3apos that promotes the expression of RNA; uTR sequence and application thereof in mRNA drugs

Abstract

The invention relates to a 3 'UTR (Untranslated Region) sequence for promoting RNA (Ribonucleic Acid) expression and application of the 3' UTR sequence in mRNA (Messenger Ribonucleic Acid) medicines. The 3 'UTR sequence comprises polynucleotide containing any one of SEQ ID NO.1-37. The 3 'UTR provided by the invention has a stronger function of promoting mRNA expression. The 3 'UTR fragment provided by the invention plays a role in promoting translation in different types of host cells, has broad spectrum in action, and shows an application prospect in RNA (Ribonucleic Acid) therapeutic drugs or mRNA (Messenger Ribonucleic Acid) vaccines. The 3 'UTR obtained through optimization has a better target protein expression effect and can be used for mRNA vaccine development or RNA therapeutic drugs.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering preventive or therapeutic vaccines and drugs, and specifically relates to a 3'UTR molecule disclosed by the invention, and belongs to the field of nucleic acid technology. Background Art

[0002] Messenger RNA (mRNA) therapy is a new treatment method with great potential. The development of messenger RNA drugs is mainly concentrated in the fields of immunotherapy (tumor immunity, preventive vaccines, allergy immunity, etc.), protein replacement therapy, regenerative medicine therapy, etc. Among them, the application of tumor immunity and preventive vaccines in immunotherapy is the most developed and mature track. Compared with protein-based biological preparations, the advantages of messenger RNA therapy include that messenger RNA can synthesize proteins through the body's own cells, without the need for complex protein synthesis and purification processes or production lines; intracellular and membrane-bound proteins can be used as therapeutic targets; it can be quickly industrialized under cell-free GMP conditions, and the cycle from research and development to product is short. However, the application of messenger RNA therapy is limited by factors such as easy degradation in vitro and in vivo environments, short half-life of expressed proteins, and low protein expression, which limits the progress of related drug research and development.

[0003] Messenger RNA (mRNA) consists of five parts: the 5′ cap structure, the 5′ untranslated region (5′ UTR), the protein-coding open reading frame (ORF), the 3′ untranslated region (3′ UTR) and the poly(A) tail. The 3′ untranslated region is located downstream of the mRNA coding region and is usually longer than the 5′ UTR (average length of 1200 nucleotides). In eukaryotes, the natural mRNA 3′ UTR includes cis-regulatory elements, such as AU-rich sequences (Adenylate-uridylate-rich elements, AREs), Alu sequences and miRNA binding sequences, as well as polyadenylation sites. The 3′ UTR of eukaryotic mRNA is closely related to post-transcriptional regulation, and affects the translation efficiency, stability, and mRNA localization of mRNA in the cytoplasm through the binding of other regulatory factors.

[0004] It has been shown that human α- and β-globin 3′-UTRs can improve mRNA stability. α-Globin 3′UTR recruits α-complex, which stabilizes polybinding protein (PABP) to protect the PolyA tail and prevent deadenylation, resulting in a shortened PolyA tail, thereby enhancing mRNA stability and translation efficiency. Moderna's mRNA-1273 uses α-globin 3′UTR. β-Globin is highly enriched in immature red blood cells, and its mRNA half-life can reach 16-48 hours, while most eukaryotic mRNA half-lives only have a few minutes to hours in cells. β-Globin is highly enriched in immature red blood cells, and its mRNA half-life can reach 16-48 hours, while most eukaryotic mRNA half-lives only have a few minutes to hours in cells. Now β-globin mRNA 3′UTR is widely used. In addition, the stability of β-globin mRNA has been clearly dependent on the specific sequence of its 3′UTR. In human dendritic cells (DC), studies have shown that the use of a 2-fold repeat of the 3′UTR sequence of β-globin can further enhance mRNA stability and improve gene expression. BioNTech's patent uses two 3′UTRs of β-globin in series. Orlandini et al. gradually enriched synthetic mRNA with a longer half-life through several cycles of reverse transcriptase: transfection into human dendritic cells (DC), transcription inhibition, and mRNA purification. This method screened out 3′UTR sequences that can increase protein expression of mRNA in DC cells, such as the amino-terminal split enhancer (AES) and mtRNR1 3′UTR. The 3′UTR part of the new crown vaccine BNT162b2 of BioNTech / Pfizer uses AES 3′UTR and mtRNR1 3′UTR in series.

