5 'UTR sequences for high expression of RNA

Abstract

The present invention relates to the stabilization of RNA, in particular mRNA, and the improvement of mRNA translation. In particular, the present invention relates to the modification of RNA, in particular in vitro transcription RNA, which results in increased transcript stability or translation efficiency. According to the present invention, the stability and the translation efficiency are improved according to the certain sequences of the 5 '-untranslated region (UTR) of the RNA molecule, and compared with the prior art, the 5' UTR obtained through optimization has the excellent target protein expression effect, and can be used for DNA and mRNA vaccine development or other protein treatment drug mining.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering preventive or therapeutic vaccines and drugs, and specifically relates to a 5'UTR sequence capable of enhancing high expression of mRNA and its application, and belongs to the field of nucleic acid technology. Background Art

[0002] The discovery of messenger RNA (mRNA) began in 1961. It is a single-stranded long RNA that can transmit genetic information in DNA and synthesize proteins through translation. Due to the instability of mRNA, the development of mRNA-related drugs has been slow. In recent years, with the continuous maturity of mRNA in vitro synthesis and delivery technology, the stability and translation efficiency of mRNA have been greatly improved. mRNA technology has become an ideal platform for the treatment of many disease areas. Based on its greatly shortened and reduced cycle and cost of new drug development, many leading pharmaceutical companies are actively planning and developing mRNA vaccines and mRNA drugs.

[0003] In eukaryotes, the translation of mRNA is a continuous cycle, which mainly includes three steps: translation initiation, peptide chain extension, and peptide chain termination. The m7G cap-dependent ribosome scanning mechanism is the main form of translation initiation in eukaryotes and is also recognized as the most classic molecular mechanism. This mechanism was first proposed by Kozak in 1989. After continuous summary and improvement, its process mainly includes the assembly of the 43S pre-initiation complex, the binding of the 43S pre-initiation complex to the m7G cap structure, the selection of the start codon, the addition of the 60S ribosomal subunit, and the formation of the 80S subunit. According to the scanning model of translation initiation, the ribosome binds to the 5' cap structure of the mRNA and begins to scan along the 5' UTR until it finds a suitable translation initiation site (TIS), at which point it will begin the protein assembly process. During this process, the 5' UTR length, secondary structure, and upstream AUG codon can regulate the scanning and number of ribosomes on the mRNA; secondly, the translating ribosomes can usually protect the mRNA from degradation. In summary, the 5' UTR sequence is an important component of the mRNA structure and plays a vital role in the translatability and stability of the mRNA molecule.

[0004] The 5'UTR structure includes pseudo dots (Pseudoknot), hairpins, RNA G-quadruplexes (RG4s), upstream open reading frames (uORFs), upstream start codons (uAUGs), RNA binding proteins (RBPs), long noncoding RNAs (lncRNAs), internal ribosome entry sites (IRESs), and Kozak sequences around the start codon. Pseudoknot, hairpins, and RNA G-quadruplexes (RG4s), as well as upstream open reading frames (uORFs) and upstream start codons (uAUGs) in the 5'UTR structure mainly inhibit the translation of downstream target proteins. Kozak sequences are preferred for translation initiation, and optimized Kozak sequences can significantly enhance the expression of target genes.

[0005] The physicochemical properties of the 5'UTR sequence mainly include: the length of the 5'UTR sequence, the negative folding free energy (ΔG) level, the GC content, the secondary structure configuration, and the ribosome loading rate. If the 5'UTR is too long and the GC content is too high, a large number of secondary structures with large structures will be formed, and the negative folding free energy will increase, which will lead to low ribosome scanning efficiency and reduced initiation efficiency. Secondly, it is necessary to maintain a dynamic balance between the ribosome loading rate and stability in the 5'UTR sequence in order to achieve a stronger translation level of the target protein.

[0006] Since the target protein of mRNA in cells has a stronger translation level depending on the 5'UTR, screening for 5'UTR sequences that can enhance the high expression of mRNA is an important means to increase the production of antigenic proteins and the final antibody titer, and has become one of the prerequisites for the development of mRNA drug platforms. Therefore, screening for 5'UTR sequences that stably express mRNA is of great significance for the application of mRNA therapy.

