5 'UTR (Untranslated Region) optimized sequence and application thereof

Abstract

The invention discloses a 5 'UTR (Untranslated Region) optimized sequence and application thereof, and the nucleotide sequence of the 5' UTR optimized sequence is shown as any one of SEQ ID NO: 2 and SEQ ID NO: 10. The 5 'UTR optimized sequence provided by the invention can effectively improve mRNA transcription and protein expression efficiency, can be used for improving mRNA transcription and / or protein expression efficiency, and is especially suitable for improving mRNA transcription and protein expression efficiency of tumor neoantigen. The tumor neoantigen mRNA constructed and synthesized by using the 5 'UTR optimized sequence provided by the invention can be effectively applied to the field of tumor treatment, such as preparation of anti-tumor mRNA vaccines and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a 5'UTR optimized sequence and application thereof. Background Art

[0002] Messenger RNA (mRNA), as a gene delivery molecule, is often used as a substitute for plasmid DNA in tumor immunotherapy and stem cell-based biomedical research. In addition, it is often used to treat various diseases such as cancer, rare genetic diseases and infectious diseases. mRNA includes an exogenous open reading frame (ORF) flanked by 5′UTR and 3′UTR, where 5′UTR and 3′UTR are derived from naturally abundant mRNA in tissues.

[0003] The 5' untranslated region (5'UTR) refers to the region of mature mRNA located upstream of the coding region (CDS) that is not translated into protein. It is located at the beginning of the mRNA molecule, upstream of the start codon that initiates protein translation. The 5' untranslated region is an mRNA domain that contains a large number of elements, such as upstream ORFs (uORFs), internal ribosome entry sites, protein binding sites, and structural components involved in the regulation of mRNA stability, pre-mRNA splicing, and translation initiation.

[0004] For a given gene, the 5′UTR is involved in regulating the stability and translation of mRNA. Variations in the 5′UTR can serve as an important switch for regulating gene expression, affecting the efficacy of mRNA-based therapy. This shows the functional importance of the 5′UTR in regulating gene expression. Current mRNA therapies often rely on standard UTR sequences, which are often unstable and have low protein expression levels. Therefore, it is necessary to screen and optimize better UTR sequences to stabilize therapeutic mRNA and / or increase protein synthesis levels. Summary of the invention

[0005] The object of the present invention is to provide a 5'UTR optimized sequence and application thereof to solve at least one of the above technical problems.

[0006] The inventors found in the research process that the luciferase mRNA constructed and synthesized with the 5′UTR of HBA alpha-globin as the 5′UTR could not be successfully reverse transcribed. After repeatedly adjusting the reaction parameters, the inventors chose to optimize the 5′UTR of HBA alpha-globin. Finally, through a large number of experimental screenings, 6 nucleotide sequences as shown in any one of SEQ ID NO: 2 to 7 were obtained. When used in the mRNA in vitro reverse transcription synthesis process, the mRNA transcription ratio can be significantly improved, and the transcripts can obtain high protein expression levels in different cell lines. 5′UTR optimized sequences. In addition, the inventors also optimized the 5′UTR sequence of Moderna's new crown vaccine mRNA-1273, and finally screened out 3 5′UTR optimized sequences as shown in any one of SEQ ID NO: 8 to 9. Through further research, it was found that the application of the 5′UTR optimized sequence provided by the present invention to the construction and synthesis of tumor neoantigen mRNA can significantly improve the transcription ratio of tumor neoantigen mRNA and the expression efficiency of the target protein, so that it can be used to prepare anti-tumor vaccines based on tumor neoantigen mRNA and effectively used in the prevention and treatment of tumors. Based on the above findings, the present invention is completed.

[0007] According to one aspect of the present invention, a 5′UTR optimized sequence is provided, the nucleotide sequence of which is shown in any one of SEQ ID NOs: 2-10.

[0008] The present invention uses the wild 5′UTR of HBA alpha-globin as shown in SEQ ID NO:1 and the 5′UTR sequence of Moderna's new crown vaccine mRNA-1273 as shown in SEQ ID NO:11 as the basis, and optimizes both to obtain a series of 5′UTR optimized sequences. By carrying the luciferase gene to construct and synthesize multiple mRNAs for in vitro transcription and cell experiments, it is confirmed that the 5′UTR optimized sequence provided by the present invention, as shown in any one of SEQ ID NO:2 to 10, can significantly improve the transcription ratio of mRNA, and after mRNA is transfected into different cells (Macrophage, DC cells, RD cells, 293T and C2C12 cells), high levels of protein expression can be obtained.