[0005] For synthetic mRNAs, by screening and optimizing the 3′UTR sequence of in vitro transcribed mRNA, selecting the appropriate 3′UTR can enhance the protein expression level of mRNA, thereby increasing the chance of mRNA becoming a drug. Summary of the invention

[0006] The object of the present invention is to provide RNA with improved stability and translation efficiency. The present invention relates to stabilizing RNA, especially mRNA, and improving the translation of mRNA. According to the present invention, it has been shown that novel 3'UTR sequences improve stability and translation efficiency in RNA molecules. The present invention relates to the use of the RNA of the present invention for treatment, especially for vaccination, and vaccine compositions. The stability of mRNA in cells depends to a large extent on the 3'UTR. Therefore, screening for 3'UTR sequences that can enhance the stability of mRNA is an important means to increase the yield 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 3'UTR sequences that stably express mRNA is of great significance for the application of mRNA therapy.

[0007] Based on the above invention, the object is to screen a group of novel 3′UTR sequences that can enhance mRNA stability and thus promote mRNA expression, wherein the 3′UTR sequence includes or contains a polynucleotide of any one of SEQ ID NO.1-37; or

[0008] The 3'UTR sequence comprises a polynucleotide of any one of SEQ ID NOs. 1-37.

[0009] As a preferred technical solution, the 3'UTR includes a sequence of any one of SEQ ID NO.1-37, or a sequence having at least 80% homology with any one of SEQ ID NO.1-37. Preferably, the 3'UTR includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any one of SEQ ID NO.1-37.

[0010] The present invention also provides an expression vector or a host cell comprising or containing the 3'UTR.

[0011] The present invention also provides a DNA or RNA vaccine comprising or containing the 3'UTR.

[0012] The present invention also provides a nucleic acid product for treatment or prevention, or a protein product for treatment or prevention, comprising or containing the 3'UTR.

[0013] The present invention also provides the use of the 3'UTR in the expression of encoded proteins.

[0014] The present invention also provides mRNA, which comprises in the 5′ to 3′ direction: at least one 3′UTR element, the 3′UTR element comprises a polynucleotide containing any one of SEQ ID NOs 1-37, and at least one open reading frame encoding a peptide or protein, the mRNA further comprising a 5′UTR sequence and a PolyA sequence.

[0015] 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.

[0016] The present invention also provides a pharmaceutical composition, comprising the 3'UTR sequence described in the above technical solution and the mRNA described in the above technical solution.

[0017] As a preferred technical solution, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0018] In some specific embodiments of the present invention, the protein coding region or non-coding region of the present invention is Fluc gene and hEPO gene, which verifies the feasibility of the technical solution of the present invention.

[0019] The present invention provides the use of the mRNA molecule in the preparation of RNA therapeutic drugs or mRNA vaccines.

[0020] Compared with the prior art, the advantages of the present invention are: the 3′UTR provided by the present invention has a stronger function of promoting mRNA expression. Compared with the AES-mtRNR UTR sequence used by BioNTech, the insertion of the 3′UTR fragment provided by the present invention increases the expression of luciferase in 293T cells. Compared with the control group, the insertion of 3′UTR-3 and 3′UTR-6 fragments increases the expression of luciferase in 293T cells by about 2 times. Compared with the control group, the insertion of the 3′UTR fragment provided by the present invention increases the expression of hEPO protein in A549 cells, and the insertion of 3′UTR-6 and 3′UTR-10 increases by about 2 times compared with the control group. The 3′UTR fragment provided by the present invention plays a translation-promoting function in different types of host cells, has a broad spectrum of action, and shows the application prospects in RNA therapeutic drugs or mRNA vaccines.