[0007] The purpose of the present invention is to screen out a 5'UTR sequence that can enhance mRNA stability and increase protein expression, thereby promoting mRNA expression. Summary of the invention

[0008] In the present invention, we designed more than 70 different novel 5'UTR sequences with different lengths and secondary structures, and used the Biopython toolkit to calculate the GC content of the sequences, the ViennaRNA toolkit to calculate the RNA secondary structure and minimum free energy, and generated the corresponding secondary structure diagram. We demonstrated that different 5'UTR sequences regulate the yield of proteins translated from mRNA encoding reporter genes and encoding the same protein in vitro to varying degrees. In addition, we found that compared with known 5'UTR sequences, multiple 5'UTR sequences of the present invention resulted in higher transcription and translation efficiencies, while compared with other 5'UTR sequences, the translation and transcription efficiencies of some 5'UTR sequences were significantly reduced. These novel 5'UTR sequences will be used to improve or regulate the translation and transcription of mRNA and genes used for mRNA vaccines, mRNA therapy, and gene therapy.

[0009] The object of the present invention is to provide a group of novel 5'UTR sequences, which include polynucleotides containing any one of SEQ ID NOs. 1-73.

[0010] As a preferred technical solution, the 5'UTR comprises a sequence of any one of SEQ ID NOs 1-73, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NOs 1-73.

[0011] As a preferred technical solution, the 5'UTR sequence has a high GC content, for example, a GC content of not less than 30%. Further preferably, the 5'UTR sequence has a GC content of about 30%, 40%, 50%, 60% or 70%.

[0012] More preferably, the GC content is 30%, 40% or 50%.

[0013] As a preferred technical solution, the 5'UTR includes a Kozak sequence.

[0014] In some specific embodiments of the present invention, the Kozak sequence is 5'GCCACC3'.

[0015] The present invention also provides an expression vector or a host cell comprising the 5'UTR.

[0016] The present invention also provides a DNA or RNA vaccine comprising the 5'UTR.

[0017] The present invention also provides a nucleic acid product or protein product for treatment or prevention comprising the 5'UTR.

[0018] The present invention also provides the use of 5'UTR in the expression of encoded proteins.

[0019] The present invention also provides mRNA, which comprises in the 5' to 3' direction: at least one 5'UTR element, the 5'UTR element comprising a polynucleotide containing any one of SEQ ID NOs 1-73, and at least one open reading frame encoding a peptide or protein, the mRNA further comprising a 3'UTR sequence and a PolyA sequence.

[0020] As a preferred technical solution, the peptide or protein can encode hFIX, SPB, VEGF-A, human methylmalonyl CoA variant enzyme, CFTR, cancer autoantigen, gene editing enzyme, HIV antibody, CD19 antibody, CD22 antibody, CD3 antibody, CLDN6 antibody, Luc2, GLuc, Fluc, eGFP, hEPO, Cas9 nuclease, new coronavirus S protein or tumor suppressor.

[0021] In some specific embodiments of the present invention, the protein coding region or non-coding region of the present invention is the Fluc gene, which verifies the feasibility of the technical solution of the present invention.

[0022] Compared with the prior art, the advantages of the present invention are: the 5'UTR sequence optimized in the present invention can be used for the development of DNA vaccines, mRNA vaccines, protein therapeutic drugs and other biological products. The optimized 5'UTR improves the translation efficiency of the target protein. The optimized 5'UTR sequence can be used for the construction of efficient expression vectors, improve the expression level of the target protein, and be applied to the development and application of DNA or mRNA vaccines or other therapeutic and preventive proteins and nucleic acid products. It can be used to regulate the expression of various proteins and be used to treat various diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Optimize fluorescence expression results for 5'UTR sequences;

[0024] Figure 2 Optimize fluorescence expression results for 5'UTR sequences;

[0025] Figure 3 Optimize the fluorescence expression results of the 5'UTR sequence for the second experiment. DETAILED DESCRIPTION

[0026] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0027] Example 1, 5'UTR sequence design

[0028] Table 1 lists the novel 5'UTR sequences produced and tested in this study. All of these sequences were designed independently, and multiple nucleic acid sequences of 50 nt in length were generated in a random manner. The ribosome load of the sequences generated in the first step was predicted by calculation, and the sequences with suitable ribosome loads were selected for mutation, and the ribosome load was calculated again. This process was repeated until the target ribosome load was reached, and the sequences that reached the target ribosome load were output. The GC content of the sequence was calculated using the Biopython toolkit, and the RNA secondary structure and minimum free energy were calculated using the ViennaRNA toolkit, and the corresponding secondary structure diagram was generated. The appropriate sequence was selected by filtering conditions such as GC content, minimum free energy, and RNA secondary structure.

