UTR (Untranslated Region) element NHP1 as well as construction method and application thereof

Abstract

The invention provides an UTR element NHP1 as well as a construction method and application thereof, and relates to the technical field of mRNA. A 5 'UTR with a good expression effect is designed by integrating dominant sequences of a human high-expression gene and a pathogen natural UTR, a chimeric structure NHP1 with high ribosome load is predicted through a calculation model, a DNA sequence of the NHP1 is as shown in SEQ NO 1, and an RNA sequence of the NHP1 is as shown in SEQ NO 2; an EGFP report system is adopted on the DNA level to rapidly screen UTR; the translation efficiency is quantitatively evaluated on the RNA level through luciferase mRNA (N1-methyl pseudouridine modification); and the particle size is controlled by a microfluidic technology, so that the optimized UTR-mRNA is efficiently expressed after being delivered.

Description

Technical Field

[0001] This invention relates to the field of mRNA molecular technology, specifically to a UTR element NHP1, its construction method, and its applications. 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 coding regions, research on sequence design targeting the untranslated region (UTR) in gene sequences, 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'UTRs of genes with extremely high expression efficiency in human cells, such as the 5'UTRs of human α-globin genes. The second is the natural 5'UTR of the pathogen's mRNA itself. However, the secondary structure of natural 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, natural 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 proposes a UTR element NHP1, its construction method, and its application, specifically implemented through the following technical solutions: In a first aspect, the present invention proposes a UTR element NHP1, the DNA sequence of which is shown in SEQ NO1 and the RNA sequence of which is shown in SEQ NO2.

[0005] Secondly, the present invention proposes a method for constructing the aforementioned UTR element NHP1, comprising the following steps: The 5'UTR sequences of the influenza NS1 gene and the HIV TAT 202 gene were obtained from the NCBI database. The 5'UTR sequence of the influenza NS1 gene was modified by adding a GA sequence to its 5' end as a 5'cap structure and adding a kozak sequence to its 3' end, as shown in SEQ NO 3; The 5'UTR sequence of the HIV TAT 202 gene was optimized using the PaddleHelix platform, and the optimized sequence HTAT 202 P1 was obtained. The modified NS1 5'UTR sequence was concatenated with the HTAT 202 P1 sequence to construct the NHP1 element.

[0006] Furthermore, the HIV TAT 202 gene is numbered LOC129008191 in the NCBI database; The influenza NS1 gene is identified as XM_021929452.2 in the NCBI database.

[0007] Thirdly, the present invention proposes an mRNA molecule comprising the aforementioned NHP1 element, target gene coding sequence, 3'UTR sequence, and polyA tail sequence.

[0008] 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.

[0009] 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.

[0010] In a sixth aspect, the present invention provides a pharmaceutical composition characterized by comprising the aforementioned mRNA-LNP complex and pharmaceutically acceptable excipients.

[0011] In a seventh aspect, the present invention proposes the use of the described element, the described mRNA molecule, the described mRNA-LNP complex, or the described pharmaceutical composition in the preparation of vaccines for the prevention or treatment of viral infections.

[0012] Compared with the prior art, the present invention has the following advantages: This invention designs a 5′UTR with good expression by integrating the advantageous sequences of highly expressed human genes and the natural UTRs of pathogens. The chimeric structure NHP1 is predicted to carry ribosomes using a computational model (PaddleHelix platform). At the DNA level, the EGFP reporter system is used to rapidly screen UTRs. At the RNA level, the translation efficiency is quantitatively assessed by luciferase mRNA (N1-methylpseudouridine modified). Furthermore, microfluidic technology is used to control the particle size to ensure that the optimized UTR-mRNA is expressed efficiently after delivery. Attached Figure Description

[0013] 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.

[0014] Figure 1 The secondary structure prediction diagram of NS1 RNA in an embodiment of the present invention is shown; Figure 2 The diagram shows a predicted secondary structure of HTAT 202 P1 RNA in an embodiment of the present invention. Figure 3 The image shows the expression effect of strong green fluorescent protein (EGFP) in HEK293T cells at 48 hours in an embodiment of the present invention. Figure 4 The luciferase activities of HTAT 202 and HTAT 202P1 in HEK293T cells 24 hours after mRNA transfection in an embodiment of the present invention are shown. Figure 5The luciferase activities of HTAT 202 and HTAT 202P1 in HEK293T cells 24 hours after mRNA transfection in an embodiment of the present invention are shown. Figure 6 The luciferase activity of NS1 in HEK293T cells 24 hours after mRNA transfection is shown in an embodiment of the present invention. Figure 7 The luciferase activity of NS1 in HEK293T cells 48 hours after mRNA transfection is shown in this embodiment of the invention. Figure 8 The secondary structure prediction diagram of the NS1-HTAT202 P1 chimera (NHP1) in an embodiment of the present invention is shown; Figure 9 The luciferase activity of HEK293T cells 24 hours after NHP1 transfection is shown in an embodiment of the present invention. Figure 10 The luciferase activity of HEK293T cells after NHP1 transfection in an embodiment of the present invention is shown. Figure 11 The results of NHP1 mRNA expression in HEK293T cells measured using the Westren bolt (quantitative) method in an embodiment of the present invention are shown. Figure 12 The following is an example of the H1N1 grayscale analysis results of the NHP1-WB band in an embodiment of the present invention; Figure 13 The following is an example of the H3N2 grayscale analysis results of the NHP1-WB band in an embodiment of the present invention; Figure 14 The results of the NHP1-WB band grayscale analysis (BIV) in an embodiment of the present invention are shown. Detailed Implementation

