A 5'UTR sequence and its application in improving mRNA translation efficiency

Abstract

The present invention discloses a 5'UTR sequence and its application in improving mRNA translation efficiency. The present invention belongs to the field of biotechnology. The 5'UTR sequence designed in the present invention has an excellent function of promoting mRNA expression. Compared with the commonly used HBA1 gene 5'UTR, the expression level of FLuc mRNA fused with the 5'UTR in the present invention can be increased by 2 to 3.5 times.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a 5'UTR sequence and an application thereof in improving mRNA translation efficiency. Background Art

[0002] In recent years, nucleic acid drugs, represented by DNA and RNA, have gradually become a hot topic in precision medicine and disease treatment. Compared with traditional drugs, nucleic acid drugs offer advantages such as a rich selection of candidate targets, simplified design, shortened development cycles, strong target specificity, high expression efficiency, and prolonged duration of action. Nucleic acid drugs can be broadly divided into two categories: DNA drugs and RNA drugs. Compared with DNA drugs, RNA drugs have a lower risk of genomic integration and induced mutagenesis, higher expression efficiency, and no risk of cumulative toxicity, offering broader application prospects in disease prevention and treatment.

[0003] A complete mRNA molecule includes a 5' cap structure, a 5' untranslated region (5'UTR), an antigen-coding sequence, a 3' untranslated region (3'UTR), and a polyadenylation tail. In addition to the antigen-coding sequence, other structural elements are crucial to the stability and transcription efficiency of mRNA. Among them, the 5'UTR is the main site of ribosome assembly during mRNA translation. The main obstacle to the development of UTR sequences is the difficulty in predicting the impact of any UTR sequence, because some of the cis-acting elements can affect multiple molecular processes through interactions with RNA-binding proteins and microRNAs, and the same UTR sequence can even be expressed differently in different tissues. As the main determinant of translation efficiency, engineering optimization of the 5'UTR is very necessary. Summary of the Invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0005] The present invention provides a 5'-UTR element, which is used to construct an mRNA template and improve the translation efficiency of the coding region in the mRNA transcribed from the mRNA template. The sequence of the 5'-UTR element is shown in SEQ ID NO: 1-9.

[0006] Furthermore, the sequence of the 5'-UTR element is shown in SEQ ID NO: 1, 2, and 9.

[0007] The present invention provides a nucleotide construct comprising a nucleotide sequence encoding the aforementioned 5'-UTR element.

[0008] Furthermore, the nucleotide construct further comprises any one or more of an enzyme cleavage site and a promoter element.

[0009] In some embodiments, a "restriction site" refers to a specific sequence of bases on DNA that a restriction endonuclease can recognize and cut into two segments.

[0010] In some embodiments, a "promoter element," "promoter," or "promoter sequence" refers to a DNA sequence located at the 5' end (i.e., before) of the protein coding region of a DNA polymer. Most naturally known promoters are located before the transcription region. Promoters act as switches for gene expression, activating gene expression. If a gene is activated, it is said to be transcribed, or to participate in transcription. Transcription involves the synthesis of mRNA from a gene. Thus, a promoter acts as a transcriptional regulatory element and also provides a start site for transcription of a gene into mRNA. A promoter may be derived in its entirety from a natural gene, or include different elements derived from different promoters found in nature, or even include synthetic DNA fragments. It is understood by those skilled in the art that different promoters can direct gene expression in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is also recognized that, since the exact boundaries of regulatory sequences have not been fully defined in most cases, some variant DNA fragments may have the same promoter activity. Promoters that cause a gene to be expressed in most cell types at most times are generally referred to as "constitutive promoters." New and diverse types of promoters that can be used in plant cells are constantly being discovered.

[0011] The present invention provides an mRNA construct, wherein the construct has the above-mentioned 5'-UTR element.

[0012] Furthermore, the mRNA construct has the following structure: a 5'-UTR element, an alternative coding region, and a 3'-UTR element are sequentially connected.

[0013] Furthermore, the mRNA construct further comprises one or more of an IRES sequence and a polyA element.

[0014] In some embodiments, an "IRES sequence" refers to an internal ribosome entry site that is capable of causing ribosomes to bind to the interior of an RNA to initiate translation.

