UTR (Untranslated Region) element Hh as well as construction method and application thereof

Abstract

The invention provides an UTR element Hh and a construction method and application thereof, and relates to the technical field of mRNA. Ribosome load prediction and secondary structure optimization are respectively carried out on 5 'UTR of a gene of which the NCBI number is AF339412.1 and 5' UTR of a gene of which the NCBI number is MK475748.1 through a PaddleHelix platform, and an inhibitory hairpin structure is avoided by the obtained optimized sequences, so that the luciferase expression quantity is obviously improved compared with that of natural UTR. On the basis of sequence optimization, HBB and hbb are directionally assembled through a molecular cloning technology, an HBB-hbb chimera (Hh) is constructed, the DNA sequence of the Hh is as shown in SEQ NO.1, the RNA sequence of the Hh is as shown in SEQ NO.2, and the protein expression quantity is further improved.

Description

Technical Field

[0001] This invention relates to the field of mRNA molecular technology, specifically to a UTR element Hh, its construction method, and its application. Background Technology

[0002] In recent years, mRNA technology has made groundbreaking progress in vaccine development, gene therapy, and protein replacement therapy. The gene expression structure of mRNA molecules typically consists of multiple functional elements, including the promoter (which may contain enhancers), the 5' untranslated region (5'UTR), the protein-coding region, the 3'UTR, and the PolyA signaling domain. Among these, the 5'UTR, as a key region regulating mRNA translation efficiency and stability, directly affects ribosome loading and translation initiation. However, compared to the optimization of the coding region, research on sequence design targeting the UTR (especially the 5'UTR) remains relatively insufficient, limiting further improvements in the efficacy of mRNA therapies.

[0003] Currently, most commonly used 5'UTRs are derived from hemoglobin genes. Studies have shown that designing new UTR sequences can further enhance protein expression levels. 5'UTR sequences primarily serve two functions: improving mRNA stability and initiating translation. In the development of mRNA vaccines, there are four strategies for optimizing 5'UTR sequences. The first is to directly select the 5'UTR of genes with extremely high expression efficiency in human cells, such as the 5'UTR of the human α-globin gene. The second is the native 5'UTR of the pathogen's mRNA itself. However, the secondary structure of native UTRs may inhibit translation (e.g., stem-loop structures adjacent to the cap structure), and they are unstable in different genes or cell types; furthermore, native sequences have poor universality, requiring re-screening for different target genes. The third is to achieve the systematic evolution of 5'UTRs through logarithmic enrichment; this method was actually first used for 3'UTR screening. The fourth is to use deep computational models for high-throughput screening from massive databases, but existing models rely on limited structural and expression data, resulting in insufficient predictive accuracy, and still require extensive experimental validation. Moreover, the latter two methods require a significant amount of time and present certain technical challenges. Furthermore, due to the significant differences in the expression mechanisms of plasmids and mRNA (such as nuclear transcription versus direct cytoplasmic translation), the screening results have a low correlation with the actual mRNA expression efficiency. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a UTR element Hh, its construction method, and its application, specifically implemented through the following technical solutions: In a first aspect, the present invention proposes a UTR element Hh, wherein the DNA sequence of Hh is shown in SEQ NO.1 and the RNA sequence is shown in SEQ NO.2.

[0005] Secondly, this invention proposes a method for constructing the aforementioned UTR element Hh, comprising the following steps: obtaining: Obtain the 5' UTR sequence HBB of Homo sapiens haplotype T7 delta-globin and the 5' UTR sequence hbb of Homo sapiens voucher Baka_10_1 hemoglobin subunit beta from the NCBI database; Ribosome loading and secondary structure prediction were performed on HBB and hbb sequences using the PaddleHelix platform. Optimized HBB 5'UTR and hbb 5'UTR were obtained based on the prediction results. Based on the optimized HBB 5'UTR and hbb 5'UTR, they are cascaded together. A GA sequence is added to the 5' end of the cascaded HBB-hbb as a 5'cap structure, and a Kozak sequence is added to the 3' end to construct the UTR element Hh.

