide selected from the following: A (6Me-A, Z), U (Psi, N1Me-Psi, 5OMe-U, 3Me-5OMe-U), C (5Me-C), and G (N7Me-G). Among them, N1Me-pUTP and pUTP can significantly improve the protein-coding ability of mRNA, while other modified nucleotides weaken the coding efficiency of mRNA, but may have a promoting effect on reducing the immunogenicity of mRNA and improving its structural stability. Attached Figure Description

[0118] The above and other objects, features, and advantages of exemplary embodiments of the present disclosure will become readily apparent upon reading the following detailed description with reference to the accompanying drawings. In the drawings, several embodiments of the present disclosure are illustrated by way of example and not limitation, and like or corresponding reference numerals denote like or corresponding parts, wherein:

[0119] Figure 1: Overexpression of tRNA increases Spike protein expression levels.

[0120] (a) Codon usage frequency analysis of Spike (B.1.1.529) mRNA sequence;

[0121] (b) Codon stability analysis of Spike (B.1.1.529) mRNA sequence;

[0122] (c) Combined analysis of codon usage frequency and stability coefficient of Spike (B.1.1.529) mRNA sequence to determine which codons contribute significantly to mRNA stability;

[0123] (d) The tRNA library that may increase Spike protein expression levels, as analyzed in Figure 1(ac);

[0124] (e) Schematic diagram of the method for evaluating whether tRNA can promote Spike protein expression: tRNA plasmid and Spike protein plasmid are co-transfected into HEK293T cells in a certain ratio. After 48 hours, the cells are lysed, the protein is extracted, and protein quantification is performed.

[0125] (f) Quantitative analysis results of tRNA promoting Spike protein expression.

[0126] Figure 2: Functional verification results of mRNAs modified with different nucleotides.

[0127] Figure 2(a) Green fluorescence results of cells transfected with EGFP-mRNA modified with different nucleotides;

[0128] Figure 2(bc) shows the flow cytometry results of EGFP-mRNA transfected with different nucleotides in cells;

[0129] Figure 2(d) shows the results of protein quantification analysis after cells were transfected with EGFP-mRNA modified with different nucleotides.

[0130] Figure 3: Functional validation results of tRNAs modified at single and multiple points.

[0131] Figure 3(a) Schematic diagram of the decoding function of site-directed modified Leu-stRNA-CUA;

[0132] Figure 3(b) Schematic diagram of the decoding function of site-directed modified Leu-tRNA-CAG;

[0133] Figure 3(c) Green fluorescence recovery results after co-transformation of EGFP39UAG-mRNA and site-modified Leu-StRNA-CUA into cells;

[0134] Figure 3(d) Quantitative analysis of green fluorescent protein after co-transformation of EGFP39UAG-mRNA and site-modified Leu-StRNA-CUA into cells;

[0135] Figure 3(e) shows the expression of mCherry fluorescent protein in cells co-transfected with 5×CUG-mCherry-mRNA and site-modified Leu-tRNA-CAG;

[0136] Figure 3(f) Flow cytometry analysis results of mCherry fluorescent protein after co-transfection of 5×CUG-mCherry-mRNA and site-modified Leu-tRNA-CAG cells.

[0137] Figure 4: Immunological evaluation results of LNP-co-encapsulated tRNA+mRNA vaccines.

[0138] Figure 4(a) Schematic diagram of LNP co-encapsulating site-modified tRNA and Spike-mRNA;

[0139] Figure 4(b) Size and intensity distribution of LNP particles;

[0140] Figure 4(c) Schematic diagram of the process of immunizing mice with tRNA+mRNA vaccine;

[0141] Figure 4(d) Results of humoral immunity evaluation of tRNA+mRNA vaccine;

[0142] Figure 4(e) shows the cellular immunity evaluation results of the tRNA+mRNA vaccine.

[0143] Figure 5: tRNA increases the expression of the monoclonal antibody sacituzumab.

[0144] Figure 5(a) ELISA detection of changes in the expression level of monoclonal antibody sacituzumab after overexpression of Val-tRNA-AAC-3-1 and Ile-tRNA-AAT-1-1;

[0145] Figure 5(b) shows the changes in expression level and quality of monoclonal antibody sacituzumab after overexpression of Val-tRNA-AAC-3-1 and Ile-tRNA-AAT-1-1 by immunoblotting.

