Use of tRNA in promoting the protein-coding ability of mRNA

Abstract

The present invention relates to the use of tRNA in promoting the protein-coding ability of mRNA, and the expression level of a target protein is increased by overexpressing tRNA, and the codon corresponding to the tRNA can promote or enhance the stability of the mRNA. The present invention also provides a novel tRNA+mRNA immune-enhanced vaccine, and the antigen protein-coding ability of the mRNA vaccine is enhanced by introducing one or more tRNA molecules, thereby eliciting a stronger humoral immune and cellular immune response in vivo. The present invention also provides a recombinant cell for producing an antibody, and the recombinant cell overexpresses tRNA capable of increasing the antibody expression level, and the tRNA includes the tRNA isodecoder family. The present invention also provides a recombinant cell for producing or packaging recombinant AAV, characterized in that the recombinant cell overexpresses tRNA capable of increasing the AAV packaging efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid vaccines, and particularly relates to the use of tRNA in promoting the protein-coding ability of mRNA and improving mRNA expression, and particularly relates to an enhanced tRNA+mRNA vaccine in which a lipid nanoparticle (LNP) co-encapsulates tRNA and mRNA. Background Art

[0002] A nucleic acid vaccine directly introduces an exogenous gene (DNA or RNA) encoding a certain antigen protein into animal somatic cells, synthesizes the antigen protein through the expression system of the host cell, and induces the host to produce an immune response against the antigen protein, so as to achieve the purpose of preventing and treating diseases. Although the research on nucleic acid vaccines began in the 1980s and 1990s, no nucleic acid vaccine was approved for marketing before 2020. The infection of SARS-CoV-2 has significantly promoted the research and development process of nucleic acid vaccines. With the wide application of SARS-CoV-2 mRNA vaccines, many adverse reactions related to mRNA and lipid nanoparticles (LNPs) have been gradually reported, such as stroke, myocardial infarction, pulmonary embolism, etc., suggesting the urgency of enhancing the coding ability of mRNA and optimizing the components of LNP.

[0003] Protein synthesis is one of the most energy-consuming processes in cells, and efficient translation is crucial for cell adaptation and function. In recent years, in order to achieve higher protein yields, researchers have modified mRNA, including screening new cap structure analogs with higher initiation probabilities and modified nucleotides with lower immunogenicity and higher decoding abilities; optimizing the codon distribution and composition; optimizing the secondary structure, length and sequence of 5’UTR and 3’UTR to achieve stronger ribosome binding activity, higher stability and accurate subcellular localization; designing a polyadenylate tail with branched chemical modifications to enhance the stability and translation ability of mRNA. In addition, circular RNA (CircRNA) and self-amplifying RNA (saRNA) or trans-amplifying RNA represent the next generation of RNA-based therapies. However, the methodologies for the design, synthesis, purification, delivery and safety evaluation of these RNAs still need to be further studied. Generally speaking, we expect to develop a translation accelerator to produce more target proteins under the same 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 main decoder of the genetic code and the central and dynamic component of translation. The tRNA pool in metazoan cells is dynamically changing, and the accessibility of tRNA (abundance and modification level) affects mRNA and ribosomes in a translation-dependent manner, which is related to cell differentiation or proliferation, response to cell stress, and tumor metastasis. Upregulating the accessibility of tRNA can increase the optimality of corresponding codons and improve the translation rate and stability of mRNA rich in corresponding codons. tRNA is considered an accelerator of translation and can endow mRNA with stronger protein-coding ability, indicating that the combination of mRNA and tRNA can be used as a feasible strategy to achieve efficient protein production, called the "tRNA+" strategy. The "tRNA+" strategy can be applied to 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 effect of RNA therapies and the immune response effect of RNA vaccines. Based on this, we propose a novel tRNA+mRNA immune-enhanced vaccine. Summary of the Invention

[0005] Aiming at the technical problems of poor coding ability, low expression level, and poor immune effect of mRNA vaccines in the prior art, the object of the present invention is to apply the translation element tRNA to improve the expression level of proteins.

[0006] Brief Description of the Invention

[0007] The present invention first proposes that the "tRNA+" strategy can increase the protein-coding ability of mRNA and can be used as a general strategy to increase the yield of target proteins.

[0008] The present invention first develops a novel tRNA+mRNA immune-enhanced vaccine, and the enhanced vaccine enhances the antigen protein-coding ability of the mRNA vaccine by introducing one or more tRNA molecules, thereby stimulating stronger humoral and cellular immune responses in the body.