[0006] Furthermore, the NCBI number of the Homo sapiens haplotype T7 delta-globin is AF339412.1; the NCBI number of the Homo sapiens voucher Baka_10_1 hemoglobin subunit beta is MK475748.1.

[0007] Furthermore, the Kozak sequence is GCCACCATG.

[0008] Thirdly, the present invention proposes an mRNA molecule comprising the Hh element as its 5'UTR.

[0009] Fourthly, this invention proposes an mRNA-LNP complex comprising: The mRNA molecule mentioned above; Lipid nanoparticles comprising SM102, DMG-PEG2000, DSPC, and cholesterol in weight percentages of 50%, 1.5%, 10%, and 38.5%, respectively.

[0010] Fifthly, the present invention provides a method for preparing the mRNA-LNP complex, comprising the following steps: The mRNA molecule was dissolved in 50 mM citrate buffer at pH 4.0 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 at 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.

[0011] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the aforementioned mRNA-LNP complex and pharmaceutically acceptable excipients.

[0012] In a seventh aspect, the present invention proposes the use of the UTR element Hh, the mRNA molecule, the mRNA-LNP complex, or the pharmaceutical composition in the preparation of vaccines for the prevention or treatment of viral infections.

[0013] Compared with the prior art, the present invention has the following advantages: This invention successfully developed a 5'UTR sequence (Hh) through the rational design and optimization of naturally occurring highly expressed gene elements. Dual verification at the DNA level (EGFP reporter system) and the RNA level (luciferase reporter mRNA) confirmed the Hh sequence's strong ability to promote gene expression. Particularly at the RNA level, which is closer to therapeutic mRNA applications, its effect on driving luciferase expression is significant, demonstrating its excellent translation initiation efficiency. Finally, in vivo imaging experiments in mice demonstrated that mRNA containing the Hh UTR can maintain efficient and stable protein expression even under complex physiological conditions in vivo. This means that when producing the same dose of mRNA drugs or vaccines, a lower mRNA dose can be used to achieve the desired therapeutic effect, directly reducing raw material and production costs. This invention is not a simple sequence extraction or blind screening, but rather a rational design combining bioinformatics tools (such as PaddleHelix's ribosome load prediction and secondary structure prediction). Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 The secondary structure prediction diagram of HBB RNA in an embodiment of the present invention is shown. Figure 2 The diagram shows the secondary structure prediction map of hbb RNA in an embodiment of the present invention. Figure 3 The image shows the expression effect of strong green fluorescent protein (EGFP) in 293T cells at 48 hours in an embodiment of the present invention. Figure 4 The luciferase activity of mRNA transfected into HEK293T cells 24 hours after the invention is shown in this embodiment. Figure 5 The luciferase activity of mRNA transfected into HEK293T cells in this embodiment of the invention is shown 48 hours later. Figure 6 The diagram shows the modification of Hh (HBB-hbb chimera) and the predicted RNA secondary structure in an embodiment of the present invention. Figure 7 The luciferase activity of HEK293T cells 24 hours after Hh transfection is shown in an embodiment of the present invention. Figure 8 The luciferase activity of Hh transfected HEK293T cells 48 hours after the invention was presented in this embodiment. Figure 9 This shows an in vivo imaging image of a mouse 24 hours after vaccination with the Hh-luciferase mRNA-LNP vaccine in an embodiment of the present invention; Figure 10 The bioluminescent signal intensity of the Hh-mRNA vaccine in an embodiment of the present invention is shown. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. 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. Example 1

[0017] The 5'UTR (named HBB in this invention) of Homo sapiens haplotype T7 delta-globin (β-globin) (NCBI number AF339412.1) and the 5'UTR (named hbb in this invention) of Homo sapiens voucher Baka_10_1 hemoglobin subunit beta (NCBI number MK475748.1) were obtained from the NCBI database.