[0146] Figure 6: tRNA increases AAV2 packaging.

[0147] Figure 6(a) Fluorescence results showing the effect of Arg-tRNA-TCT-3-2 on AAV2 packaging;

[0148] Figure 6(b) Detection of virus titer using the freeze-thaw method;

[0149] Figure 6(c) Fluorescence image of HEK293T cells infected with the same volume of viral stock solution. Detailed Implementation

[0150] 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 embodiments of the present invention, and not all embodiments. Based on the 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.

[0151] Example 1: Overexpression of tRNA increases Spike protein expression level

[0152] We developed a method for screening enhanced tRNAs by combining codon usage frequency and stability coefficient analysis (Figure 1a-c). We evaluated the contribution of each codon to the stability of SARS-CoV-2 Spike mRNA (B.1.1.529) (for specific analysis and evaluation methods, please refer to Vladimir Presnyak et al. Codon optimality is a major determinant of mRNA stability. Cell. 2015 Mar 12; 160(6):1111-24.). The tRNAs corresponding to codons with high scores were considered to be able to increase the translation level of the target mRNA (Figure 1d).

[0153] We co-transfected Spike protein expression plasmids and tRNA expression plasmids into HEK293T cells at a certain ratio, with the control group receiving an empty vector plasmid. After 48 hours, the cells were lysed, proteins were extracted, and protein quantification was performed to compare the Spike protein expression levels in the tRNA-overexpressing group and the control group (Figure 1e). Experimental results: As shown in Figure 1f, tRNA overexpression significantly increased the expression level of Spike protein, with a maximum increase of 5-9 times, indicating that the tRNA+ strategy is a feasible strategy for promoting efficient protein production. The names and sequences of the enhancing tRNAs that can enhance Spike protein expression are shown in Table 1.

[0154] Table 1: Names and sequences of enhancing tRNAs

[0155] Example 2: Protein expression results of mRNA with a single nucleotide complete substitution

[0156] We performed a complete substitution of a single nucleotide modifier on mRNA via in vitro transcription. The modified nucleotides included: A (6Me-A, Z), U (Psi, N1Me-Psi, 5OMe-U, 3Me-5OMe-U), C (5Me-C), and G (N7Me-G). The complete substitution of a single nucleotide modifier on mRNA required one of the following conditions to be met: enhanced protein-coding capacity of the mRNA, improved translational fidelity, prolonged half-life, or reduced immunogenicity of the mRNA. We performed the complete substitution of a single nucleotide modifier on the EGFP-mRNA sequence. First, we obtained a linearized in vitro transcription DNA template through enzyme digestion and gel extraction. Second, the in vitro transcription reaction system was prepared according to the following requirements and incubated at 37°C for 2 hours:

[0157] EGFP-mRNA (A, U, C, G);

[0158] EGFP-mRNA-pUTP(A, pU, C, G);

[0159] EGFP-mRNA-N1Me-pUTP (A, N1Me-pU, C, G);

[0160] EGFP-mRNA-5OMe-UTP (A, 5OMe-U, C, G);

[0161] EGFP-mRNA-3Me-5OMe-UTP (A, 3Me-5OMe-U, C, G);

[0162] EGFP-mRNA-5Me-CTP (A, U, 5Me-C, G);

[0163] EGFP-mRNA-6Me-ATP (6Me-A, U, C, G);

[0164] EGFP-mRNA-A+6Me-ATP (A:6Me-A 1:1, U, C, G);

[0165] EGFP-mRNA-6Me-ATP-PA (6Me-A, U, C, G), polyA tail is ATP;

[0166] EGFP-mRNA-ZTP(Z,U,C,G);

[0167] EGFP-mRNA-ZTP-PA (Z, U, C, G), polyA tail is ATP;

[0168] EGFP-mRNA-N7Me-GTP (A, U, C, N7Me-G).

[0169] Finally, the final mRNA product was obtained by purification using RNA clean magnetic beads.

[0170] We transfected HEK293T cells with different modified EGFP-mRNAs. After 24 hours, we detected the fluorescence intensity and protein expression level of EGFP and used this to evaluate the function of the modified nucleotides.

