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

Abstract

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

Description

[0001] This application is a divisional application of the invention entitled "A 5'UTR sequence and its application in improving mRNA translation efficiency", filed on February 6, 2025, with application number 202510132465.0. Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to a 5'UTR sequence and its application in improving mRNA translation efficiency. Background Technology

[0003] 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 have advantages such as abundant candidate targets, simple design, short development cycle, strong targeting specificity, high expression efficiency, and long 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 mutation induction, higher expression efficiency, and no risk of continuous cumulative toxicity, making them more promising for disease prevention and treatment.

[0004] A complete mRNA molecule consists of a 5' cap, a 5' untranslated region (5'UTR), an antigen-coding sequence, a 3' untranslated region (3'UTR), and a polyadenylated tail. Except for the antigen-coding sequence, the other structural elements are crucial for mRNA stability and transcription efficiency. Among these, the 5'UTR is the main site of ribosome assembly during mRNA translation. The most significant obstacle in UTR sequence development is the difficulty in predicting the impact of arbitrary UTR sequences, as some cis-acting elements can influence multiple molecular processes through interactions with RNA-binding proteins and microRNAs. Even the same UTR sequence can exhibit different expressions in different tissues. As the 5'UTR is a major determinant of translation efficiency, its engineering optimization is essential. Summary of the Invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0006] This 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 selected from the nucleotide sequence shown in SEQ ID NO:3 or its complementary sequence.

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

[0008] Furthermore, the nucleotide construct also includes one or more of the following: an enzyme cleavage site and a promoter element.

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

[0010] In some implementations, "promoter element," "promoter," or "promoter sequence" refers to the 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 a switch to activate gene expression. If a gene is activated, it is said to be transcribed, or involved in transcription. Transcription involves the synthesis of mRNA from a gene. Therefore, promoters serve as transcriptional regulatory elements and also provide a starting site for the transcription of a gene into mRNA. Promoters may be derived entirely from a natural gene, or may comprise different elements derived from different naturally found promoters, or may even comprise 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, because the exact boundaries of the regulatory sequence are not fully defined in most cases, some variant DNA fragments may have the same promoter activity. Promoters that cause gene expression in most cell types most of the time are generally referred to as "constitutive promoters." Many new types of promoters that can be used in plant cells are constantly being discovered.

[0011] This invention provides an mRNA construct having the aforementioned 5ˋ-UTR element.

[0012] Furthermore, the mRNA construct has the following structure: a 5ˋ-UTR element, a replaceable coding region, and a 3ˋ-UTR element sequentially linked together.

[0013] Furthermore, the mRNA construct also includes one or more of the IRES sequence and polyA element.

[0014] In some implementations, the "IRES sequence" refers to an internal ribosome entry site that enables ribosomes to bind to the RNA interior in order to initiate translation.

[0015] In some implementations, the polyA element refers to a "polyA tail," or polyA sequence, which includes a polyA tail region structure on mRNA or a structure corresponding to a coding sequence on a DNA template. The addition of a polyA sequence contributes to mRNA stability and transport, 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 may contain non-adenine nucleotides. In any form, a sequence is considered a poly(A) sequence as long as it is functionally equivalent to a conventional poly(A) sequence, i.e., it provides similar biological functions as a conventional poly(A) sequence, such as affecting mRNA stability, translation efficiency, or ribosome binding. 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 functionally considered equivalent to conventional poly(A) sequences. In this disclosure, the polyA sequence has a length of 20-500 adenine nucleotides, for example, 25, 50, 100, 150, 175, 200, 300, 400 or 500 adenine nucleotides.

[0016] In some embodiments, the polypeptide or protein encoded by the replaceable coding region is used for the prevention and / or treatment of infectious diseases, rare genetic diseases, neurodegenerative diseases, retinal diseases, cancer, or tumors.

[0017] In this invention, the term "infectious disease" includes diseases caused by various organisms, such as bacteria, mycoplasma, protozoa, fungi, and viruses (such as HIV, hepatitis viruses, especially HBV or HCV).