[0029] Table 1 is the 5'UTR sequence of the present invention

[0030]

[0031]

[0032]

[0033] Example 2, plasmid construction

[0034] 1. Firefly Lucierase (hereinafter referred to as FLuc) was used as a reporter gene, and the 5'UTR sequence designed in Example 1 was used in combination with the T7 promoter to construct an expression module (the construction order of the expression sequence was T7, 5'UTR, Kozak, FLuc, 3'UTR, and 120Poly (A) were connected to form the expression sequence; here T7 refers to the T7 promoter coding sequence, Kozak refers to the Kozak sequence (5'GCCACC3'), and FLuc refers to the FLuc coding sequence and the T7 promoter coding sequence).

[0035] 2. Amplification of target fragment and construction of recombinant plasmid.

[0036] (1) Prepare the reaction system according to the following table:

[0037] Components volume <![CDATA[RNase-free H2O]]> Up to 50ul 2x Buffer 25ul UTR-F primer 1.5ul F Primer 1.5ul R Primer 1.5ul dNTP 1ul Phanta max super-fidelity DNA polymerase 1ul

[0038] (2) Perform PCR amplification according to the following table for 30 cycles:

[0039] Pre-denaturation 95℃3min transsexual 95℃30s annealing 62℃30s extend 70℃30s Final extension 70℃5min

[0040] (3) Gel excision, purification and recovery of target DNA fragment

[0041] (4) Vector digestion, and the reaction system was prepared as shown in the table below;

[0042] Components volume pDNA 5ug Ncol 3ul EcoRI 3ul 10x Buffer 5ul <![CDATA[RNase-free H2O]]> Up to 50ul

[0043] (5) Double enzyme digestion of vector DNA, gel excision, purification and recovery;

[0044] (6) Prepare the reaction system according to the table below and carry out the recombination reaction;

[0045] Components volume Double restriction enzyme digestion of pDNA 100ng Target DNA fragment 200ng 2x Clone Express Mix 5ul <![CDATA[RNase-free H2O]]> Up to 20ul

[0046] (7) Place the above recombination reaction system in a water bath and incubate at 50°C for 30 min.

[0047] 3. Recombinant plasmid transformation and plating

[0048] (1) Add 10ul of the recombinant product to 100ul of NEB-Stable competent cells and incubate on ice for 30min;

[0049] (2) Heat shock in water bath for 90 seconds, remove and place in ice bath for 3 minutes;

[0050] (3) Add 600 μl of antibiotic-free LB medium and incubate on a shaker for 1 h;

[0051] (4) Centrifuge at 6000 rpm for 4 min, remove 500 μl of supernatant, gently blow up the precipitate with a pipette, and take 100 μl of bacterial solution to coat the corresponding resistance plate;

[0052] (5) Place in a constant temperature incubator and culture for 16-20 hours;

[0053] (6) Pick a single clone and place it in a shaking incubator for 16-17 hours.

[0054] 4. Plasmid extraction and identification

[0055] (1) Plasmids were extracted by alkaline lysis, eluted with DEPC-treated water, and then the concentration, 260 / 280 ratio, 260 / 230 ratio and size of the extracted plasmids were determined by UV spectrophotometry and agarose gel electrophoresis;

[0056] (2) Send the plasmid for sequencing to determine whether the 5'UTR sequence is correct and whether Poly(A) is missing.

[0057] 5. Plasmid Linearization and Purification

[0058] (1) Prepare HindIII single enzyme digestion system as shown in the table below:

[0059] Components <![CDATA[RNase-free H2O]]> Up to 100ul 10×Buffer 10ul HindIII 12ul pDNA 10ug

[0060] (2) The above reaction solutions were mixed evenly, centrifuged briefly, incubated at 37°C for 2.5 h, and then purified by column.