[0021] The present invention can be used for the development of DNA vaccines, mRNA vaccines, protein therapeutic drugs and other biological products. The optimized 3′UTR can improve the translation efficiency of the target protein, 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. In addition, it can also be used to regulate the expression of various proteins and be used to treat various diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the electrophoresis diagram of Luciferase-3′UTR plasmid;

[0023] Figure 2 Agarose electrophoresis diagram of Luciferase-3′UTR plasmid after linearization and purification;

[0024] Figure 3 is the agarose electrophoresis diagram of Luciferase-3′UTR-mRNA;

[0025] Figure 4 is the electrophoresis diagram of hEPO-3′UTR plasmid;

[0026] Figure 5 This is the agarose electrophoresis diagram of hEPO-3′UTR plasmid after linearization and purification;

[0027] Figure 6 is the agarose electrophoresis diagram of hEPO-3′UTR-mRNA;

[0028] Figure 7 Optimize fluorescence expression results for 3′UTR sequence;

[0029] Figure 8 Figure 3 shows the hEPO expression results for 3′UTR sequence optimization. DETAILED DESCRIPTION

[0030] 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.

[0031] Example 1. Sequence design of 3′UTR

[0032] AU-rich sequences (Adenylate-uridylate-rich elements, AREs) in the 3′UTR of mRNA can induce translational inhibition, dealkenylation (poly-A tail shortening), decapping or cleavage, and lead to mRNA decay. The presence of adenylate / uridylate (AU)-rich sequences (AREs) in the natural 3′UTR is associated with the regulation of mRNA stability and is a cis-acting sequence that activates rapid mRNA decay. Tristetraprolin (TTP) and its paralogs, butyrate response factors 1 and 2 (BRF-1 / 2), bind to AU-rich elements through their two zinc finger domains and promote mRNA degradation. The number and position of AUUUA repeats on the ARE have a key influence on the shortening of the poly(A) tail and RNA degradation, while another GU-rich sequence (GRE) in this region can bind to the CELF1 protein in mammalian cells to accelerate mRNA decay. Some AU-rich elements are mainly located in a certain class of mRNAs, which mainly encode short-term factors including cytokines, lymphokines, growth factors and oncogenes. MiRNAs usually target miRNA binding sequences in the 3′UTR of mRNAs to promote mRNA decay. It is generally believed that shorter 3′UTRs are more conducive to transcript stability, which may be due to the lack of microRNA (miRNA) binding sites, thereby avoiding mRNA degradation. Studies have shown that compared with similarly proliferating untransformed cell lines, cancer cell lines usually express a large number of mRNA isoforms with shorter 3′UTRs. These shorter isoforms are usually caused by alternate cleavage and polyadenylation (APA). These shorter mRNA isoforms show higher stability and typically produce ten times more protein, partly due to the loss of miRNA-mediated repression. MiRNAs or binding sites for miRNAs as defined above can be removed from the 3′-UTR or introduced into the 3′-UTR to customize the expression of nucleic acids.

[0033] When designing IVT mRNA, it is necessary to predict and exclude such sequences that interfere with mRNA stability. Online software such as TargetScan 7.2 (http: / / www.targetscan.org / vert_72 / ), miRanda (http: / / www.microma.org / microma / home.do), DIANATools (http: / / diana.imis.athena-innovation.gr / DianaTools / index.php?r=site / page&view=software), miRDB (http: / / www.mirdb.org / miRDB / ), star Base (http: / / starbase.sysu.edu.cn / index.php) and miRWalk (http: / / mirwalk.umm.uni-heidelberg.de / ) can be used to detect the sites targeted by 3′UTR miRNA.

[0034] When designing 3'UTR sequences, 3'UTRs of genes that may have high expression efficiency or large protein molecular weight were screened from the UTR database UTRdb (http: / / utrdb.ba.itb.cnr.it / ) or AURA (http: / / aura.science.unitn.it / about / ), and the 3'UTR miRNA-targeted sites were detected through the website to find AU-rich sequences (AREs) and GU-rich sequences (GREs), and avoid or delete AU-rich sequences (AREs) and GU-rich sequences (GREs) on the 3'UTR. Table 1 lists the novel 3'UTR sequences produced and tested in this study.

