UTR (Untranslated Region) element H2202 P1-G as well as construction method and application thereof

Abstract

The invention provides an UTR (Untranslated Region) element H2202 P1-G as well as a construction method and application thereof, and relates to the technical field of mRNA (messenger ribonucleic acid). According to the present invention, the ribosome load prediction and the secondary structure optimization are performed on the natural 5 'UTR of the HIV TAT 202 gene through the BaidleHelix platform, and the obtained HTAT 202 P1 sequence avoids the inhibitory hairpin structure so as to significantly improve the luciferase expression quantity compared to the natural UTR; an ncRNA sequence without a secondary structure is introduced on the basis of the HTAT 202 P1, translation inhibition of a 5 'cap region is further relieved, and the protein expression quantity of the constructed H2202 P1-G mutant (the DNA sequence of the H2202 P1-G is as shown in SEQ NO 1, and the RNA sequence is as shown in SEQ NO 2) is further improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of mRNA molecules, in particular to a UTR element H202 P1-G and a construction method and application thereof. BACKGROUND

[0002] In recent years, mRNA technology has made breakthrough progress in the fields of vaccine development, gene therapy and protein replacement therapy. The gene expression structure of mRNA molecule is usually composed of multiple functional elements, including promoter (may contain enhancer), 5' untranslated region (5'UTR), protein coding region, 3'UTR and PolyA signal. Among them, 5'UTR as a key region to regulate mRNA translation efficiency and stability, directly affects the loading and translation initiation process of ribosome. However, compared with the optimization of coding region, the sequence design research of UTR (especially 5'UTR) is still relatively insufficient, which limits the further improvement of the efficacy of mRNA therapy.

[0003] The commonly used 5'UTR at present is mostly derived from hemoglobin gene. Studies have shown that the protein expression level can be further improved by designing new UTR sequences. The 5'UTR sequence has two main functions: improving mRNA stability and initiating translation. In the development process of mRNA vaccine, there are four strategies to optimize the 5'UTR sequence. The first is to directly select the 5'UTR of the gene with extremely high expression efficiency in human cells, such as the 5'UTR of human alpha-globin gene, for direct application. The second is the natural 5'UTR of pathogen mRNA itself. However, the secondary structure of natural UTR may inhibit translation (such as the stem-loop structure adjacent to the cap structure), and it is unstable in different genes or cell types; in addition, the universality of natural sequence is poor, and it needs to be reselected for different target genes. The third is to realize the systematic evolution of 5'UTR through number enrichment. In fact, this method is first used for the screening of 3'UTR. The fourth is to perform high-throughput screening from a large amount of database through deep calculation model, but the existing model relies on limited structure and expression data, and the prediction accuracy is insufficient, which still needs a large number of experimental verification. Moreover, the latter two methods need to consume a large amount of time and have certain technical difficulty. And due to the significant difference between plasmid and mRNA in expression mechanism (such as nuclear transcription and cytoplasmic direct translation), the correlation between the screening results and the actual mRNA expression efficiency is low. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application provides a UTR element H202 P1-G and a construction method and application thereof, which are realized by the following technical solutions: In a first aspect, the present application provides a UTR element H202 P1-G, wherein a DNA sequence of the H202 P1-G is shown as SEQ NO 1, and a RNA sequence of the H202 P1-G is shown as SEQ NO 2.

[0005] In a second aspect, the present application provides a construction method of the UTR element H202 P1-G, comprising the following steps: obtaining a natural 5'UTR of an HIV TAT 202 gene from a NCBI database; performing ribosome load prediction and secondary structure prediction on the natural UTR, and screening an optimized UTR sequence HTAT 202 P1 according to a prediction result; on the basis of the optimized HTAT 202 P1 sequence, retaining a 5'cap structure, inserting a ncRNA sequence without secondary structure in front of the 5'UTR, and constructing the H202 P1-G.

[0006] Further, the HIV TAT 202 is numbered as LOC129008191 in the NCBI database.

[0007] Further, the ncRNA sequence without secondary structure is shown as SEQ NO 3.

[0008] In a third aspect, the present application provides an mRNA molecule, comprising the H202 P1-G element, a target gene coding sequence, a 3'UTR sequence and a polyA tail sequence.