[0015] In some embodiments, the polyA element refers to a "polyA tail," or a poly(A) sequence, which includes the structure of the poly(A) tail region on an mRNA or the structure corresponding to the coding sequence on a DNA template. The addition of a poly(A) sequence contributes to the stability and transport of mRNA, prevents its degradation, and plays an important role in post-transcriptional modification. This poly(A) sequence can be a continuous chain of pure adenine nucleotides or contain non-adenine nucleotides. In any form, as long as it is functionally equivalent to a traditional poly(A) sequence, that is, it can provide similar biological functions as traditional poly(A) sequences, such as affecting mRNA stability, translation efficiency, or ribosome binding, the sequence is considered a poly(A) sequence. This includes, but is not limited to, known variants such as the human growth hormone (hGH) poly(A) sequence and the simian virus 40 (SV40) poly(A) sequence, which may differ in nucleotide composition but are considered functionally equivalent to traditional poly(A) sequences. In the present disclosure, the poly A sequence has a length of 20-500 adenine nucleotides, for example, a length of 25, 50, 100, 150, 175, 200, 300, 400 or 500 adenine nucleotides.

[0016] In some embodiments, the polypeptide or protein encoded by the alternative coding region is used to prevent and / or treat infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathy, cancer or tumors.

[0017] The term "infectious diseases" in the present invention includes diseases caused by various organisms, such as bacteria, mycoplasmas, protozoa, fungi and viruses (such as HIV, hepatitis viruses, especially HBV or HCV).

[0018] In some embodiments, the cancer or tumor type includes but is not limited to colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, endometrial uterine cancer, ependymoma, epithelioid hemangioendothelioma (EHE), esophageal cancer, Ewing's sarcoma family of tumors, Ewing's sarcoma in the Ewing's tumor family, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, gallbladder cancer, gastric (stomach) cancer, Carcinoma, gastric carcinoid, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, brain stem glioma, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, acute lymphoblastic leukemia (also known as acute lymphoblastic leukemia), acute myeloid leukemia (also known as acute myeloid leukemia) , chronic lymphocytic leukemia (also known as chronic lymphocytic leukemia), leukemia, chronic myeloid leukemia (also known as chronic myeloid leukemia), hairy cell leukemia, lip and oral cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphoma (AIDS-related), lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanoma, Merkel cell carcinoma , squamous cell carcinoma of the neck with occult metastasis from the primary site, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma (bone marrow cancer) in children, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative diseases, chronic myeloid leukemia, myxoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer.

[0019] In some embodiments, the neurodegenerative disease includes but is not limited to neurological disorders (e.g., migraine; epilepsy; Alzheimer's disease; Parkinson's disease; brain injury; stroke; cerebrovascular disease (including cerebral arteriosclerosis, cerebral amyloid angiopathy, hereditary cerebral hemorrhage and cerebral hypoxia-ischemia); spinal muscular atrophy; lateral sclerosis; multiple sclerosis; cognitive disorders (including amnesia, senile dementia, HIV-related dementia, Alzheimer's disease-related dementia, Huntington's disease-related dementia, Lewy body dementia, vascular dementia, drug-induced dementia, delirium and mild cognitive impairment); cognitive dysfunction associated with Parkinson's disease and depression; mental deficits (including Down syndrome and fragile X syndrome); psychosis (including schizophrenia (e.g., continuous or episodic, paranoid, juvenile, catatonic, poorly differentiated and residual schizophrenia), schizoaffective disorder, schizophreniform psychosis and delusional disorder).

[0020] In some embodiments, the construct or sequence can be modified at the level of the nucleic acid linkage (e.g., phosphorothioate, H-phosphate, alkyl phosphate), at the level of the backbone (e.g., α-oligonucleotide or PNA or 2′-O-alkyl ribose). Each of these modifications can occur in combination, as long as at least one phosphate is present in the nucleic acid. The nucleic acid can be natural or synthetic, an oligonucleotide, a polynucleotide, a nucleic acid fragment, a ribosomal RNA, a messenger RNA, a transfer RNA, or a nucleic acid obtained by enzymatic amplification techniques.