[0035] The 3′UTR structure further screened the 3′UTR sequence and conducted subsequent experiments based on the effect of the 3′UTR structure on the translation and stability of mRNA. Control-BioNTech was used as the control, and the sequence was selected from the source AES-mtRNR1. The sequence information is as follows.

[0036] cTggTacTgcaTgcacgcaaTgcTagcTgccccTTTcccgTccTgggTaccccgagTcTcccccgaccTcgggTcccaggTaTgcTcccaccTccaccTgccccacTcaccaccTcTgcTagTTccagacaccTcccaa gcacgcagcaaTgcagcTcaaaacgcTTagccTagccacacccccacgggaaacagcagTgaTTaaccTTTagcaaTaaacgaaagTTTaacTaagcTaTacTaaccccagggTTggTcaaTTTcgTgccagccacacc

[0037] Table 1 shows the 3′UTR sequence of the present invention.

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048] Example 2, plasmid construction

[0049] Firefly Luciferase (hereinafter referred to as FLuc) and hEPO were used as reporter genes, and the 3′UTR sequence designed in Example 1 was used in combination with the T7 promoter to construct an expression module. Then, primers were designed and synthesized. Primers were designed using Snapgene software and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0050] 1. Amplification of target fragment and construction of recombinant plasmid.

[0051] (1) Prepare the reaction system according to the following table:

[0052] Components volume <![CDATA[RNase-free H2O]]> Up to 50ul 2x Buffer 25ul Primers 1ul each dNTP 1ul Phanta max super-fidelity DNA polymerase 1ul

[0053] (2) Perform PCR amplification according to the following table for 30 cycles:

[0054]

[0055]

[0056] (3) Gel excision, purification and recovery of target DNA fragment

[0057] (4) Vector digestion, and the reaction system was prepared as shown in the table below;

[0058] Components volume pDNA 5ug SacI 2ul BglII 2ul 10x Buffer 5ul <![CDATA[RNase-free H2O]]> Up to 50ul

[0059] (5) Double enzyme digestion of vector DNA, gel excision, purification and recovery;

[0060] (6) Prepare the reaction system according to the table below and carry out the recombination reaction;

[0061] 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

[0062] (7) Place the above recombination reaction system in a water bath and incubate at 50°C for 30 min.

[0063] 2. Recombinant plasmid transformation and plating

[0064] (1) Add 10ul of the recombinant product to 100ul of DH5a competent cells and incubate on ice for 30min;

[0065] (2) Heat shock in a 42°C water bath for 90 seconds, then remove and place in an ice bath for 3 minutes;

[0066] (3) Add 600 μl of antibiotic-free LB medium and incubate at 37°C in a shaker for 1 h;

[0067] (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;

[0068] (5) Place in a constant temperature incubator at 37°C for 16-20 hours;

[0069] (6) Pick a single clone and place it in a shaking incubator and culture at 37°C for 16-17 h.

[0070] 3. Plasmid extraction and identification

[0071] (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;

[0072] (2) Send the plasmid for sequencing to determine whether the 3′UTR sequence is correct. Figure 1 This is the electrophoresis diagram of Luciferase-3′UTR plasmid. Figure 4 This is the electrophoresis diagram of hEPO-3′UTR plasmid.

[0073] 4. Plasmid Linearization and Purification

[0074] (1) Prepare HindIII single enzyme digestion system as shown in the table below:

[0075] Components <![CDATA[RNase-free H2O]]> Up to 100ul 10×Buffer 10ul HindIII 12ul pDNA 10ug

[0076] (2) The above reaction solutions were mixed evenly, centrifuged briefly, incubated at 37°C for 2.5 h, and then purified by column. Figure 2 As shown in the agarose electrophoresis diagram of Luciferase-3′UTR plasmid after linearization and purification, Figure 5 As shown, agarose electrophoresis of hEPO-3′UTR plasmid after linearization and purification

[0077] 5. Co-transcriptional capping experiment

[0078] (1) Prepare the co-transcription capping system according to the following table:

[0079] 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 ——

[0080] (2) The reaction mixture was mixed evenly, centrifuged briefly, and incubated at 37°C for 2 h;

[0081] (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;

[0082] (4) RNA purification was performed using lithium chloride precipitation. Figure 3 This is the agarose electrophoresis diagram of Luciferase-3′UTR-mRNA. Figure 6 This is the agarose electrophoresis diagram of hEPO-3′UTR-mRNA.