[0171] Experimental Results: mRNAs modified with different nucleotides exhibited varying protein-coding capabilities, ranked from highest to lowest efficiency as follows: N1Me-pUTP, pUTP, UTP, 5Me-CTP, 5OMe-UTP, ZTP-PA, ZTP, A+6Me-ATP, 3Me-5OMe-UTP, 6Me-ATP, 6Me-ATP-PA, and N7Me-GTP. Among all modified nucleotides, N1Me-pUTP and pUTP-modified mRNAs showed superior protein-coding capabilities, while A+6Me-ATP, 3Me-5OMe-UTP, 6Me-ATP, 6Me-ATP-PA, and N7Me-GTP significantly reduced mRNA protein-coding capabilities. This may be related to the fact that these modified nucleotides disrupted the interaction between mRNA and ribosomes, interfering with the recognition of codons and tRNA anticodons (Figures 2a-d).

[0172] Example 3: Introducing single- or multi-site modified nucleotides into chemically synthesized Leu-tRNA

[0173] We introduce specific modifying nucleotides into specific sites on tRNA via solid-phase chemical synthesis. These nucleotide modifications at specific sites on tRNA must meet one of the following conditions: enhance tRNA decoding ability, improve translation fidelity, improve tRNA structural stability, or reduce tRNA immunogenicity. Modifications on the anticodon loop, such as 2'O-methylation of C34 (Cm), are among the commonly used wobble modifications in human tRNA, enhancing the interaction between the codon and anticodon. 5-methylation of C34 (m5C) protects tRNA from endonuclease cleavage. Modifications at G37, typically N1-methylguanosine (m1G), located at the Watson-Crick interface, stabilize tRNA-ribosome interactions and prevent +1 frameshift reading. Furthermore, modifications outside the anticodon loop can perform many other tRNA-related functions; for example, Gm18 (D loop) modification reduces the immunostimulatory activity of tRNA and synergistically promotes tRNA structural stability with Ψ55 (T loop) modification. m1A58 (T loop) modification is present in almost all human tRNAs and can enhance the affinity of tRNA for translation elongation factor EF1A, increasing the efficiency of tRNA delivery to the ribosomal A site.

[0174] 3.1 We co-transfected HEK293T cells with IVS-Leu-stRNA-CUA and IVT-EGFP-39UAG-mRNA (N1Me-Psi full replacement) with different modifications. EGFP-39UAG-mRNA can be read through by stRNA-CUA, and its readthrough efficiency can indicate the function of the modification and partially represent the decoding ability of Leu-tRNA-CAG (Figure 3a).

[0175] Experimental results showed that Cm34-modified Leu-stRNA-CUA exhibited the highest reading efficiency among single-site modified tRNAs, arranged from highest to lowest as follows: Cm34, m5C34, m1G37, m1A58, Ψ55, Gm18. Combinations of modified bases further enhanced its reading efficiency, such as Cm34+m1G37, Cm34+m1G37+m1A58, and Gm18+Cm34+m1G37+Ψ55+m1A58. Among all site-modified Leu-stRNA-CUA, Cm34-modified tRNAs showed superior reading efficiency compared to m5C34-modified tRNAs (Figures 3c-d). The sequences of modified tRNAs Lst-1 to Lst-16 based on Leu-stRNA-CUA are shown in Table 2.

[0176] Table 2: Modified tRNAs Lst-1 to Lst-16 based on Leu-stRNA-CUA

[0177] 3.2 We co-transfected HEK293T cells with IVS-Leu-tRNA-CAG and IVT-5×CUG-mCherry-mRNA (N1Me-Psi full replacement) with different modifications. Under the same tRNA concentration, the stronger the decoding ability of the tRNA, the faster the translation rate of 5×CUG-mCherry-mRNA, and the higher the expression level of mCherry protein (Figure 3b).