[0018] After preliminary modifications to HBB and hbb sequences, ribosome load prediction was performed using the Baidu PaddleHelix platform. The preliminary modification method involved capping the HBB and hbb sequences at the 5' end and adding a kozak sequence to the 3' end. The BioNTech 5' UTR (Pfizer bivalent expression vector BNT162b2, NCBI ID 0R134577.1), commonly used in existing technologies, was used as a control. Ribosome load prediction (i.e., zx corresponding to HBB and hbb) was performed using the preliminarily modified hbb 5'UTR and HBB 5'UTR sequences as the original sequences. Ribosome load prediction (i.e., zx corresponding to BioNTech) was performed using the original BioNTech 5'UTR sequence as a control. The platform-optimized sequences of hbb 5'UTR, HBB 5'UTR, and BioNTech 5'UTR were PP1~PP5, respectively. Candidate sequences were screened based on the zx and PP1~PP5 predicted ribosome load values ​​of hbb 5'UTR, HBB 5'UTR, and BioNTech 5'UTR.

[0019] The selection criteria are as follows: if the difference in ribosome loading between the automatically optimized sequence (PP1~PP5) and the preliminarily modified original sequence (zx) is >0.4, then the sequence with the highest ribosome loading among the system-optimized sequences (PP1-PP5) is selected; if the difference is <0.4, then the preliminarily modified original sequence zx is selected.

[0020] The results are shown in Table 1 (the values ​​in Table 1 represent the ribosome binding potential of different UTR sequences; the higher the value, the stronger the ribosome binding potential of the corresponding UTR sequence).

[0021] Table 1

[0022] As shown in Table 1, the predicted ribosome loading values ​​of each optimized sequence of hbb and HBB (PP1-PP5) are higher than those of the original hbb 5'UTR sequence and the original HBB 5'UTR sequence after preliminary modification, but the difference is less than 0.4. Therefore, the zx sequences corresponding to hbb and HBB (i.e., the original hbb 5'UTR sequence and the original HBB 5'UTR sequence after preliminary modification) are determined as the target versions for subsequent optimization to ensure that the optimization direction is directly related to the ribosome binding efficiency.

[0023] Simultaneously, referring to the BioNTech 5'UTR secondary structure, RNA secondary structure was used as one of the screening criteria. The secondary structure of the pre-modified hbb 5'UTR and HBB 5'UTR original sequences was predicted using the unafold.org website. Sequences with high predicted ribosome binding efficiency and moderate secondary structure complexity were selected as candidates. The secondary structure prediction results of HBB RNA are shown in Figure 1 (dG=-4.50, no obvious inhibitory structural features), and the secondary structure prediction results of hbb RNA are shown in Figure 2 (dG=-9.80, no stem-loop inhibitory structure adjacent to the cap structure), both meeting the requirements for subsequent assembly. Example 2

[0024] Validation at the DNA level was performed using an enhanced green fluorescent protein (EGFP) reporter system: 1. Plasmid construction: Blank control group: Original pCMV-EGFP blank plasmid without insertion of the target 5'UTR; BioNTech positive control group: control recombinant plasmid with pCMV-EGFP inserted into the 5'UTR of BioNTech; Experimental group: including HBB and hbb; HBB: HBB 5'UTR and hbb5'UTR are inserted into the preset site of the pCMV-EGFP reporter plasmid using molecular cloning technology to obtain the recombinant plasmid pCMV-HBB 5'UTR-EGFP; hbb: The hbb5'UTR is inserted into the preset site of the pCMV-EGFP reporter plasmid using molecular cloning technology to obtain the recombinant plasmid pCMV-hbb 5'UTR-EGFP.

[0025] 2. Plasmid amplification: The pCMV-EGFP blank plasmid, control recombinant plasmid, recombinant plasmid pCMV-HBB 5'UTR-EGFP, and recombinant plasmid pCMV-hbb 5'UTR-EGFP were transformed into TOP10 competent cells for amplification. At the same time, the amplified plasmids were verified by DNA sequencing to confirm that the HBB 5'UTR and hbb 5'UTR sequences were correctly inserted into the preset sites of the pCMV-EGFP plasmid, and there were no abnormalities such as base mutations, deletions, or reverse insertions.