[0009] The present invention relates to a delivery system of lipid nanoparticles (LNPs) co-encapsulating tRNA and mRNA. The tRNA molecule used in the present invention contains modified nucleotides with single-point or multi-point combinations at positions 18, 26, 34, 37, 47d, 46, 55, and 58, and has higher aminoacylation efficiency, lower immunogenicity, and higher decoding ability compared to unmodified tRNA. The mRNA molecule used in the present invention contains full 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).

[0010] The "tRNA+" strategy proposed by the present invention involves a method for joint analysis of codon usage frequency and stability coefficient, which evaluates the contribution value of each codon to the stability of the target mRNA. The tRNA corresponding to the codon with a high score is considered to be able to increase the translation level of the target mRNA. The tRNA described above has been verified at the protein expression level and proven to be able to increase the expression level of the target protein.

[0011] The novel tRNA+mRNA immune-enhanced vaccine described in the present invention enhances the antigen protein coding ability of the mRNA vaccine by introducing one or more tRNA molecules, thereby stimulating stronger humoral and cellular immune responses in the body. The tRNA+mRNA vaccine comprises three major components: lipid nanoparticles (LNPs) (delivery vector), mRNA (template for antigen coding), and tRNA (promoter for enhancing antigen protein expression).

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

[0013] The present invention relates to mRNA encoding an antigen protein, characterized in that: the mRNA contains full 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] The present invention relates to single-point or multi-point modified tRNAs capable of promoting mRNA translation, characterized in that: the tRNAs carry leucine (Leu), and the single-point or multi-point modified nucleotides included therein are as follows: the 18th position is replaced by Gm (Gm18); the 26th position is replaced by m2,2G (m2,2G26); the 34th position is replaced by m5C (m5C34); the 34th position is replaced by Cm (Cm34); the 37th position is replaced by m1G (m1G37); the 47d position is replaced by m3C (m3C47d); the 46th position is replaced by m7G (m7G46); the 55th position is replaced by Ψ (Ψ55); the 58th position is replaced by m1A (m1A58); multi-point replacement of Gm18 and Ψ55; multi-point replacement of m5C34 and m1G37; multi-point replacement of Cm34 and m1G37; multi-point replacement of m5C34, m1G37 and m1A58; multi-point replacement of Cm34, m1G37 and m1A58; multi-point replacement of Gm18, m5C34, m1G37, Ψ55 and m1A58; multi-point replacement of Gm18, Cm34, m1G37, Ψ55 and m1A58. Detailed Description of the Invention

[0016] The present invention provides the use of tRNA in promoting the protein-coding ability of mRNA, and the expression level of the target protein is increased by overexpressing tRNA. A novel tRNA+mRNA immune-enhanced vaccine is also provided, which enhances the antigen protein-coding ability of the mRNA vaccine by introducing one or more tRNA molecules, thereby eliciting stronger humoral and cellular immune responses in the body. The tRNA+mRNA vaccine uses a lipid nanoparticle (LNP) delivery system; the tRNA molecule contains modified nucleotides with single-point or multi-point combinations at positions 18, 26, 34, 37, 47d, 46, 55, and 58, and has higher aminoacylation efficiency, lower immunogenicity, and higher decoding ability compared to unmodified tRNA; the mRNA contains complete substitutions of the following single modified nucleotides, including A (6Me-A, Z), U (Psi, N1Me-Psi, 5OMe-U, 3Me-5OMe-U), C (5Me-C), G (N7Me-G). The tRNA+mRNA immune-enhanced vaccine can be applied to the preparation of viral mRNA vaccines and tumor mRNA vaccines with stronger immune effects, the development of protein replacement mRNA therapies with higher protein expression levels, and can also be applied to empower other forms of RNA therapies and vaccines, such as circular RNA, self-replicating RNA, etc. The present invention also provides a recombinant cell for producing antibodies, and the recombinant cell overexpresses tRNA capable of increasing the antibody expression level, and the tRNA includes the tRNA isodecoder family, preferably Val-tRNA-AAC, Val-tRNA-CAC, Ile-tRNA-AAT, Gln-tRNA-CTG. The present invention also provides a recombinant cell for producing or packaging recombinant AAV, characterized in that the recombinant cell overexpresses tRNA capable of increasing the AAV packaging efficiency, and the tRNA includes Asp-GTC-2-1, Ile-AAT-1-1, Ile-GAT-1-1, Leu-TAA-1-1, Arg-TCT-3-2.