[0021] The present invention provides a cell comprising the nucleotide construct as described above.

[0022] The term "cell" as used herein refers to a cell into which a vector containing a polynucleotide sequence encoding an antibody can be introduced for cloning or gene expression. Suitable cells for cloning or expressing the DNA in the vector of the present invention are prokaryotes, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia, e.g., Serratia marcescans; and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces.

[0023] In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for expressing anti-coronavirus N proteins. Saccharomyces cerevisiae or common baker's yeast is the most commonly used lower eukaryotic host microorganism. However, a variety of other genera, species, and strains are generally available and suitable for use in the present invention, such as Schizosaccharomyces pombe; Kluyveromyces hosts, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402 402); and K. ,226); Pichiapastoris (EP 183 ,070); Candida; Trichoderma reesia (EP 244 ,234); Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium and Aspergillus hosts, such as A. nidulans and A. niger.

[0024] Vertebrate cells have also attracted considerable interest, and propagating vertebrate cells in culture (tissue culture) has become routine. Examples of suitable mammalian host cell lines are monkey kidney CV1 strain transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR; mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human liver tumor line (Hep G2).

[0025] The present invention provides a method for producing optimized mRNA for preparing mRNA drugs, comprising the steps of: culturing the cells as described above to obtain a cell culture containing a nucleotide construct containing a 5'-UTR element; isolating the nucleotide construct from the cell culture, and transcribing the nucleotide construct to obtain optimized mRNA.

[0026] Furthermore, the optimized mRNA is purified and / or modified.

[0027] The present invention provides an optimized mRNA, which is prepared by the method described above. The optimized mRNA structure is as follows: a 5'-UTR element, a replaceable coding region, and a 3'-UTR element are sequentially connected.

[0028] Furthermore, the optimized mRNA also includes elements and / or modifications that stabilize the mRNA.

[0029] The present invention provides a method for preparing an mRNA pharmaceutical composition, which comprises: mixing the optimized mRNA as described above with a pharmaceutically acceptable adjuvant to obtain the mRNA pharmaceutical composition.

[0030] The present invention provides a pharmaceutical composition comprising the aforementioned mRNA construct or the aforementioned optimized mRNA, and a pharmaceutically acceptable adjuvant.

[0031] Pharmaceutically acceptable adjuvants used in the compositions of the present invention may include, but are not limited to, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles (e.g., sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection or Ringer's dextrose and lactate injection), non-aqueous vehicles (e.g., fixed oils of plant origin, cottonseed oil, corn oil, sesame oil or peanut oil), antimicrobial agents, isotonic agents (e.g., sodium chloride or dextrose), buffers (e.g., phosphate or citrate buffer), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending / dispersing agents (e.g., sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifiers (e.g., polysorbate 80 (Tween-80)), diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof. Suitable components may include, for example, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, or emulsifiers.

[0032] The term "pharmaceutically acceptable" as used in the present invention refers to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, and in accordance with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0033] The present invention provides the use of the aforementioned 5'-UTR element, wherein the 5'-UTR element is used to construct an mRNA construct, thereby improving the mRNA translation efficiency.

[0034] The present invention provides a method for preparing a polypeptide or protein, comprising: replacing the 5'-UTR element of an mRNA transcription template of the polypeptide or protein with the aforementioned 5'-UTR element, and translating the replaced mRNA transcription template to obtain the polypeptide or protein.

[0035] The term "5'UTR" as used herein generally refers to the sequence from the 5' end of an mRNA molecule to the translation start codon, which is capable of recruiting the ribosome complex and initiating translation of the mRNA. The 5'UTR includes the structure of the 5'UTR region on the mRNA or the structure corresponding to the coding sequence on the DNA template. The 5'UTR regulates post-transcriptional modification, the formation and stability of the translation initiation complex, and other processes by interacting with transcription factors, ribosomes, and other transcriptional regulatory proteins. Sequence design and optimization of this region are crucial for improving the efficiency of post-transcriptional modification and protein expression. As used herein, the terms "5'UTR structure," "5'UTR," "5'UTR sequence," and "5'UTR element" are used interchangeably and refer to 5'UTR elements that can enhance the expression of a target gene, obtained through extensive screening by the inventors. The 5'UTR sequence has a sequence selected from the group consisting of the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-9, or its complementary sequence, or a nucleic acid sequence that has at least 80% homology to the nucleotide sequence set forth in any one of SEQ ID NOs: 1-9, or its complementary sequence. Furthermore, the 5'UTR sequence of the present invention has the nucleic acid sequence shown in SEQ ID NO: 1, 2, and 9. The 5'UTR element of the present invention can be used for the design of mRNA molecular structure and DNA molecular template for mRNA therapy, mRNA vaccine and personalized immunotherapy to improve translation efficiency and enhance the expression of target genes.