[0009] The use of the 5′UTR optimized sequence provided by the present invention can effectively improve the efficiency of mRNA transcription and / or protein expression.

[0010] In some embodiments, the mRNA may be a tumor neoantigen mRNA. Thus, the 5′UTR optimized sequence provided by the present invention can be used to prepare a tumor neoantigen mRNA vaccine.

[0011] In some embodiments, the target of tumor neoantigens can be K-Ras.

[0012] According to another aspect of the present invention, a tumor neoantigen mRNA is provided, the target of which is K-Ras, and the nucleotide sequence of which is shown in any one of SEQ ID NOs: 13 to 15.

[0013] Taking K-Ras as the target, targeting its six different mutant epitope genes (G12D, G12C, G12V, G13D, G12R, G12A), using the 5'UTR optimized sequence provided by the present invention as the 5'UTR, synthesized tumor neoantigen mRNA with a nucleotide sequence as shown in any one of SEQ ID NOs: 13 to 15, and detecting the expression and translation level of the G12D protein in the six mutant epitopes in the tumor neoantigen mRNA by in vitro cell experiments, confirming that the 5'UTR optimized sequence provided by the present invention can make the target protein expressed to different degrees, and the tumor neoantigen mRNA transcription ratio is high, so that the tumor neoantigen mRNA constructed and synthesized using the 5'UTR optimized sequence provided by the present invention can be effectively applied to the field of tumor treatment, such as in the preparation of anti-tumor mRNA vaccines.

[0014] In some embodiments, the tumor can be selected from at least one of non-small cell lung cancer, colorectal cancer, and pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the plasmid map of the pAB401 vector;

[0016] Figures 2 to 6 The following are the expression levels of Luciferase in C2C12, 293T, Macrophage, DC and RD cells 24 hours after transfection of mRNA constructed and synthesized using different 5′UTRs in Example 1 of the present invention;

[0017] Figures 7 to 9 The plasmid maps of plasmids W4, W5, and W6 are shown in order;

[0018] Fig.10 This is the result of G12D protein expression level detection. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with the embodiments. The examples are only for explanation and are not intended to limit the present invention in any way. Unless otherwise specified, the raw materials and reagents used in the examples are conventional products that can be obtained commercially; the experimental methods in the examples where specific conditions are not specified are usually carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0020] Example 1

[0021] The original 5′UTR of HBA alpha-globin (SEQ ID NO: 1) was loaded with the luciferase gene (XM_031473197.1), and the necessary elements such as codons, 3′UTR, and PolyA were combined to construct and synthesize an mRNA, and then the corresponding DNA was inserted into the pAB401 vector (its map is shown in Figure 1 A plasmid was constructed in the presence of 5′UTR as shown in SEQ ID NO: 12, and the plasmid was named Sequence 1. Then, the 5′UTR sequence in Sequence 1 was replaced with the 5′UTR optimized sequence as shown in SEQ ID NO: 2 to 10, and the 5′UTR sequence of Moderna's new crown vaccine mRNA-1273 as shown in SEQ ID NO: 11, to construct a series of plasmids, which were named Sequence 2 to 11.

[0022] 1. IVT transcription

[0023] The materials required for the in vitro transcription (IVT) test, such as nucleotides and enzymes, are all commercially available. The IVT pilot test (two replicates for each sample) and the 20 μL reaction system are shown in Table 1 below:

[0024] Table 1 20 μL IVT transcription reaction system

[0025]

[0026]

[0027] Experimental steps:

[0028] (1) Incubate at 37°C for 3 h;

[0029] (2) Add 2 μL DNase I to each tube and react at 37°C for 20 min;

[0030] (3) Add LiCl solution (7.5 M) to adjust the final concentration to about 2.5 M and place at -20 °C for 30 min;

[0031] (4) Centrifuge at 12000 rpm for 15 min, discard the supernatant, add pre-cooled 70% ethanol to wash the precipitate, centrifuge for 5 min and discard the supernatant; repeat the washing once;

[0032] (5) Dry the precipitate in the tube, dissolve it in 100 μL of water, and measure the RNA concentration using Nanodrop.

[0033] The results are shown in Table 2.

[0034] Table 2 IVT transcription results

[0035]

[0036]

[0037] From the results in Table 2, it can be seen that reverse transcription cannot be successfully performed using the original 5′UTR of HBA alpha-globin, while the optimized 5′UTR sequence provided by the present invention significantly improves the transcription ratio.