[0017] Specifically:

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

[0019] Furthermore, the use of tRNA in promoting the protein-coding ability of mRNA or increasing the expression of mRNA according to the present invention is characterized in that the mRNA encodes an antibody, preferably a monoclonal antibody; the tRNA increases the expression level of the antibody.

[0020] Furthermore, the use of the tRNA of the present invention to promote the protein-coding ability of mRNA or increase the expression of mRNA 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 of the present invention to promote the protein-coding ability of mRNA or increase the expression of mRNA is characterized in that the mRNA encodes AAV packaging-related proteins, preferably encodes AAV capsid proteins, recombinant foreign genes; the tRNA increases the packaging efficiency of recombinant AAV viruses.

[0022] Furthermore, the use of the tRNA of the present invention to promote the protein-coding ability of mRNA or increase the expression of mRNA 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, Arg-TCT-3-2.

[0023] Furthermore, the tRNA of the present invention contains single-point or multi-point modified nucleotides selected from the following: positions 18, 26, 34, 37, 47d, 46, 55, 58 on the tRNA molecule; and the modified nucleotides are selected from: Gm, m2,2G, m5C, Cm, m3C, m1G, m7G, Ψ, m1A.

[0024] Preferably, the single-point or multi-point modified nucleotides contained in the tRNA are selected from the following group:

[0025] (1) The 18th position is replaced with Gm (Gm18);

[0026] (2) The 26th position is replaced with m2,2G (m2,2G26);

[0027] (3) The 34th position is replaced with m5C (m5C34);

[0028] (4) The 34th position is replaced with Cm (Cm34);

[0029] (5) The 37th position is replaced with m1G (m1G37);

[0030] (6) The 47th position is replaced by m3C (m3C47d);

[0031] (7) The 46th position is replaced by m7G (m7G46);

[0032] (8) The 55th position is replaced by Ψ (Ψ55);

[0033] (9) The 58th position is replaced by m1A (m1A58);

[0034] (10) The 18th position is replaced by Gm (Gm18) + the 55th position is replaced by Ψ (Ψ55);

[0035] (11) The 34th position is replaced by m5C (m5C34) + the 37th position is replaced by m1G (m1G37);

[0036] (12) The 34th position is replaced by Cm (Cm34) + the 37th position is replaced by m1G (m1G37);

[0037] (13) The 34th position is replaced by m5C (m5C34) + the 37th position is replaced by m1G (m1G37) + the 58th position is replaced by m1A (m1A58);

[0038] (14) The 34th position is replaced by Cm (Cm34) + the 37th position is replaced by m1G (m1G37) + the 58th position is replaced by m1A (m1A58);

[0039] (15) The 18th position is replaced by Gm (Gm18) + the 34th position is replaced by m5C (m5C34) + the 37th position is replaced by m1G (m1G37) + the 55th position is replaced by Ψ (Ψ55) + the 58th position is replaced by m1A (m1A58);

[0040] (16) The 18th position is replaced by Gm (Gm18) + the 34th position is replaced by Cm (Cm34) + the 37th position is replaced by m1G (m1G37) + the 55th position is replaced by Ψ (Ψ55) + the 58th position is replaced by m1A (m1A58).

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

[0042] Furthermore, the use of the tRNA of the present invention to promote the protein-coding ability of mRNA or increase the expression of mRNA is characterized in that the mRNA comprises all substitutions of a single modified nucleotide, preferably including the following modifications:

[0043] (1) All A on the mRNA are replaced with modified nucleotides selected from 6Me-A and Z;

[0044] (2) All U on the mRNA are replaced with modified nucleotides selected from Psi, N1Me-Psi, 5OMe-U, and 3Me-5OMe-U;

[0045] (3) All C on the mRNA are replaced with the modified nucleotide 5Me-C;

[0046] (4) All G on the mRNA are replaced with the modified nucleotide N7Me-G.

[0047] Furthermore, the use of the tRNA of the present invention to promote the protein-coding ability of mRNA or increase the expression of mRNA is characterized in that the tRNA and the mRNA adopt the same delivery system, preferably co-encapsulated and delivered using lipid nanoparticles LNP;

[0048] Wherein, the mass ratio of the tRNA to the 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%.

[0049] In a second aspect, the present invention provides the use of tRNA in the preparation of mRNA vaccines. The tRNA is used as an expression promoter to increase the expression level of mRNA, and the codons corresponding to the tRNA can promote or increase the stability of the mRNA; preferably, the tRNA further comprises single-point or multi-point modified nucleotides selected from the following: positions 18, 26, 34, 37, 47d, 46, 55, and 58 on the tRNA molecule; and the modified nucleotides are selected from: Gm, m2,2G, m5C, Cm, m1G, m7G, Ψ, m1A.