[0009] In a fourth aspect, the present application provides an mRNA-LNP complex, comprising: the mRNA molecule; a lipid nanoparticle (LNP) comprising 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid 1-octyl nonyl ester (SM102), 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), distearoyl phosphatidylcholine (DSPC) and cholesterol, and the mass percentages of the SM102, the DMG-PEG2000, the DSPC and the cholesterol are 50%, 1.5%, 10% and 38.5% respectively.

[0010] In a fifth aspect, the present application provides a method for preparing the mRNA-LNP complex, comprising the following steps: dissolving the mRNA molecule in a 50mM citric acid buffer solution with a pH of 4.0 to obtain an mRNA solution; dissolving the lipid nanoparticle in anhydrous ethanol to form an LNP solution; The mRNA solution is mixed with the LNP solution at a volume ratio of 3:1 to form a mixed solution; The mixed solution is diluted with citric acid buffer and then ultrafiltration purification is performed, and the purified solution is replaced into Tris-HCl buffer to obtain the mRNA-LNP complex.

[0011] In a sixth aspect, the present application provides a pharmaceutical composition, characterized by comprising the mRNA-LNP complex and pharmaceutically acceptable adjuvants.

[0012] In a seventh aspect, the present application provides use of the UTR element H202 P1-G, the mRNA molecule, the mRNA-LNP complex or the pharmaceutical composition in the preparation of a vaccine for preventing or treating viral infection.

[0013] Compared with the prior art, the present application has the following advantages: Based on the deep learning method of the PaddleHelix platform, the present application predicts and optimally designs the ribosome binding capacity of the candidate UTR sequence, establishes a double-dimensional screening system, uses the enhanced green fluorescent protein (EGFP) reporter system on the DNA level and the luciferase expression system on the RNA level for double verification. For the UTR sequence with high expression efficiency, base modification and combination optimization strategies are used for structure improvement, and microfluidic chip technology is used to accurately control the particle size distribution (80-100 nm) and encapsulation efficiency (>90%) of the lipid nanoparticles (LNP). In vitro transfection experiments show that the optimized UTR makes the expression of the reporter gene better. The present application provides a new technical path for the subsequent UTR engineering of vaccines. Through the PaddleHelix platform, the ribosome load of the HIV TAT 202 natural 5'UTR sequence is predicted and the secondary structure is optimized, and the obtained HTAT 202 P1 sequence avoids the inhibitory hairpin structure, so that the luciferase expression is obviously improved compared with the natural UTR. The ncRNA sequence without secondary structure is introduced on the basis of HTAT 202 P1, which further releases the translation inhibition of the 5'cap region, and the constructed H202 P1-G mutant further improves the protein expression. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0015] Figure 1A secondary structure prediction map of the HTAT 202 RNA in the embodiment of the present application is shown; Figure 2 A secondary structure prediction map of the HTAT 202 P1 RNA in the embodiment of the present application is shown; Figure 3 An expression effect map of a strong green fluorescent protein (EGFP) in HEK293T cells in the embodiment of the present application is shown; Figure 4 Luciferase activity 24H after mRNA transfection of HEK293T cells in the embodiment of the present application is shown; Figure 5 Luciferase activity 48H after mRNA transfection of HEK293T cells in the embodiment of the present application is shown; Figure 6 A secondary structure prediction result of the H202 P1-G mutant in the embodiment of the present application is shown; Figure 7 Luciferase activity 24H after H202 P1-G transfection of HEK293T cells in the embodiment of the present application is shown; Figure 8 Luciferase activity 48H after H202 P1-G transfection of HEK293T cells in the embodiment of the present application is shown; Figure 9 An expression result of H202 P1-G mRNA in HEK293T cells measured by the westren bolt (quantitative) method in the embodiment of the present application is shown; Figure 10 H1N1 result of H202 P1-G-WB band gray scale analysis in the embodiment of the present application is shown; Figure 11 H3N2 result of H202 P1-G-WB band gray scale analysis in the embodiment of the present application is shown; Figure 12 BIV result of H202 P1-G-WB band gray scale analysis in the embodiment of the present application is shown. DETAILED DESCRIPTION

[0016] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application are described below in a clear and complete manner with reference to the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application. Embodiment 1

[0017] The 5'UTR of the gene with extremely high expression efficiency in human cells and the natural 5'UTR sequence of the pathogen mRNA itself were found through the NCBI database. The ribosome loading of the natural 5'UTR of HIV TAT 202 gene (NCBI number: LOC129008191) was predicted, automatically optimized and designed, and the secondary structure was predicted (secondary structure prediction website: unafold.org) through the PaddleHelix platform. The UTR with better prediction effect was selected for verification.

