3' UTR for enhancing expression of exogenous gene, and use thereof

Abstract

The present application relates to the field of biotechnology, and relates to a 3' UTR for enhancing the expression of an exogenous gene, and the use thereof. The 3' UTR obtained in the present application can effectively improve the expression of an exogenous protein, has strong stability, and can relatively persistently express an exogenous mRNA in a cell.

Description

3'utr for enhancing expression of exogenous genes and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a 3'UTR for enhancing expression of exogenous genes and application thereof. BACKGROUND

[0002] mRNA drugs are a new class of drugs that can be used to prevent and treat a variety of diseases. The two highly effective mRNA vaccines produced by Moderna and Pfizer / BioNTech have been successful in preventing COVID-19, highlighting the great potential of mRNA technology in changing life sciences and medical research.

[0003] RNA therapy represented by mRNA has the following advantages compared to traditional treatment methods:

[0004] 1. RNA drugs can be used for "non-druggable" molecular targeted therapy, while small molecule drugs are difficult or impossible to achieve this.

[0005] 2. Compared with proteins, the design and manufacturing process of RNA products, especially mRNA, is simple, low cost and fast.

[0006] 3. The safety risk of RNA drugs is relatively low. Since DNA drugs can enter the nucleus and may integrate into the host genome, there are certain safety problems. mRNA will not integrate into the genome and will be completely degraded within a certain period of time, without the safety risk of inserting the genome.

[0007] However, due to the immaturity of mRNA technology, there are still some key problems to be solved, for example: mRNA faces certain challenges in stability, immunogenicity, in vivo delivery and the ability to cross various biological barriers, among which the stability of mRNA itself is a problem to be solved. In other words, improving the stability and translation efficiency of mRNA is a barrier that must be overcome for mRNA to achieve more widespread clinical applications. Currently, mRNA structure chemical optimization is an important way to improve the stability and translation efficiency of mRNA.

[0008] mRNA structure optimization mainly involves the following aspects: 5' cap, 5'UTR, 3'UTR, poly(A) tail, codon optimization, nucleotide modification, etc.

[0009] Among them, the main role of 3'UTR is to regulate the translation of mRNA, interact with protein complexes, and mediate the transport, stability and translation process of mRNA. The 3'UTR sequence is crucial for targeting transcripts to specific cellular compartments in cells, especially highly polarized or differentiated cells.

[0010] Several studies have shown that 3'UTR is a very important regulatory element, for example: miRNA silences mRNA expression by binding to this region. Some studies show that in rapidly proliferating cells, mRNA exhibits shorter 3'UTR sequences, which can reduce miRNA binding sites, and promote protein expression; in addition, by reducing the non-structured sequence in the 3'UTR sequence, the poly(A) tail can be combined with the translation element to improve protein translation efficiency.

[0011] In summary, since the stability and half-life of mRNA in cells depend largely on the 3'UTR, screening 3'UTR sequences that can enhance mRNA stability is an important means to improve the yield of exogenous proteins, and has become one of the prerequisites for the development of mRNA drug platforms. Therefore, screening 3'UTR sequences for stable expression of mRNA is of great significance for the application of mRNA drugs. SUMMARY

[0012] Based on the deficiencies of the prior art, the following technical solutions are requested to be protected in this application:

[0013] In a first aspect, the present application relates to a nucleic acid molecule comprising, in the 5'-3' transcription direction:

[0014] 1) a promoter;

[0015] 2) a transcribable nucleic acid sequence or a nucleic acid sequence for introducing a transcribable nucleic acid sequence; and

[0016] 3) a template sequence of a 3'-untranslated region (3'UTR) which, when transcribed under the control of the promoter, is transcribed into a 3'-untranslated region (3'UTR) in the transcript, the 3'-untranslated region (3'UTR) comprising a sequence selected from the group consisting of:

[0017] a nucleic acid sequence or a fragment thereof as shown in SEQ ID NO: 1-3 or having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1-3, or a variant of the nucleic acid sequence or the fragment thereof.

[0018] wherein the transcribable nucleic acid sequence or the nucleic acid sequence for introducing a transcribable nucleic acid sequence under the control of the promoter and the template sequence of the 3'-untranslated region (3'UTR) can be transcribed to produce a co-transcript, and the 3'-untranslated region can improve the translation efficiency and / or stability of the transcribable nucleic acid sequence or the nucleic acid sequence for introducing a transcribable nucleic acid sequence.

[0019] In some embodiments, the template sequence of the 3'UTR is non-naturally attached to the transcribable nucleic acid sequence or the nucleic acid sequence used to introduce the transcribable nucleic acid sequence.

[0020] In a second aspect, the present application provides a DNA molecule transcribable into an mRNA, the DNA molecule comprising:

[0021] (1) an exogenous protein or polypeptide coding region; and

[0022] (2) a template sequence of a 3'-untranslated region (3'UTR) downstream of the polypeptide coding region.

[0023] In certain specific embodiments, the 3'UTR comprises a sequence selected from the group consisting of:

[0024] a nucleic acid sequence as set forth in SEQ ID NO: 1-3 or a nucleic acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 1-3 or a fragment thereof, or a variant of said nucleic acid sequence or fragment thereof.

[0025] In a third aspect, the present application relates to a method of obtaining a peptide or protein, comprising:

[0026] (i) providing the nucleic acid molecule described in the present application;

[0027] (ii) transcribing into RNA using the nucleic acid molecule as a template; and

[0028] (iii) translating the RNA.

[0029] In a fourth aspect, the present application further relates to a method of obtaining an RNA, comprising:

[0030] (i) providing the nucleic acid molecule described in the present application; and

[0031] (ii) transcribing RNA using the nucleic acid molecule as a template.