[0083] Example 3: Optimization of 3′UTR sequence to regulate the translation efficiency of FLuc gene

[0084] Transfect 293T cells when they are cultured to 50-60% confluence, dilute Lipofectamine 3000 reagent with MEM medium according to the design instructions, mix thoroughly, dilute mRNA to a certain concentration with MEM medium, prepare mRNA premix, mix thoroughly, add diluted mRNA to each tube of diluted Lipofectamine 3000 reagent (1:1 ratio). Incubate at room temperature for 15 minutes, add mRNA-liposome complex to the cells, culture the infected 293T cells in a CO2 incubator, and detect mRNA expression after 16 hours of culture. Figure 7 As shown, Control-BioNTech is the 3′UTR of Biontech. Compared with the 3′UTR of Biontech, the sequence significantly improved the translation efficiency of the coding sequence. Figure 7 The results showed that the new 3′UTR sequence had a higher translation efficiency than the control sequence. The insertion of 3′UTR-3 and 3′UTR-6 fragments increased the luciferase expression in 293T cells by about 2 times.

[0085] Example 4: 3′UTR sequence optimization to regulate hEPO protein expression.

[0086] A549 cells were seeded in a 24-well culture plate at a number of 10,000 cells per well and cultured overnight in an incubator at 37°C and 5% CO2. Lipo3000 and 1200ng of RNA were diluted with Opti-MEM medium (or serum-free DMEM medium) respectively; the diluted Lipo3000 and RNA were directly mixed at room temperature, and incubated at room temperature for 10 minutes after mixing evenly; after discarding the medium from the cells cultured overnight, the mixture of incubated Lipo3000 and RNA was added, and Opti-MEM medium (or serum-free DMEM medium) was supplemented to 1mL, and cultured in an incubator at 37°C and 5% CO2 for 4h. After transfection, the cells discarded the medium, added 1ml of complete medium, and continued to culture for 18-20h. After the culture was completed, the cell culture supernatant was aspirated, and the concentration of hEPO protein in the supernatant was detected using the hEPO Elisa detection kit. Figure 8 As shown, Control-BioNTech is the 3′UTR of Biontech. Compared with the 3′UTR of Biontech, the sequence significantly improved the translation efficiency of the coding sequence. Figure 8 All of them showed higher translation efficiency than the control sequence, and the insertion of 3′UTR-6 and 3′UTR-10 increased the protein expression by about 2 times compared with the control group.

Claims

1. A set of 3'UTR sequences that promote RNA expression, characterized in that: This includes a polynucleotide containing any one of SEQ ID NOs. 1-37.

2. The 3'UTR sequence of claim 1, wherein the 3'UTR sequence comprises a sequence of any one of SEQ ID NOs. 1-37, or a sequence having at least 80% homology to any one of SEQ ID NOs. 1-37.

3. An expression vector, host cell, DNA or RNA vaccine comprising the 3'UTR of claim 1.

4. A nucleic acid product for treatment or prevention, or a protein product for treatment or prevention comprising the 3'UTR of claim 1.

5. Use of the 3'UTR of claim 1 in the expression of encoded proteins.

6. mRNA, characterized in that It comprises in the 5' to 3' direction: at least one 3'UTR element, the 3'UTR element comprises a polynucleotide containing any one of SEQ ID NOs 1-37, and at least one open reading frame encoding a peptide or protein, and the mRNA further comprises a 5'UTR sequence and a PolyA sequence.

7. A pharmaceutical composition, characterized in that The method comprises the 3'UTR sequence according to claims 1 to 5 and the mRNA according to claim 6.

8. The mRNA according to claim 7, characterized in that The pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

9. Use of the mRNA molecule according to claim 7 or 8 in the preparation of RNA therapeutic drugs or mRNA vaccines.

Images