[0050] Furthermore, the use of the tRNA of the present invention in the preparation of mRNA vaccines is characterized in that the tRNA stimulates the body to generate stronger humoral immune responses and cellular immune responses by increasing the expression level of mRNA.

[0051] Furthermore, the use of the tRNA of the present invention in the preparation of mRNA vaccines is characterized in that: the single-point or multi-point modified nucleotides comprised in the tRNA are selected from the following group:

[0052] (1) The 18th position is replaced with Gm (Gm18);

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

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

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

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

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

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

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

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

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

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

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

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

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

[0066] (15) Replace the 18th position with Gm (Gm18) + replace the 34th position with m5C (m5C34) + replace the 37th position with m1G (m1G37) + replace the 55th position with Ψ (Ψ55) + replace the 58th position 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 single or multiple modified nucleotides based on Leu-StRNA-CUA shown in SEQ ID NO:1, or contains single or multiple modified nucleotides based on Leu-tRNA-CAG shown in SEQ ID NO:2.

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

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

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

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

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

[0074] Furthermore, the use of the tRNA of the present invention in the preparation of mRNA vaccines is characterized in that the tRNA and the mRNA adopt the same delivery system, preferably co-encapsulated and delivered using lipid nanoparticles LNP;

[0075] Among them, the mass ratio of the tRNA to the 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%.

[0076] In the third aspect, the present invention provides a vaccine composition, comprising:

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

[0078] (2) tRNA, the corresponding codon of which can promote or improve the stability of the mRNA and thus can increase the expression level of the mRNA, preferably the tRNA further comprises single or multiple modified nucleotides;

[0079] And, optionally

[0080] (3) A delivery system.

[0081] Furthermore, the vaccine composition of the present invention is characterized in that: the single or multiple modified nucleotides comprised by the tRNA are selected from the following group:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0096] (15) Replace the 18th position with Gm (Gm18) + Replace the 34th position with m5C (m5C34) + Replace the 37th position with m1G (m1G37) + Replace the 55th position with Ψ (Ψ55) + Replace the 58th position 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 single or multiple modified nucleotides based on Leu-StRNA-CUA shown in SEQ ID NO: 1, or contains single or multiple modified nucleotides based on Leu-tRNA-CAG shown in SEQ ID NO: 2.

[0099] Furthermore, the vaccine composition of the present invention is characterized in that the mRNA contains all substitutions of single modified nucleotides, and preferably, includes the following modifications:

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

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

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

[0103] (4) Replace all G 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 adopt the same delivery system, preferably co-encapsulated and delivered using lipid nanoparticles LNP;

[0105] Among them, the mass ratio of the tRNA to the 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%.

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

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

[0108] (2) Calculate the R value (R-value), that is, the correlation coefficient between the codon usage frequency and the stability of the target mRNA, using the Pearson correlation between the frequency of codons appearing on the transcript and the half-life of the target mRNA;

[0109] (3) Evaluate the contribution value of each codon to the stability of the target mRNA, select the codons with high contribution value to the stability of the target mRNA or promoting the stability of the target mRNA, and use the corresponding tRNA as the enhanced tRNA.

[0110] Further, the method for screening enhanced tRNA 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] In a fifth aspect, the present invention provides a recombinant cell for producing an antibody, characterized in that the recombinant cell overexpresses a tRNA that can increase the antibody expression level, 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.

[0112] In a sixth aspect, the present invention provides a recombinant cell for producing or packaging recombinant AAV, characterized in that the recombinant cell overexpresses a tRNA that can increase the AAV packaging efficiency, and the tRNA includes 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, Glu-tRNA-TTC.