[0038] 2. Cell experiments

[0039] Five cell lines (Macrophage, DC cells, RD cells, 293T and C2C12 cells) were used in a 96-well culture plate. Each sample was transfected with 5 replicates, and the transfection sequence was 2 to 11. Each well was transfected with 50 ng of the mRNA obtained in the above step "1, IVT transcription", corresponding to 0.3 μL of Lipofectamine messenger MAX, and the cell transfection operation was performed according to the instructions of Lipofectamine messenger MAX transfection reagent (manufacturer name: Thermo Fisher). After transfection, the 96-well culture plate was placed at 37°C and 5% CO 2 Incubate in an incubator, and after 24 hours, detect the expression level of Luciferase according to the instruction manual of Novozyme Bio-Lite, and calculate the results.

[0040] The results are as follows Figures 2 to 6 shown.

[0041] The results showed that the mRNA constructed and synthesized using the 5′UTR optimized sequence provided by the present invention as the 5′UTR can highly express Luciferase in five different cell lines after transfection, among which the mRNA synthesized using the nucleotide sequence as the 5′UTR optimized sequence shown in SEQ ID NO:7 has a better expression level of Luciferase in the five cell lines.

[0042] Example 2

[0043] Taking K-Ras as the target, the nucleotide sequence is shown in SEQ ID NO:7, the 5′UTR optimized sequence is 5′UTR, which is matched with 3 different codons, and carries 6 tumor neoantigens, namely 6 different mutant epitope genes of K-Ras (G12D, G12C, G12V, G13D, G12R, G12A), as well as 3′UTR, PolyA and other necessary elements, to construct and synthesize 3 mRNAs (tumor neoantigen mRNAs), whose nucleotide sequences are shown in SEQ ID NO:13 to 15, and then the corresponding DNAs are inserted into the 15-Thermo-M vector (plasmid map is shown in Figures 7 to 9 The plasmids were constructed and recorded as W4, W5 and W6. Then, the 5′UTR optimized sequence of the present invention was replaced with the 5′UTR sequence of Moderna's new crown vaccine mRNA-1273, and three control plasmids were constructed and synthesized in the same way, recorded as W1, W2 and W3.

[0044] The experiment was performed with reference to the methods of “1. IVT transcription” and “2. Cell experiment” in Example 1.

[0045] 1. IVT transcription

[0046] The results are shown in Table 3.

[0047] Table 3 IVT transcription results

[0048]

[0049]

[0050] It can be seen from the results in Table 3 that the tumor neoantigen mRNA constructed and synthesized using the 5′UTR optimized sequence provided by the present invention can be successfully reverse transcribed, and has a similar transcription ratio to the tumor neoantigen mRNA constructed and synthesized using the 5′UTR sequence of Moderna's new crown vaccine mRNA-1273.

[0051] 2. Cell experiments

[0052] Six tumor neoantigen mRNA stock solutions were used for cell transfection experiments. After 293T cells were plated on a 24-well culture plate, each well was transfected with 1 μg of IVT-transcribed mRNA, corresponding to 1.5 μL of Lipofectamine messenger MAX. Cell transfection was performed according to the instructions of Lipofectamine messenger MAX transfection reagent (manufacturer name: Thermo Fisher). At the same time, a blank control group was set up, that is, no treatment was performed on the cells. After transfection, the 24-well culture plate was kept at 37°C and 5% CO. 2After incubation in the incubator for 24 h, the cells were collected, and the expression level of the G12D epitope protein was detected by western blot using GAPDH protein as an internal reference.

[0053] The results are as follows Fig.10 shown.

[0054] Fig.10 The results showed that the tumor neoantigen mRNA constructed and synthesized using the 5′UTR sequence of Moderna's new crown vaccine mRNA-1273 was not expressed in 293T cells, while the tumor neoantigen mRNA constructed and synthesized using the 5′UTR optimized sequence provided by the present invention was able to obtain different levels of translation expression in 293T cells.

[0055] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. 5'UTR optimized sequence, characterized in that: The nucleotide sequence thereof is shown in any one of SEQ ID NOs: 2 to 10.

2. Use of the 5'UTR optimized sequence according to claim 1 in improving mRNA transcription and / or protein expression efficiency.

3. The use according to claim 2, characterized in that: The mRNA is tumor neoantigen mRNA.

4. The use according to claim 3, characterized in that: The target of the tumor neoantigen is K-Ras.

5. Tumor neoantigen mRNA, characterized in that: The nucleotide sequence thereof is shown in any one of SEQ ID NOs: 13 to 15.

6. Use of the tumor neoantigen mRNA according to claim 5 in the preparation of anti-tumor mRNA vaccines.

7. The use according to claim 6, characterized in that: The tumor is selected from at least one of non-small cell lung cancer, colorectal cancer, and pancreatic cancer.

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