[0178] Experimental results showed that the m1G37-modified Leu-tRNA-CAG exhibited the highest decoding efficiency among tRNAs with a single modification, arranged from highest to lowest as follows: m1G37, m1A58, Ψ55, m5C34, Cm34, Gm18. Combinations of modified bases further enhanced its decoding efficiency, for example, m5C34+m1G37, Cm34+m1G37+m1A58, Cm34+m1G37+m1A58, Gm18+m5C34+m1G37+Ψ55+m1A58, and Gm18+Cm34+m1G37+Ψ55+m1A58. Among all site-specific modified Leu-tRNA-CAGs, the m5C34-modified tRNA showed superior decoding ability compared to the Cm34-modified tRNA (Figures 3e-f). The sequences of modified tRNAs Lt-1 to Lt-16 based on Leu-tRNA-CAG are shown in Table 3.

[0179] Table 3: Modified tRNAs Lt-1 to Lt-16 based on Leu-tRNA-CAG

[0180] Example 4: Immunological evaluation results of LNP co-encapsulated tRNA+mRNA vaccine

[0181] Based on the experimental results described in Examples 2 and 3, we further evaluated the immunogenicity of the tRNA+mRNA vaccine in mice by using lipid nanoparticles (Figure 4a-b) to co-package site-modified tRNA with strong decoding ability and N1Me-Psi fully replaced SARS-CoV-2 Spike (B.1.1.529) mRNA (Figure 4c).

[0182] The experimental results showed that, compared with the control group (T1: null tRNA + mRNA), the experimental groups (T2 / T3 / T6 / T7) all exhibited higher endpoint Spike IgG antibody titers and more IL-2 ELISA spots, indicating that the humoral and cellular immunity of the experimental groups was stronger than that of the control group. Among the experimental groups, T7 showed the highest antibody titer and the highest number of IL-2 ELISA spots, which is directly related to the strongest decoding ability of the site-modified tRNA used in the T7 group.

[0183] Example 5: Screening for tRNAs that can increase antibody expression

[0184] tRNAs that can increase the expression of the antibody sacituzumab were screened according to the method in Example 1. Antibody yield was detected by ELISA (Figure 5a), and antibody quality was detected by Western blotting (Figure 5b).

[0185] We overexpressed different tRNAs into CHO-K1 engineered cells stably expressing the monoclonal antibody sacituzumab, or co-transfected different tRNAs and sacituzumab light and heavy chain expression plasmids into CHO-K1, CHO-S, or Expi-293F engineered cells commonly used for antibody expression at a certain ratio. The blank control group used empty vector plasmids. Taking the increase in antibody expression by Val-tRNA-AAC-3-1 and Ile-tRNA-AAT-1-1 as an example: Overexpression of Val-tRNA-AAC-3-1 and Ile-tRNA-AAT-1-1 in CHO-K1 engineered cells stably expressing sacituzumab, ELISA and Western blotting experiments showed that Val-tRNA-AAC-3-1 and Ile-tRNA-AAT-1-1 could increase sacituzumab expression by 1.3 and 4.2 times, respectively (Figures 5a-b).

[0186] tRNAs that can increase antibody expression were screened and included the following tRNA isodecoder families: Val-tRNA-AAC, Val-tRNA-CAC, Ile-tRNA-AAT, Gln-tRNA-CTG, Ala-tRNA-AGC, Asn-tRNA-GTT, Asp-tRNA-GTC, Gly-tRNA-GCC, Lys-tRNA-CTT, Ser-tRNA-AGA, Ser-tRNA-GCT, and Thr-tRNA-AGT.

[0187] Example 6: Screening for tRNAs that can increase AAV packaging efficiency

[0188] Screening with tRNA-stabilized cell lines can increase the amount of AAV2-packaged tRNA, as shown in Figure 6.

[0189] We co-transfected different tRNA expression plasmids and the transgenic vector plasmid pScAAV-EGFP for packaging AAV2 virus, the helper plasmid pAAV2, and pAdhelper into HEK293T cells. Taking the increase in AAV2 packaging in HEK293T cells using Arg-tRNA-TCT-3-2 as an example: since the transgenic vector plasmid carries the EGFP reporter gene, the expression of green fluorescent protein during packaging indirectly reflects the AAV2 packaging efficiency of HEK293T cells. We used fluorescence microscopy to photograph the packaging efficiency and real-time quantitative PCR to detect the genomic titer (Figures 6a-b). The EGFP reporter gene is expressed after normal viral infection of cells, and the expression level is positively correlated with the number of infected viruses. Therefore, we infected HEK293T cells with the same volume of viral stock solution and photographed the cells 72 hours after infection (Figure 6c). The results showed that Arg-tRNA-TCT-3-2 could increase AAV2 packaging by 3.35-fold.