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

[0114] The present invention is based on the law of mRNA translation in eukaryotic cells, where the accessibility of tRNA is one of the important factors affecting protein production efficiency, and proposes the "tRNA+" strategy. The core idea of this strategy is that when the accessibility of a certain tRNA increases, it can promote the translation of mRNA rich in homologous codons, thereby increasing the expression level of the target protein. By combining the analysis of codon usage frequency and stability coefficient, the contribution of each codon to the stability of the target mRNA was evaluated, and an enhancing tRNA that can promote the expression of the target protein was screened through protein expression function experiments. Secondly, by in vitro transcription, modified nucleotides were introduced into mRNA, and the favorable modified nucleotide types that can improve the protein-coding ability of mRNA were verified. Further, referring to the modification paradigm of natural tRNA, the influence of specific modified nucleotides at specific positions on the decoding ability of tRNA was evaluated, and favorable modification sites and modified nucleotide types were screened therefrom. Moreover, by analyzing the mutual cooperation between nucleotides at different positions on tRNA, combinations of modified nucleotides at different positions were made, and a combination paradigm of more functional modification sites and nucleotides was screened and verified. Finally, a tRNA+mRNA vaccine preparation was obtained by co-encapsulating site-specifically modified tRNA and SARS-COV-2 Spike (B.1.1.529) mRNA with lipid nanoparticles, and its humoral and cellular immune response effects were 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 by the present invention is experimentally verified to show that overexpressing tRNA can significantly promote the expression level of the target protein. The present invention provides a novel tRNA+mRNA immune-enhanced vaccine. The enhanced vaccine enhances the antigen protein-coding ability of the mRNA vaccine by introducing one or more tRNA molecules, thereby stimulating a stronger humoral immune and cellular immune response in vivo, indicating that tRNA+mRNA is an immune-enhanced vaccine.

[0116] Second, the present invention discovers that different modified nucleotides at different sites have different effects on the decoding ability of tRNA. The tRNA molecules used in the present invention contain modified nucleotides with single or multiple combinations at positions 18, 26, 34, 37, 47d, 46, 55, and 58, and have higher aminoacylation efficiency, lower immunogenicity, and higher decoding ability compared to unmodified tRNA. Among all site-directed modified Leu-stRNA-CUA, Cm34 can significantly enhance the readthrough efficiency of tRNA, and Cm34+m1G37, Cm34+m1G37+m1A58, Gm18+Cm34+m1G37+Ψ55+m1A58 further enhance its readthrough efficiency. Among all site-directed modified Leu-stRNA-CUA, tRNA containing Cm34 modification shows better readthrough ability than tRNA modified with m5C34. Among all site-directed modified Leu-tRNA-CAG, tRNA modified with m1G37 shows the highest decoding efficiency among single-site modified tRNAs, and m5C34+m1G37, Cm34+m1G37, m5C34+m1G37+m1A58, Cm34+m1G37+m1A58, Gm18+m5C34+m1G37+Ψ55+m1A58, Gm18+Cm34+m1G37+Ψ55+m1A58 further enhance its decoding efficiency. Among all site-directed modified Leu-tRNA-CAG, tRNA containing m5C34 modification shows better decoding ability than tRNA modified with Cm34.

[0117] Third, the present invention discovers that different modified nucleotides have different effects on the protein-coding ability of mRNA. The mRNA molecules used in the present invention contain full substitutions of single modified nucleotides selected from the following, including A (6Me-A, Z), U (Psi, N1Me-Psi, 5OMe-U, 3Me-5OMe-U), C (5Me-C), 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. Brief Description of the Drawings

[0118] By reading the following detailed description with reference to the accompanying drawings, the above and other objects, features, and advantages of the exemplary embodiments of the present disclosure will become readily understood. In the drawings, several embodiments of the present disclosure are shown in an exemplary rather than restrictive manner, and the same or corresponding reference numerals represent the same or corresponding parts, wherein:

[0119] Figure 1 Overexpressing tRNA increases the expression level of Spike protein.

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

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

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

[0123] (d) According to Figure 1 The tRNA library that may increase the expression level of Spike protein obtained from the analysis in (a-c);

[0124] (e) Schematic diagram of the method for evaluating whether tRNA can promote the expression of Spike protein: Co-transfect the tRNA plasmid and Spike protein plasmid into HEK293T cells at a certain ratio. After 48 hours, lyse the cells, extract the protein, and perform protein quantitative analysis.

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

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

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

[0128] Figure 2 (b-c) Flow cytometry quantitative analysis results after transfection of EGFP-mRNA modified with different nucleotides into cells;

[0129] Figure 2 (d) Protein quantitative analysis results after transfection of EGFP-mRNA modified with different nucleotides into cells.

[0130] Figure 3 : Functional verification results of single-point and multi-point modified tRNA.

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

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

[0133] Figure 3(c) Results of green fluorescence recovery after co - transfection of EGFP39UAG - mRNA and site - specific modified Leu - StRNA - CUA into cells;

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

[0135] Figure 3 (e) Results of mCherry fluorescent protein expression after co - transfection of 5×CUG - mCherry - mRNA and site - specific modified Leu - tRNA - CAG into cells;

[0136] Figure 3 (f) Results of flow cytometry - based quantitative analysis of mCherry fluorescent protein after co - transfection of 5×CUG - mCherry - mRNA and site - specific modified Leu - tRNA - CAG into cells.