[0018] In some implementations, the cancer or tumor types include, but are 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 tumor sarcoma family, Ewing sarcoma within the Ewing tumor family, extracranial germ cell tumors, gonadal germ cell tumors, extrahepatic bile duct cancer, ocular cancer, intraocular melanoma, gallbladder cancer, and gastric cancer. Cancer, gastric carcinoid tumor, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, brainstem glioma, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, 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, primary liver cancer, non-small cell lung cancer, small cell lung cancer, lymphoma (AIDS-related), lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma, melanoma, Merkel cell carcinoma Occult metastatic squamous cell carcinoma of the neck, oral cancer, multiple endocrine neoplasia syndrome, childhood multiple myeloma (bone marrow carcinoma), multiple myeloma / plasma cell vegetation, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, chronic myeloid leukemia, myxoma, nasal and paranasal sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer.

[0019] In some implementations, the neurodegenerative diseases include, but are not limited to, neurological disorders (e.g., migraine; epilepsy; Alzheimer's disease; Parkinson's disease; brain injury; stroke; cerebrovascular diseases (including cerebral arteriosclerosis, cerebral amyloid angiopathy, hereditary cerebral hemorrhage, and cerebral hypoxic-ischemic encephalopathy); spinal muscular atrophy; lateral sclerosis; multiple sclerosis; cognitive impairments (including amnesia, senile dementia, HIV-related dementia, Alzheimer's disease-related dementia, Huntington's disease-related dementia, Lewy body dementia, vascular dementia, drug-related dementia, delirium, and mild cognitive impairment); cognitive impairments 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, hebephrenic, catatonic, dysdifferentiated, and residual schizophrenia), affective schizophrenia, schizophrenia-like psychosis, and paranoia).

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

[0021] The present invention provides a cell containing the nucleotide constructs as described above.

[0022] The term "cell" as used in this invention refers to a cell into which a vector containing a polynucleotide sequence encoding an antibody can be introduced for cloning or gene expression. Cells suitable for cloning or expressing DNA in the vectors of this invention are prokaryotic, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as those in the family Enterobacteriaceae, such as *Escherichia* (e.g., *Escherichia coli*), *Enterobacter*, *Erwinia*, *Klebsiella*, *Proteus*, *Salmonella* (e.g., *Salmonella typhimurium*), and *Serratia* (e.g., *Serratia myxoides*). The genera *Bacillus* include *Bacillus subtilis* and *Bacillus licheniformis*; *Pseudomonas*, such as *P. aeruginosa*; and *Streptomyces*.

[0023] Besides prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable cloning or expression hosts for expressing the anti-coronavirus N protein. Saccharomyces cerevisiae or common baking yeast are among the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and suitable for use in this invention, such as *Schizosaccharomyces pombe*; hosts of the genus *Kluyveromyces*, such as *Kluyveromyces lactis*, *Kluyveromyces fragilis* (ATCC 12,424), *Kluyveromyces bulgaricus* (ATCC 16,045), *Kluyveromyces wickeramii* (ATCC 24,178), *Kluyveromyces waltii* (ATCC 56,500), *Kluyveromyces drosophilarum* (ATCC 36,906), *Kluyveromyces thermomotolerans*, and *Kluyveromyces marxianus*; *Yarrowia* (EP402,226); and *Pichiapastoris* (EP402,226). 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as hosts of Neurospora, Penicillium, Tolypocladium, and Aspergillus, such as Aspergillus nidulans and Aspergillus niger.

[0024] Vertebrate cells have also attracted great attention, and propagating vertebrate cells in cultures (tissue cultures) has become a routine method. Examples of suitable mammalian host cell lines include: SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); young 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 cancer cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human hepatocytes (Hep G2, HB 8065); and mouse mammary tumors (MMT). 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human liver tumor line (Hep G2).

[0025] This invention provides a method for generating optimized mRNA for the preparation of mRNA drugs, the method comprising the steps of: culturing cells as described above to obtain a cell culture containing a nucleotide construct containing a 5ˋ-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] This invention provides an optimized mRNA, which is prepared by the method described above. The optimized mRNA has the following structure: a 5ˋ-UTR element, a replaceable coding region, and a 3ˋ-UTR element sequentially linked together.