[0190] tRNAs that can enhance AAV2 packaging were screened and included the following tRNA isodecoder families: Asp-tRNA-GTC, Ile-tRNA-AAT, Ile-tRNA-GAT, Leu-tRNA-TAA, Arg-tRNA-TCT, Phe-tRNA-GAA, Leu-tRNA-CAG, Leu-tRNA-GAG, Val-tRNA-CAC, Ala-tRNA-AGC, Tyr-tRNA-GTA, Thr-tRNA-AGT, Gly-tRNA-GCC, Pro-tRNA-GGG, Asn-tRNA-ATT, Lys-tRNA-TTT, and Glu-tRNA-TTC.

[0191] The preferred embodiments of the present invention have been described above to make the spirit of the present invention clearer and easier to understand, and are not intended to limit the present invention. All modifications, substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope summarized by the appended claims.

Claims

1. The use of tRNA in the preparation of mRNA vaccines, characterized in that, The tRNA acts as an expression promoter to increase the expression level of mRNA, and the codon corresponding to the tRNA can promote or improve the stability of the mRNA; preferably, the tRNA contains single or multiple modifying nucleotides selected from the following: positions 18, 26, 34, 37, 47d, 46, 55, and 58 on the tRNA molecule; and the modifying nucleotides are selected from: Gm, m2, 2G, m5C, Cm, m3C, m1G, m7G, Ψ, and m1A.

2. The use of tRNA in the preparation of mRNA vaccines as described in claim 1, characterized in that, The tRNA, by increasing the expression level of mRNA, stimulates the body to produce a stronger humoral immune response and cellular immune response.

3. The use of tRNA in the preparation of mRNA vaccines as described in claim 1 or 2, characterized in that, The mRNA comprises complete substitutions of a single modifying nucleotide, preferably including at least one of the following modifications: (1) Replace all A's on the mRNA with modified nucleotides selected from 6Me-A and Z; (2) Replace all the Us on the mRNA with modified nucleotides selected from Psi, N1Me-Psi, 5OMe-U, 3Me-5OMe-U; (3) Replace all Cs on the mRNA with the modified nucleotide 5Me-C; (4) Replace all the Gs on the mRNA with the modified nucleotide N7Me-G.

4. The use of tRNA in the preparation of mRNA vaccines as described in claim 3, characterized in that, The mRNA in question is SARS-CoV-2 B.1.1.529 Spike mRNA.

5. The use of tRNA in the preparation of mRNA vaccines as described in claim 4, characterized in that, Replace all Us on the mRNA with N1Me-Psi.

6. The use of tRNA in the preparation of mRNA vaccines as described in claim 1 or 2, characterized in that, The tRNA contains single or multiple site-modifying nucleotides selected from the following group: (1) Replace the 18th bit with Gm (Gm18); (2) Replace the 26th bit with m2,2G(m2,2G26); (3) Replace the 34th bit with m5C (m5C34); (4) Replace the 34th bit with Cm (Cm34); (5) Replace the 37th bit with m1G (m1G37); (6) Replace the 47d position with m3C (m3C47d); (7) Replace the 46th bit with m7G (m7G46); (8) Replace the 55th position with Ψ(Ψ55); (9) Replace the 58th bit with m1A (m1A58); (10) Replace the 18th bit with Gm (Gm18) + replace the 55th bit with Ψ (Ψ55); (11) Replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37); (12) Replace the 34th bit with Cm (Cm34) + replace the 37th bit with m1G (m1G37); (13) Replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37) + replace the 58th bit with m1A (m1A58); (14) Replace the 34th bit with Cm (Cm34) + replace the 37th bit with m1G (m1G37) + replace the 58th bit with m1A (m1A58); (15) Replace the 18th bit with Gm (Gm18) + replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37) + replace the 55th bit with Ψ (Ψ55) + replace the 58th bit with m1A (m1A58); (16) Replace the 18th position with Gm (Gm18) + replace the 34th position with Cm (Cm34) + replace the 37th position with m1G (m1G37) + replace the 55th position with Ψ (Ψ55) + replace the 58th position with m1A (m1A58).