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

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

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

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

[0141] Figure 4 (d) Humoral immune evaluation results of tRNA + mRNA vaccines;

[0142] Figure 4 (e) Cellular immune evaluation results of tRNA + mRNA vaccines.

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

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

[0145] Figure 5(b) Detection of the expression level and quality changes 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 the packaging of AAV2.

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

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

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

[0150] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

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

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

[0153] We co-transfected the Spike protein expression plasmid and the tRNA expression plasmid into HEK293T cells at a certain ratio, and the control group was the empty vector plasmid. After 48 hours, the cells were lysed, the proteins were extracted, and protein quantitative analysis was carried out to compare the Spike protein expression levels between the group overexpressing tRNA and the control group. Figure 1e). Experimental results: From Figure 1 f, it can be seen that overexpression of tRNA can significantly increase the expression level of Spike protein, with a maximum increase of 5-9 times, indicating that the tRNA+ strategy is a feasible strategy to promote efficient protein production. The names and sequences of the enhanced tRNAs that can enhance Spike protein expression are shown in Table 1.

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

[0155]

[0156] Example 2: Protein expression results of mRNA with single nucleotide substitutions

[0157] We performed a full substitution of single modified nucleotides on mRNA by in vitro transcription. The modified nucleotides include: A (6Me-A, Z), U (Psi, N1Me-Psi, 5OMe-U, 3Me-5OMe-U), C (5Me-C), G (N7Me-G). The full substitution of single modified nucleotides on mRNA needs to meet one of the following conditions: enhancing the protein-coding ability of mRNA, improving translation fidelity, extending the half-life, or reducing the immunogenicity of mRNA. We performed a full substitution of single modified nucleotides in the sequence of EGFP-mRNA. First, we obtained a linearized in vitro transcription DNA template by enzymatic digestion and gel extraction. Secondly, prepare the in vitro transcription reaction system according to the following requirements and react at 37 °C for 2 hours:

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

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

[0160] EGFP-mRNA-N1Me-pUTP (A, N1Me-pU, C, G);

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

[0162] EGFP-mRNA-3Me-5OMe-UTP (A, 3Me-5OMe-U, C, G);

[0163] EGFP-mRNA-5Me-CTP (A, U, 5Me-C, G);

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

[0165] EGFP-mRNA-A+6Me-ATP (A: 6Me-A 1:1, U, C, G);

[0166] EGFP-mRNA-6Me-ATP-PA (6Me-A, U, C, G), with polyA tail being ATP;

[0167] EGFP-mRNA-ZTP (Z, U, C, G);

[0168] EGFP-mRNA-ZTP-PA (Z, U, C, G), with polyA tail being ATP;

[0169] EGFP-mRNA-N7Me-GTP (A, U, C, N7Me-G).

[0170] Finally, the finished mRNA product was obtained by purification with RNA clean magnetic beads.

[0171] We transfected EGFP-mRNA with different modifications into HEK293T cells. After 24 hours, the fluorescence intensity of EGFP and the protein expression level were detected, and the functions of the modified nucleotides were evaluated based on this.

[0172] Experimental results: mRNAs modified with different nucleotides showed different protein-coding abilities. The protein-coding efficiencies ranked from high to low are as follows: N1Me-pUTP, pUTP, UTP, 5Me-CTP, 5OMe-UTP, ZTP-PA, ZTP, A+6Me-ATP, 3Me-5OMe-UTP, 6Me-ATP, 6Me-ATP-PA, N7Me-GTP. Among all the modified nucleotides, mRNAs modified with N1Me-pUTP and pUTP both showed better protein-coding abilities, while A+6Me-ATP, 3Me-5OMe-UTP, 6Me-ATP, 6Me-ATP-PA, N7Me-GTP significantly reduced the protein-coding ability of mRNA, which may be related to these modified nucleotides disrupting the interaction between mRNA and ribosome and interfering with processes such as the recognition between codons and tRNA anticodons ( Figure 2 a-d).