[0018] The ribosome loading prediction results of the PaddleHelix platform for the HIV TAT (hereinafter abbreviated as HTAT) 202 gene are shown in Table 1: Table 1

[0019] The prediction results of the secondary structure of HTAT 202 RNA and the secondary structure of HTAT 202 P1 RNA are shown in Figure 1 and Figure 2 It can be seen from Table 1 that the ribosome loading prediction value of HTAT 202 and HTAT 202 P1 designed by PaddleHelix is high and the secondary structure is obviously different, so HTAT 202 P1 is selected for subsequent verification. Example 2

[0020] A DNA-RNA two-dimensional verification system was established.

[0021] (1) The DNA level uses an enhanced green fluorescent protein (EGFP) reporter system for verification: Plasmid amplification: After the pCMV-EGFP reporter plasmid was amplified by TOP10 competent cells, DNA sequencing was performed; Transfection and detection: HEK293T cells were transfected using Lipofectamine 3000 transfection reagent, and the expression efficiency was evaluated by fluorescence microscope.

[0022] The results are shown in Figure 3 It can be seen that the EGFP plasmid with UTR produces more green fluorescence, and in the DNA level, the green fluorescence expression effect of HTAT 202 is the best.

[0023] (2) The RNA level uses a luciferase expression system: Plasmid construction: ① The target gene fragment and pUC57 vector were double-digested with NcoI / XhoI restriction endonuclease (NEB), and PUC57-LUC luciferase plasmid was constructed using NEB T4 ligase. The ligation product was transformed into TSINGKE TSC-C01 Trelief® 5α competent cells by heat shock method, and coated on LB plate containing 100 μg / mL ampicillin, and incubated at 37°C for 16 hours. After selecting single colonies, sequencing was performed; ② The target fragment containing UTR and PUC57-LUC vector were double-digested with AseI / NcoI, and T4 ligase was used to construct pUC57 reporter vector containing T7 promoter, 5'UTR-H202 / HTAT 202 P1, Luciferase, 3'UTR, polyA. After transformation into DH5α competent cells by heat shock method, coating on LB plate containing 100 μg / mL ampicillin, and incubating at 37°C for 16 hours, single colonies were selected for sequencing; Transfection and detection: after BsaI enzyme digestion and linearization, isopropanol precipitation and purification, N1-methylpseudouridine triphosphate (N1-Me-pUTP) was used for in vitro transcription to obtain modified mRNA. After transfection, luciferase activity was detected.

[0024] As shown in Figure 4 and Figure 5 , it can be seen that the expression effect of HTAT 202 P1 is better when the cell verification is carried out at the RNA level. According to the DNA-RNA cell level results, it can be seen that the expression effect of the same UTR is not the same at DNA and RNA, therefore, subsequent experiments are only verified at the RNA level. Example 3

[0025] As shown in Example 2, HTAT 202 P1 has good expression effect, and a segment of ncRNA (AUUAACAACCCGGAG, SEQ NO 3) without secondary structure is added after the 5'cap (cap) structure and before the 5'UTR of the HTAT 202 P1 high-efficiency expression strain to construct H202 P1-G mutant (hereinafter referred to as H202 P1-G). After secondary structure prediction, plasmid construction, sequencing, amplification, linearization, in vitro transcription into mRNA, and transfection of HEK293T cells, luciferase activity was measured.

[0026] The secondary structure prediction result of H202 P1-G is shown in Figure 6 , and the luciferase activity result is shown in Figure 7 and Figure 8 , it can be seen that the luciferase activity of H202 P1-G transfected HEK293T cells is better at 24-48H.