7. The use of tRNA in the preparation of mRNA vaccines as described in claim 6, characterized in that, The tRNA molecule is Leu-tRNA-CAG or a tRNA modified based on Leu-tRNA-CAG; The nucleotide sequence of Leu-tRNA-CAG is shown in SEQ ID NO:2; the tRNA modified based on Leu-tRNA-CAG is selected from Lt-4 shown in SEQ ID NO:44, Lt-11 shown in SEQ ID NO:51, and Lt-12 shown in SEQ ID NO:

52.

8. The use of tRNA in the preparation of mRNA vaccines as described in claim 1 or 2, characterized in that, The tRNA and the mRNA use the same delivery system, preferably co-encapsulated and delivered using lipid nanoparticles (LNPs). The tRNA and mRNA have a mass ratio of 1:1, a nitrogen-phosphorus molar ratio of 3:1, a particle size between 80 and 90 nm, and an encapsulation efficiency greater than 95%.

9. A vaccine composition, characterized in that, include: (1) mRNA, wherein the mRNA encodes one or more antigenic epitopes; (2) tRNA, wherein the codon corresponding to the tRNA can promote or improve the stability of the mRNA, thereby increasing the expression level of the mRNA, and preferably the tRNA further contains single or multiple site-modifying nucleotides; And, optional (3) Delivery system.

10. The vaccine composition according to claim 9, characterized in that, The mRNA comprises complete substitutions of a single modifying nucleotide, preferably including at least one of the following modifications: (1) Replace all A's on the mRNA with modified nucleotides selected from 6Me-A and Z; (2) Replace all the Us on the mRNA with modified nucleotides selected from Psi, N1Me-Psi, 5OMe-U, 3Me-5OMe-U; (3) Replace all Cs on the mRNA with the modified nucleotide 5Me-C; (4) Replace all the Gs on the mRNA with the modified nucleotide N7Me-G.

11. The vaccine composition according to claim 9 or 10, characterized in that, The mRNA in question is SARS-CoV-2B.1.1.529Spike mRNA.

12. The vaccine composition according to claim 9, characterized in that, The tRNA contains single or multiple site-modifying nucleotides selected from the following group: (1) Replace the 18th bit with Gm (Gm18); (2) Replace the 26th bit with m2,2G(m2,2G26); (3) Replace the 34th bit with m5C (m5C34); (4) Replace the 34th bit with Cm (Cm34); (5) Replace the 37th bit with m1G (m1G37); (6) Replace the 47d position with m3C (m3C47d); (7) Replace the 46th bit with m7G (m7G46); (8) Replace the 55th position with Ψ(Ψ55); (9) Replace the 58th bit with m1A (m1A58); (10) Replace the 18th bit with Gm (Gm18) + replace the 55th bit with Ψ (Ψ55); (11) Replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37); (12) Replace the 34th bit with Cm (Cm34) + replace the 37th bit with m1G (m1G37); (13) Replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37) + replace the 58th bit with m1A (m1A58); (14) Replace the 34th bit with Cm (Cm34) + replace the 37th bit with m1G (m1G37) + replace the 58th bit with m1A (m1A58); (15) Replace the 18th bit with Gm (Gm18) + replace the 34th bit with m5C (m5C34) + replace the 37th bit with m1G (m1G37) + replace the 55th bit with Ψ (Ψ55) + replace the 58th bit with m1A (m1A58); (16) Replace the 18th position with Gm (Gm18) + replace the 34th position with Cm (Cm34) + replace the 37th position with m1G (m1G37) + replace the 55th position with Ψ (Ψ55) + replace the 58th position with m1A (m1A58).

13. The vaccine composition according to claim 9 or 12, characterized in that, The tRNA molecule is Leu-tRNA-CAG or a tRNA modified based on Leu-tRNA-CAG; The nucleotide sequence of Leu-tRNA-CAG is shown in SEQ ID NO:2; the tRNA modified based on Leu-tRNA-CAG is selected from Lt-4 shown in SEQ ID NO:44, Lt-11 shown in SEQ ID NO:51, and Lt-12 shown in SEQ ID NO:52.

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