[0173] Example 3: Introducing single or multiple modified nucleotides into chemically synthesized Leu-tRNA

[0174] We introduced specific modified nucleotides at specific sites of tRNA through solid-phase chemical synthesis. The modified nucleotides at specific sites on tRNA need to meet one of the following conditions: enhancing the decoding ability of tRNA, improving translation fidelity, enhancing the structural stability of tRNA, or reducing the immunogenicity of tRNA. Modifications on the anticodon loop, such as 2'-O-methylation of C34 (Cm), are one of the common wobble modifications in human tRNA and can enhance the interaction between codons and anticodons. 5-methylation of C34 (m5C) can protect tRNA from endonucleolytic cleavage. The modification in G37 is usually N1-methylguanosine (m1G), which is located at the Watson-Crick interface, can stabilize the interaction between tRNA and ribosome and prevent +1 frameshift reading. In addition, modifications outside the anticodon loop can perform many other tRNA-related functions. For example, Gm18 (D-loop) modification can reduce the immune-stimulatory activity of tRNA and cooperate with Ψ55 (T-loop) modification to promote the structural stability of tRNA. m1A58 (T-loop) modification is present in almost all human tRNAs and can enhance the affinity between tRNA and translation elongation factor EF1A, increasing the efficiency of tRNA delivery to the ribosomal A site.

[0175] 3.1 We co-transfected IVS-Leu-stRNA-CUA with different modifications and IVT-EGFP-39UAG-mRNA (N1Me-Psi completely replaced) into HEK293T cells. EGFP-39UAG-mRNA can be read through by stRNA-CUA, and its readthrough efficiency can indicate the function of the modification and partly represent the decoding ability of Leu-tRNA-CAG ( Figure 3 a).

[0176] The experimental results showed that: Cm34-modified Leu-stRNA-CUA showed the highest readthrough efficiency among the singly modified tRNAs, and the order from high to low was as follows: Cm34, m5C34, m1G37, m1A58, Ψ55, Gm18. Combinations of modified bases further enhanced their readthrough efficiency, such as Cm34 + m1G37, Cm34 + m1G37 + m1A58, Gm18 + Cm34 + m1G37 + Ψ55 + m1A58. Among all the site-specifically modified Leu-stRNA-CUA, the Cm34-modified tRNA showed a better readthrough efficiency than the m5C34-modified tRNA ( Figure 3 c-d). Among them, the sequences of the modified tRNAs Lst-1 to Lst-16 based on Leu-stRNA-CUA are shown in Table 2.

[0177] Table 2: Modified tRNAs Lst-1 to Lst-16 based on Leu-stRNA-CUA

[0178]

[0179] 3.2 We co-transfected HEK293T cells with IVS-Leu-tRNA-CAG with different modifications and IVT-5×CUG-mCherry-mRNA (with all N1Me-Psi substitutions). Under the condition of 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 3 b).

[0180] The experimental results showed that: m1G37-modified Leu-tRNA-CAG exhibited the highest decoding efficiency among the tRNAs with single modifications, ranked from high to low as follows: m1G37, m1A58, Ψ55, m5C34, Cm34, Gm18. Combinations of modified bases further enhanced its decoding efficiency, such as m5C34 + m1G37, Cm34 + m1G37, m5C34 + m1G37 + m1A58, Cm34 + m1G37 + m1A58, Gm18 + m5C34 + m1G37 + Ψ55 + m1A58, Gm18 + Cm34 + m1G37 + Ψ55 + m1A58. Among all site-specifically modified Leu-tRNA-CAG, tRNAs modified with m5C34 showed better decoding ability than those modified with Cm34 ( Figure 3 e-f). Among them, the sequences of the modified tRNAs Lt-1 to Lt-16 based on Leu-tRNA-CAG are shown in Table 3.

[0181] Table 3: Modified tRNAs Lt-1 to Lt-16 based on Leu-tRNA-CAG

[0182]

[0183] Example 4: Immunological evaluation results of tRNA+mRNA vaccines co-encapsulated by LNP

[0184] According to the experimental results described in Examples 2 and 3, we co-packaged site-specifically modified tRNAs with strong decoding ability and SARS-CoV-2 Spike (B.1.1.529) mRNA with all N1Me-Psi substitutions by lipid nanoparticles LNP ( Figure 4 a-b), and further evaluated the immune effect of the tRNA+mRNA vaccine in mice ( Figure 4 c).

[0185] The experimental results showed that: compared with the control group (T1: ineffective tRNA + mRNA), the experimental groups (T2 / T3 / T6 / T7) all showed higher endpoint Spike IgG antibody titers and more IL-2 enzyme-linked immunospot numbers, indicating that both the humoral immunity and cellular immunity of the experimental groups were stronger than those of the control group. Among the experimental groups, T7 showed the highest antibody titer and the highest number of IL-2 enzyme-linked immunospots, which was directly related to the strongest decoding ability of the site-directed modified tRNA used in the T7 group.

[0186] Example 5: Screening of tRNAs capable of increasing antibody expression

[0187] Refer to the method of Example 1 to screen for tRNAs capable of increasing the expression of the antibody sacituzumab. ELISA was used to detect the antibody yield ( Figure 5 a), and Western Blot was used to detect the antibody quality ( Figure 5 b).