[0026] 3. Transfection and Detection: Using plasmids verified by sequencing as experimental materials, HEK293T cells were transfected with Lipofectamine 3000 transfection reagent. Forty-eight hours after transfection, the EGFP fluorescence intensity was observed and compared under a fluorescence microscope. Results are as follows: Figure 3 As shown, the green fluorescence intensity produced by the experimental groups (HBB group, hbb group) was significantly higher than that of the blank control group and the positive control group (BioNTech group), demonstrating that at the DNA level, HBB 5'UTR and hbb 5'UTR can effectively enhance the expression efficiency of downstream EGFP genes. Example 3

[0027] Validation at the RNA level using a luciferase expression system: ① The target gene fragment and pUC57 vector were double-digested using NcoI / XhoI restriction endonuclease (NEB). The 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 and plated on LB plates containing 100 μg / mL ampicillin. The cells were incubated upside down at 37°C for 16 hours. Single clones were selected and sequenced. ② The target fragment containing the UTR and the PUC57-LUC vector were digested with AseI / NcoI double enzymes, and T4 ligase was used to construct a pUC57 reporter vector containing the T7 promoter, 5'UTR-HBB / 5'UTR-hbb, Luciferase, 3'UTR, and polyA; the vector was transformed into DTSINGKE TSC-C01 Trelief® 5α competent cells by heat shock, plated on LB plates containing 100 μg / mL ampicillin, and incubated upside down at 37°C for 16 hours. Single clones were selected and sequenced. Transfection and detection: The constructed pUC57 reporter vector was amplified by DH5α, linearized by BsaI digestion, purified by isopropanol precipitation, and then transcribed in vitro using N1-methylpseudouridine triphosphate (N1-Me-pUTP) to obtain modified mRNA. The in vitro transcribed mRNA was transfected into HEK293T cells using Lipofectamine 3000 transfection reagent, and luciferase activity was detected at 24 and 48 hours post-transfection.

[0028] The results are shown in Figure 4 (24H activity) and Figure 5 (48H activity). The luciferase activity (RLC value) of the HBB group and the hbb group was significantly higher than that of the blank control group and the BioNTech control group, proving that at the RNA level, both UTR sequences can effectively improve translation efficiency. At the same time, it was verified that the effect of the same UTR is different in DNA and RNA expression systems. Subsequent experiments will focus on RNA-level verification. Example 4

[0029] Hh chimera construction and in vitro / in vivo expression validation: Construction of the Hh chimera: Based on the significant synergistic effect demonstrated by HBB and hbb UTR elements in previous experiments, this embodiment employs a dual UTR tandem strategy. Optimized HBB and hbb sequences are directionally assembled using molecular cloning technology to form an HBB-hbb chimera (named Hh). A GA sequence is added to the 5' end of the tandem HBB-hbb structure as a 5' cap structure, and a Kozak sequence (GCCACCATG) is added to the 5' end to construct the Hh chimera (Hh element). The hbb is positioned downstream of the start codon to maximize regulatory efficiency; simultaneously, a GA sequence is added after the 5' cap structure and at the front of the 5' UTR to further enhance sequence stability.

[0030] Secondary structure verification: The secondary structure of the constructed Hh chimeric sequence was predicted, and the results are shown in Figure 6 (dG=-13.90). This structure showed no obvious inhibitory hairpins, and the ribosome-binding region was fully exposed, meeting the structural requirements for efficient translation. After completing the secondary structure prediction, plasmids were constructed and sequenced. The resulting Hh DNA and RNA sequences are shown in Table 2. Table 2

[0031] In vitro expression validation: Following the method in Example 3, a pUC57 reporter vector containing the Hh chimera was constructed. mRNA was obtained through in vitro transcription, and luciferase activity was detected after transfection into HEK293T cells. The results are shown in Figure 7 (24H activity). Figure 8 (48H activity) As shown, the luciferase activity of the Hh group was significantly higher than that of the BioNTech control group and the blank control group, and was superior to the experimental groups that used HBB or hbb alone, demonstrating the synergistic effect of the chimera.