[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 drug composition, the method comprising: mixing the optimized mRNA as described above with a pharmaceutically acceptable excipient to obtain the mRNA drug 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 excipients used in the compositions of the present invention may include, but are not limited to, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous mediators (e.g., sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, or Ringer's glucose and lactate injection), non-aqueous mediators (e.g., non-volatile plant oils, 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 buffers), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending / dispersing agents (e.g., sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, 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 excipients, 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 “pharmaceutical acceptable” as used herein refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in human and animal tissue contact without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the limits of reasonable medical judgment, and that meet a reasonable benefit / risk ratio.

[0033] This invention provides the use of the aforementioned 5ˋ-UTR element for constructing mRNA constructs, thereby improving mRNA translation efficiency.

[0034] This invention provides a method for preparing polypeptides or proteins, the method comprising: replacing the 5ˋ-UTR element of the mRNA transcription template of the polypeptide or protein with the aforementioned 5ˋ-UTR element, translating the replaced mRNA transcription template, and obtaining the polypeptide or protein.

[0035] In this invention, the term "5'UTR" generally refers to the sequence of an mRNA molecule from its 5' end to the translation initiation codon, which recruits the ribosome complex and initiates mRNA translation. The 5'UTR includes a 5'UTR region structure on the mRNA or a structure corresponding to a coding sequence on a DNA template. The 5'UTR regulates post-transcriptional modifications, translation initiation complex formation, and stability 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 modifications and protein expression. As used herein, the terms "5'UTR structure," "5'UTR," "5'UTR sequence," and "5'UTR element" are used interchangeably and all refer to 5'UTR elements obtained through extensive screening by the inventors that enhance the expression of target genes. The 5'UTR sequence has a sequence selected from the group consisting of: any one of the nucleotide sequences shown in SEQ ID NO: 1-9 or their complementary sequences, or nucleotide sequences having at least 80% homology with any one of the nucleotide sequences shown in SEQ ID NO: 1-9 or their complementary sequences. Furthermore, the 5'UTR sequence in this invention has the nucleic acid sequences shown in SEQ ID NO: 1, 2, and 9. The 5'UTR element of this invention can be used for the design of mRNA molecular structures and DNA molecular templates for mRNA therapy, mRNA vaccines, and personalized immunotherapy to improve translation efficiency and enhance the expression level of target genes.

[0036] The terms "polypeptide," "peptide," and "protein" are used interchangeably in this invention to refer to polymers of amino acid residues. The term applies 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, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues. Polypeptides can be eukaryotic, prokaryotic, or viral in origin. In some embodiments, a polypeptide can be any polypeptide used for therapeutic, preventative, or diagnostic purposes. For example, a polypeptide can be an antigen, antibody, gene-editing enzyme such as CRISPR nuclease, etc. Polypeptides can also be chimeric antigen receptors, immunomodulatory proteins, transcription factors, etc. Examples of polypeptides include, but are not limited to, luciferase, red / green fluorescent protein, human erythropoietin, and β-galactosidase.

[0037] The term “purified” as used herein refers to a polypeptide that has been removed, isolated, or separated from its natural environment or from a recombinant production source, wherein at least 60%, more preferably at least 80%, of the polypeptide is free from other components naturally associated with it, such as membranes and microsomes.

[0038] In this invention, the term "gene modification" or "modification" refers to any insertion, deletion, or mutation of the natural nucleotide or nucleic acid sequence of a cell.