[0188] We overexpressed different tRNAs in CHO-K1 engineering cells stably expressing the monoclonal antibody sacituzumab, or co-transfected different tRNAs and the expression plasmids of the light and heavy chains of sacituzumab into CHO-K1, CHO-S or Expi-293F engineering cells commonly used for antibody expression at a certain ratio. The blank control group used an empty vector plasmid. The results took Val-tRNA-AAC-3-1 and Ile-tRNA-AAT-1-1 increasing antibody expression as an example: Overexpressing Val-tRNA-AAC-3-1 and Ile-tRNA-AAT-1-1 in CHO-K1 engineering cells stably expressing sacituzumab, the ELISA and immunoblotting experimental results showed that Val-tRNA-AAC-3-1 and Ile-tRNA-AAT-1-1 could increase the expression of sacituzumab, increasing by 1.3 and 4.2 times respectively ( Figure 5 a - b).

[0189] The tRNAs capable of increasing antibody expression were screened and obtained, including 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, Thr-tRNA-AGT.

[0190] Example 6: Screening of tRNAs that can increase AAV packaging efficiency

[0191] tRNA stable cell lines were used to screen for tRNAs that can increase the packaging of AAV2. The results are as Figure 6 shown.

[0192] We co-transfected different tRNA expression plasmids, the transgenic vector plasmid pScAAV-EGFP for packaging AAV2 virus, the helper plasmid pAAV2, and pAdhelper into HEK293T cells. The result of Arg-tRNA-TCT-3-2 increasing AAV2 packaging in HEK293T cells is taken as an example: Since the transgenic vector plasmid carries the EGFP reporter gene, the expression of green fluorescent protein during the packaging process can indirectly reflect the efficiency of HEK293T cells in packaging AAV2. We used a fluorescence microscope to take pictures to detect the packaging efficiency and detected the genomic titer by real-time fluorescence quantitative PCR ( Figure 6 a-b). The EGFP reporter gene is expressed after the virus normally infects cells, and the expression level is positively correlated with the number of infected viruses. Therefore, we infected HEK293T with the same volume of virus stock solution and took pictures 72 h after infection ( Figure 6 c). The results showed that Arg-tRNA-TCT-3-2 could increase AAV2 packaging, which was increased by 3.35 times.

[0193] tRNAs that can increase the packaging of AAV2 were screened, including 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, Glu-tRNA-TTC.

[0194] The preferred embodiments of the present invention are described above, aiming to make the spirit of the present invention clearer and easier to understand, and not to limit the present invention. Any modifications, substitutions, and improvements made within the spirit and principles of the present invention shall be included within the protection scope defined by the appended claims of the present invention.

Claims

1. Use of tRNA in the preparation of mRNA vaccines, characterized in that, The mRNA is SARS-CoV-2 B.1.1.529 Spike mRNA; the tRNA molecule is Leu-tRNA-CAG or a tRNA modified on the basis of Leu-tRNA-CAG; wherein, the nucleotide sequence of Leu-tRNA-CAG is shown in SEQ ID NO:2; the tRNA modified on the basis of 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.

2. Use of the tRNA according to claim 1 in the preparation of an mRNA vaccine, characterized in that, Replace all U on the mRNA with N1Me-Psi.

3. Use of the tRNA according to claim 1 or 2 in the preparation of an mRNA vaccine, characterized in that, The same delivery system is used for the tRNA and the mRNA.

4. Use of the tRNA according to claim 3 in the preparation of an mRNA vaccine, characterized in that, Co-encapsulation and delivery are carried out using lipid nanoparticles LNP.

5. Use of the tRNA according to claim 4 in the preparation of an mRNA vaccine, characterized in that, The mass ratio of the tRNA to the 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%.

6. A vaccine composition, characterized in that, It includes: (1) SARS-CoV-2 B.1.1.529 Spike mRNA; (2) tRNA, the tRNA molecule is Leu-tRNA-CAG or a tRNA modified on the basis of Leu-tRNA-CAG; wherein, the nucleotide sequence of Leu-tRNA-CAG is shown in SEQ ID NO:2; the tRNA modified on the basis of 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; and, optionally (3) A delivery system.

7. Use of tRNA in enhancing protein expression level, characterized in that, The protein is encoded by SARS-CoV-2 B.1.1.529 Spike mRNA, and the nucleotide sequence of the tRNA is any one of SEQ ID NOs: 1-24.

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