[0036] The terms "polypeptide", "peptide", and "protein" used in the present invention are used interchangeably to refer to polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, gamma carboxylation, hydroxylation and ADP-ribosylation of glutamic acid residues. The polypeptide may be a polypeptide of eukaryotic, prokaryotic or viral origin. In certain embodiments, the polypeptide may be any polypeptide for therapeutic, preventive or diagnostic purposes. For example, the polypeptide may be an antigen, an antibody, a gene editing enzyme such as CRISPR nuclease, etc. The polypeptide may also be a chimeric antigen receptor, an immunomodulatory protein, a transcription factor, etc. Examples of polypeptides include but are not limited to: luciferase, red / green fluorescent protein, human erythropoietin, β-galactosidase.

[0037] As used herein, the term "purified" as used herein refers to a polypeptide that is removed, isolated or separated from its natural environment or from a recombinantly produced source and is at least 60%, more preferably at least 80% free from other components with which it is naturally associated, such as membranes and microsomes.

[0038] The term "genetic modification" or "modification" used in the present invention refers to any insertion, deletion or mutation of a cell's natural nucleotide sequence or nucleic acid sequence.

[0039] Advantages and beneficial effects of the present invention:

[0040] This study leverages existing 5'UTR sequence optimization design models in conjunction with specific CDS sequences to predict and optimize multiple key parameters, thereby designing 5'UTR sequences with higher translation efficiency and improved stability. The nucleic acid molecules described herein can be used as components for enhancing RNA expression levels in nucleic acid therapeutics or mRNA vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a schematic diagram of the backbone vector for Fluc mRNA in vitro transcription.

[0042] Figure 2 The figure shows the expression level of FLuc mRNA in different cells transfected with various 5'UTRs. DETAILED DESCRIPTION

[0043] Example 1, 5'UTR sequence design

[0044] Although the 5'UTR does not encode proteins, it regulates the initiation of mRNA translation and is crucial for protein synthesis. Optimizing the 5'UTR can improve mRNA translation efficiency, increase tumor antigen production, enhance immune responses, and enhance mRNA stability and duration of action, making it crucial for improving the efficacy of therapeutic mRNA vaccines for tumors. Conventional 5'UTR optimization strategies involve screening the 5'UTRs of highly expressed genes within cells, such as the 5'UTR of the commonly used α-globin gene HBA1. With the advancement of computer technology, several algorithmic models have been developed to predict the translation efficiency and stability of mRNA sequences, and even programs have emerged that can generate 5'UTR sequences from scratch that improve translation efficiency.

[0045] These algorithm models generally optimize 5'UTR sequences through the following strategies: (1) adding KOZAK sequences that can improve translation initiation efficiency; (2) by predicting and optimizing the minimum free energy (MFE) of the 5'UTR sequence, ensuring that the 5'UTR has a moderately stable secondary structure, thereby improving the stability of the mRNA; (3) based on the concept of overall functional optimization of mRNA, a joint optimization algorithm is adopted, while considering the interaction between the 5'UTR and CDS. The optimization process includes balancing parameters such as translation initiation efficiency (TIE), codon adaptation index (CAI) and minimum free energy (MFE), and achieving global sequence optimization through multiple rounds of iteration; (4) with the help of artificial intelligence models such as UTR-LM, combined with large-scale training data sets, the sequence characteristics of the 5'UTR and CDS (such as GC content, AUG context structure and secondary structure characteristics) are analyzed, and multiple key parameters (such as TIE, CAI and MFE) are optimized through model prediction, thereby generating 5'UTR sequences with higher translation efficiency and better stability.