[0015] 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

[0016] Design and verification of NHP1 components: The 5'UTR sequences of genes with extremely high expression efficiency in human cells and the 5'UTR sequences of pathogen mRNAs were found in the NCBI database. A 5'cap (GA) and kozak sequence (GCCAGG, SEQ NO 3) was added to the influenza NS1 5'UTR. The HIV TAT 202 5'UTR was automatically optimized and designed using the Baidu PaddleHelix platform. Ribosome load and secondary structure predictions were performed on the Baidu PaddleHelix platform (unafold.org). UTRs with good prediction results were selected for validation. The specific process is as follows: The 5'UTR of a gene with extremely high expression efficiency in human cells and the 5'UTR sequence of the pathogen mRNA were found in the NCBI database. The natural 5'UTRs of influenza NS1 (NCBI ID: XM_021929452.2, hereinafter referred to as NS1) and HIV TAT 202 (NCBI ID: LOC129008191, hereinafter abbreviated as HTAT 202) were obtained. A 5′ cap sequence (GA) was added to the front of the 5′ UTR sequence of influenza NS1, and a kozak sequence (GCCAGG) was added to the back. The 5'UTR of HTAT 202 and NS1 were optimized using the PaddleHelix platform to screen sequences with high ribosome loading and stable secondary structure (UNAFold verification). The ribosome loading prediction results are shown in Table 1. Table 1

[0017] The predicted results of the NS1 RNA secondary structure and the HTAT 202 P1 RNA secondary structure are as follows: Figure 1 and Figure 2 As shown in Table 1, the 5' UTR of NS1, after adding the Kozak sequence, scores close to other UTRs designed in PaddleHelix, so the original sequence is used. Among the HTAT 202 series, P1 has the highest score, and the secondary structures of the two UTRs are significantly different; therefore, HTAT 202 P1 is selected. Example 2

[0018] A DNA-RNA dual-dimensional system was established to verify the regulatory activity of the 5'UTR sequence on gene expression.

[0019] (1) Validation at the DNA level using an enhanced green fluorescent protein (EGFP) reporter system: Plasmid amplification: The pCMV-EGFP reporter plasmid was amplified in TOP10 competent cells, and the plasmid was extracted and DNA sequenced. Transfection and detection: HEK293T cells were transfected with Lipofectamine 3000 transfection reagent, and the expression efficiency was evaluated by fluorescence microscopy.

[0020] The results are as follows Figure 3 As shown, the EGFP plasmid containing UTR produces more green fluorescence than the empty vector and Biontech, and at the DNA level, HTAT 202 has the best green fluorescence expression effect.

[0021] (2) At the RNA level, a luciferase expression system was used. RNA was transfected using a Mirus kit, and luciferase activity was detected after transfection. The specific procedure is as follows: Plasmid construction: 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 vector were digested with AseI / NcoI double enzymes, and T4 ligase was used to construct a pUC57 reporter vector containing a T7 promoter, 5'UTR-H202 / HTAT 202 P1 / NS1 (please confirm that this description is correct), 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. Plasmid amplification: The recombinant plasmid was transformed into DH5α competent cells, and the plasmid was extracted after amplification; Linearization: The plasmid was digested with BsaI enzyme, and the DNA was cut downstream of polyA to generate sticky ends; mRNA preparation: The pUC57 reporter vector pUC57-T7-H202 / HTAT 202 P1 / NS1, Luciferase, 3'UTR, and polyA was constructed and linearized with BsaI before in vitro transcription (N1-Me-pUTP modification). In vitro transcription (IVT): Using linear DNA as a template, T7 RNA polymerase is used to add N1-methylpseudouridine triphosphate N1-Me-pUTP (modified uridine) for in vitro transcription to obtain modified mRNA; mRNA transfection and luciferase detection: mRNA was transfected into HEK293T cells, the transfected HEK293T cells were lysed, luciferase substrate was added, and the expression efficiency was evaluated by fluorescence microscopy.