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

[0040] This invention utilizes existing 5'UTR sequence optimization design models combined with specific CDS sequences to optimize various key parameters through model prediction, thereby designing 5'UTR sequences with higher translation efficiency and better stability. The nucleic acid molecules described in this invention can serve as elements in nucleic acid therapeutic drugs or mRNA vaccines that enhance RNA expression levels. Attached Figure Description

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

[0042] Figure 2 This is a graph showing the results of detecting the expression levels of FLuc mRNA containing various 5'UTRs in different cell transfections. Detailed Implementation

[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 simultaneously improve mRNA stability and prolong its duration of action, which is essential for improving the efficacy of therapeutic mRNA vaccines for tumors. Conventional 5' UTR optimization strategies involve screening the 5' UTRs of highly expressed genes in cells, such as the commonly used 5' UTR of the α-globin gene HBA1. With the development of computer technology, some algorithmic models have been used to predict the translation efficiency and stability of mRNA sequences, and some programs have even emerged that can generate 5' UTR sequences from scratch that improve translation efficiency.

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

[0046] Using the above algorithmic strategies, our laboratory designed 5'UTR sequences with lengths of approximately 30bp, 50bp, 70bp, and 80bp from scratch, and selected some high-scoring 5'UTR sequences. The sequence information of the obtained 5'UTRs is shown in Table 1.

[0047] Table 1. 5' UTR sequence information of the optimized design

[0048]

[0049]

[0050] Example 2: Construction of 5'UTR vector and preparation of mRNA molecules

[0051] The designed 5'UTR sequence was synthesized in full length using gene synthesis (GenScript Biotech Inc.) 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 was inserted, replacing the original HBA15'UTR sequence. After confirmation by sequencing, the inserted 5'UTR sequence was subjected to high-level purification using an endotoxin-free plasmid extraction kit (Vigras Biotechnology Co., Ltd.), and the concentration was determined using a Nanodrop micro-spectrophotometer. The resulting plasmid was then used to prepare mRNA via in vitro co-transcription.

[0052] Take 5 μg of the extracted plasmid, linearize the plasmid template with restriction endonuclease BspQI (NEB), add 2 μL of endonuclease, 10×ReactionBuffer and RNase-free water to prepare a total volume of 50 μL, shake thoroughly to mix, and then perform transient centrifugation to allow the liquid to accumulate at the bottom of the test tube. Incubate at 50℃ for 3 h, then inactivate at 80℃ for 20 min. The obtained product is further purified and recovered using magnetic beads.

[0053] DNA magnetic beads ( Prepare the DNA Clean Beads in advance, equilibrate to room temperature, and thoroughly mix the magnetic beads by inverting or vortexing. Add an equal volume of magnetic beads to the volume of the enzyme digestion product, and mix thoroughly using a pipette. Then, purify the template plasmid according to the magnetic bead recovery procedure. After purification, take 1 μL of the purified product and determine its concentration using a Nanodrop micro spectrophotometer. Then, take 50 ng of the purified product for agarose gel electrophoresis (1% TAE) to confirm whether the plasmid template is sufficiently linearized.

[0054] In this embodiment, in vitro transcription was performed using a T7 RNA polymerase co-transcription kit (Novizan Biotechnology Co., Ltd.). After adding all components as per the instructions, the mixture was thoroughly aspirated and mixed, and the liquid was collected at the bottom of the tube after a brief centrifugation. The mixture was 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 carried out at 37°C for 15 minutes. The mRNA in the transcription product was purified and recovered using VAHTS RNA CleanBeads. The amount of magnetic beads used was 2–4 times that of the co-transcription system. After thorough mixing, the operation was performed according to the magnetic bead instructions. Finally, an appropriate amount of nuclease-free water was used for elution. 1 μL of the purified RNA was taken and the concentration was detected using a micro-spectrophotometer (RNA mode). 50 ng of the purified RNA was taken for denaturing agarose gel electrophoresis (1% TBE + 6% urea) to detect the integrity and purity of the RNA product.