[0061] VI. Co-transcriptional capping experiment

[0062] (1) Prepare the co-transcription capping system according to the following table:

[0063] Components Volume(uL) Final concentration <![CDATA[RNase-free H2O]]> Up to 20 —— 10×Transcription Buffer 2 1× ATP / CTP / GTP / UTP (100 mM each) 2each 10mM each Cap1-GAG (100mM) 2 10mM Template DNA 0.5ug —— T7 RNA Polymerase Mix 2 ——

[0064] (2) The reaction mixture was mixed evenly, centrifuged briefly, and incubated at 37°C for 2 h;

[0065] (3) After the transcription reaction is completed, add 1 μL DNase I to each tube, mix well, centrifuge briefly, and incubate at 37°C for 1 h to remove the template DNA;

[0066] (4) RNA purification was performed using lithium chloride precipitation.

[0067] Example 3: 5'UTR sequence optimization to regulate the translation efficiency of the FLuc gene.

[0068] HEK-293T cells were seeded in a 96-well culture plate at a number of 3000 cells per well, and cultured in an incubator at 37°C and 5% CO2 for 24 hours. On the next day, the FLuc mRNA and Lipo3000 obtained in Example 2 were diluted with opti-MEM culture medium respectively; the diluted Lipo3000 and FLuc mRNA were directly mixed at room temperature, and incubated at room temperature for 10 minutes after mixing evenly; after discarding the culture medium of the cells cultured overnight, 50 μL of complete culture medium was added to each well, and then 50 μL of the mixture of incubated Lipo3000 and FLuc mRNA was added, and cultured in an incubator at 37°C and 5% CO2 for 24 hours. After culturing for 24 hours, 100 μL of Bright-Light reagent was added to each well, and the mixture was shaken and mixed for 3 minutes, and the luminescence value of each well was measured using an enzyme-labeled instrument. Figure 1-3 As shown, Control-BioNtech is the preferred 5'UTR for Biontech's COVID vaccine mRNA BNT162b2. Control-Moderna is the 5'UTR selected for Moderna's COVID vaccine mRNA 1273. Compared with Moderna's 5'UTR, the sequence significantly improved the translation efficiency of the coding sequence. Figure 1-2 It was shown that except for sequences 28, 33, 34, 41, 42, and 47, other 5'UTR sequences showed higher translation efficiency than Moderna's 5'UTR sequence. Figure 3 It was shown that compared with the 5'UTR sequence of BNT162b2, the new 5'UTR sequences 9, 10, 12, 23, 29, and 30 showed higher translation efficiency.

Claims

1. A set of novel 5'UTR sequences, characterized in that: The 5'UTR sequence includes a polynucleotide containing any one of SEQ ID NOs. 1-73.

2. The polynucleotide of claim 1, wherein the 5'UTR comprises a sequence of any one of SEQ ID NOs 1-73, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs 1-73.

3. The polynucleotide according to claim 1 or 2, wherein the 5'UTR sequence has a GC content of not less than 30%.

4. The polynucleotide of claim 1 or 2, wherein the 5'UTR comprises a Kozak sequence.

5. An expression vector or host cell comprising the 5'UTR of claim 1.

6. A DNA or RNA vaccine comprising the 5'UTR of claim 1.

7. A therapeutic or preventive nucleic acid product or a therapeutic or preventive protein product comprising the 5'UTR of claim 1.

8. Use of the 5'UTR of claim 1 in the expression of encoded proteins.

9. mRNA, characterized in that It comprises in the 5' to 3' direction: at least one 5'UTR element, the 5'UTR element comprises a polynucleotide containing any one of SEQ ID NOs 1-73, and at least one open reading frame encoding a peptide or protein, and the mRNA further comprises a 3'UTR sequence and a PolyA sequence.

10. The mRNA according to claim 9, characterized in that The peptide or protein can encode hFIX, SPB, VEGF-A, human methylmalonyl CoA variant enzyme, CFTR, cancer autoantigen, gene editing enzyme, HIV antibody, CD19 antibody, CD22 antibody, CD3 antibody, CLDN6 antibody, Luc2, GLuc, Fluc, eGFP, hEPO, Cas9 nuclease, new coronavirus S protein or tumor suppressor.

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