[0046] Using the above algorithm strategy, our laboratory designed 5'UTR sequences of approximately 30, 50, 70, and 80 bp in length, and screened out several 5'UTR sequences with high scores. The resulting 5'UTR sequence information is shown in Table 1.

[0047] Table 1 Optimized 5'UTR sequence information

[0048]

[0049] Example 2: 5'UTR vector construction and mRNA molecule preparation

[0050] The designed 5'UTR sequence was synthesized in full length by gene synthesis (GenScript Biotech Co., Ltd.) and ligated into the pUC57 vector backbone containing the T7 promoter sequence, HBA1 5'UTR sequence, FLuc CDS sequence, hBg 3'UTR sequence, and poly(A) sequence. Figure 1 The pIPMKC4-FLuc backbone in the plasmid was inserted into the HBA1 5'UTR, replacing the original 5'UTR sequence. After sequencing confirmed the correctness of the inserted 5'UTR sequence, it was purified using an endotoxin-free plasmid extraction kit (Viglas Biotechnology Co., Ltd.), and its concentration was measured using a Nanodrop spectrophotometer. The resulting plasmid was then used to produce mRNA via in vitro co-transcription.

[0051] Take 5µg of the extracted plasmid and use the restriction endonuclease BspQI (NEB) to linearize the plasmid template. Add 2µL of the endonuclease to 10× Reaction Buffer and RNase-free water to prepare a total volume of 50µL. After thorough vortexing and mixing, centrifuge briefly to allow the liquid to accumulate at the bottom of the test tube. React at 50℃ for 3h, then inactivate at 80℃ for 20min. The obtained product is further purified and recovered using magnetic beads.

[0052] Prepare DNA magnetic beads (VAHTSⓇ DNA Clean Beads) in advance, equilibrate to room temperature, and thoroughly mix by inversion or vortexing. Add an equal volume of magnetic beads based on the volume of the digested product. Pipette thoroughly to mix. Then, proceed with template plasmid purification according to the magnetic bead recovery procedure. After purification, measure the concentration of 1µL of the purified product using a Nanodrop spectrophotometer. Then, perform 50ng of the purified product on agarose gel electrophoresis (1% TAE) to confirm adequate linearization of the plasmid template.

[0053] In this example, in vitro transcription was performed using the T7 RNA polymerase co-transcription kit (Novozymes Biotech Co., Ltd.). After adding all components according to the instructions, the mixture was thoroughly pipetted and mixed. The liquid was collected at the bottom of the tube by brief centrifugation and incubated at 37°C for 2 hours. Then, 5 µL of DNase I was added to the reaction product to digest the DNA template, and the reaction was continued at 37°C for 15 minutes. The mRNA in the transcription product was purified and recovered using RNA magnetic beads (VAHTS RNA Clean Beads) at a dosage of 2-4 times that of the co-transcription system. After thorough mixing, the beads were processed according to the instructions. Finally, the product was eluted with an appropriate amount of nuclease-free water. 1 µL of the purified RNA was collected and its concentration was determined using an ultra-micro spectrophotometer (RNA mode). 50 ng of the purified RNA was subjected to denaturing agarose gel electrophoresis (1% TBE + 6% urea) to verify the integrity and purity of the RNA product.

[0054] Example 3: Effects of different 5'UTRs on the expression level of firefly luciferase in HepG2 and DC2.4 cells

[0055] In order to verify the effect of the designed 5'UTR sequence on the expression level of firefly luciferase in different cells, the present invention selected immortalized human hepatoma cell line (HepG2) and mouse bone marrow-derived dendritic cell line (DC2.4) for transfection test. HepG2 or DC2.4 cells with good growth status were digested, resuspended and counted, and 2×10 6Add 10 cells to a 50 mL centrifuge tube, add RPMI 1640 complete medium (Thermo Fisher Scientific) to make up to 20 mL, and adjust the cell density to 1 × 10 5 After thorough mixing, the cells were evenly distributed into 96-well white plates (Corning Company), 100 μL / well, and the number of cells plated per well was 1×10 4 The cells were gently tapped to mix, then cultured overnight at 37°C (18–24 hours). The next day, the cells were removed and transfected with Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) at a dose of 0.1 μg / well of the FLuc mRNAs prepared above, each containing a different 5'UTR. The commonly used HBA1 gene 5'UTR (HBA1 group) served as a control group, and the transfection procedures were performed according to the manufacturer's instructions.