[0027] The DNA sequence and RNA sequence of H202 P1-G obtained by sequencing are shown in Table 2: Table 2

[0028] To further verify the expression of 5'UTR at the vaccine level, the modified H202 P1-G was connected with the NA influenza vaccine sequence. The mRNA of H202 P1-G_Influenza (Influenza)_NA was dissolved in a citric acid buffer solution with a concentration of 50 mM at a pH of 4, and the final concentration was 108 ng / μL. Then a lipid mixed solution was prepared: the ionizable lipid (SM102), PEG modified lipid dimyristyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000), distearoyl phosphatidylcholine (DSPC), and cholesterol were mixed and dissolved in anhydrous ethanol solution at a ratio of 50%, 1.5%, 10%, and 38.5%, respectively, and the mRNA-citric acid solution and the lipid-ethanol solution were filtered through a 0.22 μm microporous filter, mixed using a microfluidic instrument at a volume ratio of mRNA:lipid of 3:1, and the flow rate ratio of buffer phase:ethanol phase was 15 mL / min:5 mL / min. The obtained mRNA-LNP was immediately diluted with 15 ml of the above citric acid buffer solution, and ultrafiltration was performed using a 100 KD ultrafiltration tube at a centrifugal force of 3000 g. After ultrafiltration to 1 / 4 volume, 50 mM Tris-HCl buffer (pH=7.5) was added to 15 mL, and the concentration and encapsulation rate were measured after repeating twice. Then the in vitro expression effect was verified in HEK293T cells, and Western blot analysis confirmed that the target protein was expressed well.

[0029] The results are shown in Figure 9 , and Figure 10 , Figure 11 , Figure 12 As shown in the figure, the H1N1-NA, H3N2-NA, and BIV-NA all have obvious bands, and the protein expression amount is similar to that of the BioNTech LNP control group, indicating that H202 P1-G has good expression effect at the vaccine level.

[0030] The above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A UTR element H202 P1-G, characterized in that, The DNA sequence of the H202 P1-G is shown in SEQ NO 1, and the RNA sequence is shown in SEQ NO 2.

2. A method for constructing the UTR element H202 P1-G as described in claim 1, characterized in that, Includes the following steps: get: The natural 5' UTR sequence of the HIV TAT 202 gene was obtained from the NCBI database; Ribosome loading and secondary structure were predicted from the natural 5'UTR sequence. Based on the prediction results, the optimized UTR sequence HTAT 202 P1 was obtained. Based on the optimized HTAT 202 P1 sequence, the 5'cap structure was retained, and an ncRNA sequence without secondary structure was inserted at the front of the 5'UTR to construct H202 P1-G.

3. The method for constructing the UTR element H202 P1-G according to claim 2, characterized in that, The HIV TAT202 is numbered LOC129008191 in the NCBI database.

4. The method for constructing the UTR element H202 P1-G according to claim 2, characterized in that, The ncRNA sequence without secondary structure is shown in SEQ NO 3.

5. An mRNA molecule, characterized in that, It includes the H202 P1-G element as described in claim 1, the target gene coding sequence, the 3'UTR sequence, and the polyA tail sequence.

6. An mRNA-LNP complex, characterized in that, Include: The mRNA molecule according to claim 5; Lipid nanoparticles comprising SM102, DMG-PEG2000, DSPC, and cholesterol in weight percentages of 50%, 1.5%, 10%, and 38.5%, respectively.

7. A method for preparing the mRNA-LNP complex of claim 6, characterized in that, Includes the following steps: The mRNA molecule was dissolved in citrate buffer to obtain an mRNA solution; Lipid nanoparticles were dissolved in anhydrous ethanol to form an LNP solution; The mRNA solution and LNP solution were mixed in a volume ratio of 3:1 to form a mixed solution; The mixed solution was diluted with citrate buffer and then purified by ultrafiltration. The purified solution was then replaced with Tris-HCl buffer to obtain the mRNA-LNP complex.

8. A pharmaceutical composition, characterized in that, It comprises the mRNA-LNP complex of claim 6 and pharmaceutically acceptable excipients.

9. The use of the UTR element H202 P1-G of claim 1, the mRNA molecule of claim 5, the mRNA-LNP complex of claim 5, or the pharmaceutical composition of claim 8 in the preparation of a vaccine for the prevention or treatment of viral infection.

Images