[0032] In vivo expression verification: Preparation of mRNA-LNP complex: To further verify the expression of Hh in vivo at the mRNA level, the modified Hh-luciferase plasmid was analyzed by mouse in vivo imaging. The constructed Hh-luciferase mRNA was dissolved in 50 mM citrate buffer at pH 4 to a final concentration of 108 ng / μL. Next, a lipid mixture was prepared: ionizable lipids (SM102), PEG-modified lipid dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000), distearate phosphatidylcholine (DSPC), and cholesterol were mixed and dissolved in anhydrous ethanol solution at a ratio of 50%, 1.5%, 10%, and 38.5%, respectively. The lipid-ethanol solution and the mRNA-citrate solution were filtered separately through a 0.22 μm microporous membrane at a ratio of mRNA:lipid 3:1. The volume ratio was determined using a microfluidic instrument, with a flow rate of 15 mL / min for the buffer phase and 5 mL / min for the ethanol phase. The resulting mRNA-LNP complex was immediately diluted with 15 mL of the above citrate buffer and ultrafiltered 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. This process was repeated twice, and the concentration and encapsulation efficiency were measured. The encapsulation efficiency was greater than 90%, and the LNP particle size distribution was 80-100 nm.

[0033] Animal experiments: Six- to eight-week-old female C57BL / 6 mice were randomly divided into a blank control group, a BioNTech positive control group, and an Hh group, with three mice in each group. The corresponding vaccine (mRNA-LNP complex) was injected intramuscularly into the right hind limb of each mouse, and in vivo imaging analysis was performed 24 hours after injection. The in vivo imaging images of mice 24 hours after Hh-mRNA vaccine vaccination are shown below. Figure 9 As shown, the bioluminescent signal intensity at the injection site was significantly higher in the Hh group mice than in the control group. The results of the bioluminescent signal intensity induced by the Hh-mRNA vaccine are as follows: Figure 10 As shown, the total throughput of the Hh group was significantly higher than that of the BioNTech group and the blank control group, demonstrating that the Hh chimera can still maintain efficient and stable protein expression under complex physiological conditions in vivo.

[0034] As can be seen from the above embodiments, the Hh 5'UTR sequence provided by this invention, verified by both DNA and RNA dimensions, as well as in vivo imaging experiments, has been shown to significantly improve the expression efficiency of downstream genes. This sequence design successfully achieves domestic substitution of the 5'UTR, providing a core component for the development of mRNA drugs and vaccines, and has significant application value in reducing costs and increasing efficiency.

[0035] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A UTR element Hh, characterized in that, The DNA sequence of Hh is shown in SEQ NO.1, and the RNA sequence is shown in SEQ NO.

2.

2. A method of constructing a UTR element Hh according to claim 1, wherein, Includes the following steps: get: Obtain the 5' UTR sequence HBB of Homo sapiens haplotype T7 delta-globin and the 5' UTR sequence hbb of Homo sapiens voucher Baka_10_1 hemoglobin subunit beta from the NCBI database; Ribosome loading and secondary structure prediction were performed on HBB and hbb sequences using the PaddleHelix platform. Optimized HBB 5'UTR and hbb 5'UTR were obtained based on the prediction results. Based on the optimized HBB 5'UTR and hbb 5'UTR, they are cascaded together. A GA sequence is added to the 5' end of the cascaded HBB-hbb as a 5'cap structure, and a Kozak sequence is added to the 3' end to construct the UTR element Hh.

3. The method of constructing a UTR element Hh according to claim 2, wherein, The NCBI number for Homo sapienshaplotype T7 delta-globin is AF339412.1; the NCBI number for Homo sapiens voucher Baka_10_1 hemoglobin subunit beta is MK475748.

1.

4. The method of claim 2, wherein the UTR element Hh is constructed by, The Kozak sequence is GCCACCATG.

5. An mRNA molecule, characterized in that It includes the Hh element as described in claim 1 as its 5'UTR.

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 of 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 by, It comprises the mRNA-LNP complex of claim 6 and pharmaceutically acceptable excipients.

9. The use of the UTR element Hh of claim 1, the mRNA molecule of claim 5, the mRNA-LNP complex of claim 6, or the pharmaceutical composition of claim 8 in the preparation of a vaccine for the prevention or treatment of viral infection.

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