[0055] Example 3: Effects of different 5'UTRs on the expression level of firefly luciferase in HepG2 and DC2.4 cells

[0056] To verify the effect of the designed 5'UTR sequence on the expression level of firefly luciferase in different cells, this invention used immortalized human hepatocellular carcinoma cell line (HepG2) and mouse bone marrow-derived dendritic cell line (DC2.4) for transfection testing. HepG2 or DC2.4 cells in good growth condition were collected, digested, resuspended, and counted; 2 × 10⁶ cells were collected. 6Add one cell to a 50 mL centrifuge tube, add RPMI 1640 complete medium (Thermo Fisher Scientific) to a final volume of 20 mL, and adjust the cell density to 1 × 10⁶ cells / mL. 5 After thorough mixing, distribute the cells evenly into 96-well white plates (Corning Corporation), 100 μL / well, with a cell count of 1 × 10⁶ cells per well. 4 Mix the cells thoroughly by gently tapping, then incubate overnight at 37°C (18–24 h). The next day, remove the cells and use Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) to transfect the cells with FLuc mRNA containing different 5'UTRs prepared above at a dose of 0.1 μg / well. The commonly used HBA1 gene 5'UTR (HBA1 group) was used as the control group. The transfection procedure was performed according to the manufacturer's instructions.

[0057] Luciferase activity was measured in the corresponding wells at 24h and 48h post-transfection. The Bio-Lite Luciferase Assay System kit (Novizan Biotechnology Co., Ltd.) was removed from the container beforehand and brought to room temperature, and prepared according to the instructions. The wells were then removed, the culture supernatant was removed using a pipette, and 100μL of the assay reagent was added to each well. The mixture was gently tapped to mix, and allowed to stand for 1–2 minutes to allow for complete reaction before being placed on a microplate reader (BioTek, model Synergy H1) for measurement. The results are shown below. Figure 2 As shown.

[0058] from Figure 2 The results showed that in HepG2 and DC2.4 cells, the expression levels of most FLuc mRNAs fused with the 5'UTR sequence designed in this invention were significantly higher than those of the control group. The best expression performance was observed in de novo 5'UTR sequences, with expression levels in HepG2 and DC2.4 cells being 2.2-fold and 3.5-fold higher than the control group, respectively. No significant correlation was found between UTR length and expression efficiency in these de novo designed 5'UTR sequences, indicating that the effect of 5'UTR on mRNA translation efficiency is influenced by multiple factors. Figure 2 The significance analysis is shown in Table 2.

[0059] Table 2 Figure 2 Results of data discrepancy analysis

[0060]

[0061]

[0062]

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:

3.

2. A nucleotide construct, characterized in that, The nucleotide construct comprises a nucleotide sequence encoding the 5'-UTR element of claim 1; The nucleotide construct also includes any one or more of enzyme cleavage sites and promoter elements.

3. An mRNA construct, characterized in that, The construct has the 5'-UTR element as described in claim 1; The mRNA construct has the following structure: The 5'-UTR element, the replaceable encoding area, and the 3'-UTR element are connected in sequence.

4. A cell, characterized in that, The cell contains the nucleotide construct as described in claim 2.

5. A method for generating optimized mRNA for the preparation of mRNA drugs, characterized in that, The method includes the following steps: The cells as described in claim 4 are cultured to obtain a cell culture containing a nucleotide construct with a 5'-UTR element; the nucleotide construct is isolated from the cell culture and transcribed to obtain optimized mRNA.

6. The method according to claim 5, characterized in that, The optimized mRNA is purified and / or modified.

7. An optimized mRNA, characterized in that, The optimized mRNA is prepared by the method according to claim 5 or 6, and the optimized mRNA structure is as follows: The 5'-UTR element, the replaceable encoding area, and the 3'-UTR element are connected in sequence.

8. The optimized mRNA according to claim 7, characterized in that, The optimized mRNA also includes elements and / or modifications that stabilize the mRNA.

9. A method for preparing an mRNA drug composition, characterized in that, The method includes mixing the optimized mRNA as described in claim 7 or 8 with a pharmaceutically acceptable adjuvant to obtain the mRNA pharmaceutical composition.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the mRNA construct of claim 3 or the optimized mRNA of claim 7 or 8, and a pharmaceutically acceptable adjuvant.

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

12. A method for preparing polypeptides or proteins, characterized in that, The method includes: replacing the 5'-UTR element of the mRNA transcription template of the polypeptide or protein with the 5'-UTR element as described in claim 1, translating the replaced mRNA transcription template, and obtaining the polypeptide or protein.

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