[0022] Luciferase activity assay, such as Figure 4 , Figure 5 , Figure 6 and Figure 7 As shown in the figure, BioTech represents the control group, which is the 5′ UTR used in Pfizer's COVID-19 vaccine. It can be seen that HTAT 202 P1 showed better expression at the RNA level for cell validation. Based on the DNA-RNA cell-level results, it was verified that the same UTR has different expression effects in DNA and RNA; therefore, subsequent experiments only validated at the RNA level.

[0023] according to Figure 4 , Figure 5 , Figure 6 and Figure 7 The data from the luciferase reporter system show that HTAT 202 P1 and NS1 both exhibited significant translation enhancement at the RNA level. Example 3

[0024] Because HTAT 202 P1 and NS1 showed good expression results, a high-efficiency HTAT 202 P1 and NS1 expression strain was selected and used in combination. Using molecular cloning technology, the NS1 element was directionally assembled with HTAT 202 P1 to construct the NS1-HTAT202 P1 chimera (hereinafter referred to as NHP1), and secondary structure prediction was performed (prediction results are shown in the figure). Figure 8 After prediction, plasmids were amplified, linearized, and transcribed into mRNA in vitro. After transfection into HEK293T cells, luciferase activity was measured.

[0025] The DNA and RNA sequences of NHP1 obtained from sequencing are shown in Table 2: Table 2

[0026] The results are as follows Figure 9 and Figure 10 As shown, the luciferase activity of NHP1 transfected HEK293T cells was well expressed at the mRNA level at 24H and 48H.

[0027] To further validate NHP1 expression at the vaccine level, the modified NHP1 was tandemly linked with NA influenza vaccine sequences (H1N1, H3N2) to obtain H1N1-NA, H3N2-NA, and BIV-NA. The constructed NHP1-Inliuenza_NA mRNA was dissolved in 50 mM citrate buffer at pH 4 to achieve a final concentration of 108 ng / μL. Next, a lipid mixture solution 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-citric acid solution were filtered separately through a 0.22 μm microporous membrane. The mRNA and lipids were mixed using a microfluidic instrument at a ratio of 3:1. The resulting mRNA-LNP was immediately diluted with 15 mL of the above-mentioned citrate buffer and ultrafiltered using a 100 KD ultrafiltration tube at a centrifugation force of 3000 g. After ultrafiltration to 1 / 4 volume, 50 mM Tris-HCl buffer (pH=7.5) was added to 15 mL. The process was repeated twice, and the concentration and encapsulation efficiency were measured. Then, after verifying its in vitro expression effect in 293T cells, Western blot analysis confirmed the expression of the target protein.

[0028] The results are as follows Figure 11 as well as Figure 12 , Figure 13 , Figure 14 As shown, Action is used as an internal reference protein. It can be seen that H1N1-NA, H3N2-NA, and BIV-NA all have obvious bands, and the expression levels of BioNTech LNP protein in the control group are not significantly different, indicating that NHP1 has a good expression effect at the vaccine level.

[0029] 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 NHP1, characterized in that, The DNA sequence of the UTR element NHP1 is shown in SEQ NO 1, and the RNA sequence is shown in SEQ NO 2.

2. A method for constructing the UTR element NHP1 as described in claim 1, characterized in that, Includes the following steps: The 5'UTR sequences of the influenza NS1 gene and the HIV TAT 202 gene were obtained from the NCBI database. The 5'UTR sequence of the influenza NS1 gene was modified by adding a GA sequence to its 5' end as a 5'cap structure and adding a kozak sequence to its 3' end, as shown in SEQ NO 3; The 5'UTR sequence of the HIV TAT 202 gene was optimized using the PaddleHelix platform, and the optimized sequence HTAT 202 P1 was obtained. The modified NS1 5'UTR sequence was concatenated with the HTAT 202 P1 sequence to construct the NHP1 element.

3. The method for constructing the UTR element NHP1 according to claim 2, characterized in that, The HIV TAT 202 gene is numbered LOC129008191 in the NCBI database; The influenza NS1 gene is identified as XM_021929452.2 in the NCBI database.

4. An mRNA molecule, characterized in that, It includes the NHP1 element as described in claim 1, the target gene coding sequence, the 3'UTR sequence, and the polyA tail sequence.

5. An mRNA-LNP complex, characterized in that, Include: The mRNA molecule according to claim 3; Lipid nanoparticles comprising SM102, DMG-PEG2000, DSPC, and cholesterol in weight percentages of 50%, 1.5%, 10%, and 38.5%, respectively.

6. A method for preparing the mRNA-LNP complex of claim 5, 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 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.

7. A pharmaceutical composition, characterized in that, It comprises the mRNA-LNP complex of claim 5 and pharmaceutically acceptable excipients.

8. The use of the UTR element NHP1 of claim 1, the mRNA molecule of claim 4, the mRNA-LNP complex, or the pharmaceutical composition of claim 7 in the preparation of a vaccine for the prevention or treatment of viral infection.

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