[0056] 24h and 48h after transfection, the corresponding well plates were removed for luciferase activity detection. The Bio-LiteLuciferase Assay System kit (Novozymes Biotech Co., Ltd.) was removed in advance and returned to room temperature. The kit was prepared according to the instructions. The well plates to be tested were removed, the culture supernatant was removed with a pipette, and 100μL of detection reagent was added to each well. The wells were gently tapped to mix, and the wells were allowed to stand for 1-2 minutes to allow the reaction to proceed fully. The wells were then placed on a microplate reader (BioTek, Model Synergy H1) for measurement. The test results were as follows: Figure 2 shown.

[0057] from Figure 2 The results show that in HepG2 and DC2.4 cells, the expression levels of FLuc mRNA in most of the fused 5'UTR sequences designed by the present invention were higher than those in the control group. The best expression effect was achieved by denovo80-4, whose expression levels in HepG2 and DC2.4 cells were 2.2 times and 3.5 times that of the control group, respectively. Among these de novo designed 5'UTR sequences, the transfection results did not find a significant correlation between the length of the UTR and the expression effect, indicating that the effect of the 5'UTR on the mRNA translation efficiency is affected by multiple factors. Figure 2 The significant differences were shown in Table 2.

[0058] Table 2 Figure 2 Results of data difference analysis

[0059]

[0060]

Claims

1. A 5'-UTR element, characterized in that The 5'-UTR element is used to construct an mRNA template and improve the translation efficiency of the coding region in the mRNA transcribed from the mRNA template. The sequence of the 5'-UTR element is shown in SEQ ID NO:

1.

2. A nucleotide construct, characterized in that The nucleotide construct comprises a nucleotide sequence encoding the 5'-UTR element of claim 1.

3. The nucleotide construct according to claim 2, characterized in that The nucleotide construct further comprises any one or more of an enzyme cutting site and a promoter element.

4. An mRNA construct, characterized in that The construct has the 5'-UTR element of claim 1.

5. The mRNA construct according to claim 4, characterized in that The mRNA construct has the following structure: Sequential ligation of the 5'-UTR element, the alternative coding region, and the 3'-UTR element.

6. A cell, characterized in that The cell contains the nucleotide construct of claim 2 or 3.

7. A method for producing optimized mRNA for preparing mRNA drugs, characterized in that The method comprises the steps of: Cultivating the cell according to claim 6 to obtain a cell culture containing a nucleotide construct of a 5'-UTR element; isolating the nucleotide construct from the cell culture, and transcribing the nucleotide construct to obtain optimized mRNA.

8. The method according to claim 7, characterized in that The optimized mRNA is purified and / or modified.

9. An optimized mRNA, characterized in that The optimized mRNA is prepared by the method according to claim 7 or 8, and the optimized mRNA structure is as follows: Sequential ligation of the 5'-UTR element, the alternative coding region, and the 3'-UTR element.

10. The optimized mRNA according to claim 9, characterized in that The optimized mRNA also includes elements and / or modifications that stabilize the mRNA.

11. A method for preparing an mRNA pharmaceutical composition, characterized in that: The method comprises: mixing the optimized mRNA according to claim 9 or 10 with a pharmaceutically acceptable adjuvant to obtain the mRNA pharmaceutical composition.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the mRNA construct according to claim 4 or 5 or the optimized mRNA according to claim 9 or 10, and a pharmaceutically acceptable adjuvant.

13. The use of the 5'-UTR element according to claim 1, characterized in that The 5'-UTR element is used to construct an mRNA construct, thereby improving the mRNA translation efficiency.

14. A method for preparing a polypeptide or protein, characterized in that: The method comprises: replacing the 5'-UTR element of the mRNA transcription template of the polypeptide or protein with the 5'-UTR element of claim 1, and translating the replaced mRNA transcription template to obtain the polypeptide or protein.

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