[0032] In a fifth aspect, the present application further relates to a 3'UTR element for constructing an mRNA transcript, the nucleotide sequence of the 3'UTR element being a nucleic acid sequence as set forth in SEQ ID NO: 5-7 or a nucleic acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 5-7 or a fragment thereof, or a variant of said nucleic acid sequence or fragment thereof. The 3'UTR element improves the translation efficiency of the coding region in the mRNA transcript.

[0033] In a sixth aspect, the present application also relates to an mRNA transcript having the structure of Formula I

[0034] Structure: X1-X2-X3-X4-X5-X6-X7 (I)

[0035] X1, X7 is nothing or a cleavage site;

[0036] X2 is nothing or a promoter element;

[0037] X3 is a 5' UTR element;

[0038] X4 is a gene coding region;

[0039] X5 is a 3' UTR element;

[0040] X6 is nothing or a poly(A) tail element.

[0041] The nucleotide sequence of the 3' UTR element is as set forth in SEQ ID NO: 5-7 or a nucleic acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 5-7 or a fragment thereof, or a variant of the nucleic acid sequence or fragment thereof.

[0042] In a seventh aspect, the present application also relates to an mRNA having the structure of Formula II: Y1-Y2-Y3-Y4-Y5-Y6 (II)

[0043] In the formula,

[0044] Y1 is a 5' cap element;

[0045] Y2 is nothing or an internal ribosome entry site sequence (IRES);

[0046] Y3 is a 5' UTR element;

[0047] Y4 is a gene coding region;

[0048] Y5 is a 3' UTR element;

[0049] Y6 is a poly(A) tail element.

[0050] The nucleotide sequence of the 3' UTR element is as set forth in SEQ ID NO: 5-7 or a nucleic acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 5-7 or a fragment thereof, or a variant of the nucleic acid sequence or fragment thereof.

[0051] In an eighth aspect, the present application also relates to a vector containing the nucleic acid molecule or the DNA molecule.

[0052] In a ninth aspect, the present application also relates to a host cell containing the vector.

[0053] In a tenth aspect, the present application also relates to a pharmaceutical composition containing the nucleic acid molecule, the mRNA, the DNA molecule, the vector or the host cell.

[0054] In an eleventh aspect, the present application also relates to the use of the nucleic acid molecule or the DNA molecule, the mRNA or the 3'UTR element for gene therapy.

[0055] In exemplary embodiments, the nucleic acid molecule, the mRNA molecule or the 3'UTR element of the present application is used to transfect a host cell in vitro or in vivo. The transfected host cell is used as a medicament.

[0056] The transfected host cell can be, for example, a mononuclear phagocyte, a dendritic cell, a B cell, a Langerhans cell, preferably a dendritic cell.

[0057] In a twelfth aspect, the present application also relates to the use of the 3'UTR for constructing an mRNA transcript. By employing the 3'UTR of the present application, the translation efficiency of the coding region in the mRNA transcribed from the mRNA transcript is improved.

[0058] The following explanations (where applicable) apply to the first to twelfth aspects above.

[0059] In some embodiments, the RNA can be an mRNA.

[0060] In some embodiments, the nucleotide sequence of the 3'UTR element is the nucleotide sequence from position 7 to position 284 as shown in SEQ ID NO: 1.

[0061] In some embodiments, the nucleotide sequence of the 3'UTR element is the nucleotide sequence from position 7 to position 284 as shown in SEQ ID NO: 2.

[0062] In some embodiments, the nucleotide sequence of the 3'UTR element is the nucleotide sequence from position 7 to position 284 as shown in SEQ ID NO: 3.

[0063] In some embodiments, the nucleic acid molecule further comprises a polyadenylate sequence poly(A).

[0064] In some embodiments, the nucleic acid molecule further comprises a 5' cap.

[0065] In some embodiments, the nucleic acid molecule further comprises a 5' UTR.

[0066] In certain specific embodiments, the promoter and the transcribable nucleic acid sequence are non-naturally linked.

[0067] In certain specific embodiments, the polyadenylation sequence poly(A) is polyA60, polyA90, polyA120, or polyA150.

[0068] In certain specific embodiments, the 5' cap is Cap0, Cap1, or Cap2.

[0069] In certain specific embodiments, the 5' UTR comprises: a kozak sequence (GCCACC), a human alpha-globin 5' UTR sequence, or a human beta-globin 5' UTR sequence.

[0070] In other embodiments, the 3' UTR is a 3' UTR derived from the gene ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, or NDUFB9; and the 5' UTR is a 5' UTR derived from the gene ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATP1F1, ANAPC11, CCDC12, MRPL14, or APOA1BP.

[0071] In certain specific embodiments, the 5' cap is selected from the group consisting of:

[0072] ARCA, 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pU, m7Gppp(A2'O-MOE)pG, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG, or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

[0073] In certain specific embodiments, the promoter comprises: a promoter for SP6, T3, or T7 polymerase.

[0074] In certain specific embodiments, the nucleic acid molecule is a DNA or RNA molecule.

[0075] In some more specific embodiments, the RNA molecule is an mRNA.

[0076] In one embodiment, the nucleic acid molecule is an expression vector or plasmid, such as an IVT vector.

[0077] In certain specific embodiments, the nucleic acid molecule of the application is a closed circular molecule or a linear molecule.

[0078] In certain specific embodiments, the transcribable nucleic acid sequence comprises a nucleic acid sequence encoding a peptide or a protein.

[0079] In certain specific embodiments, the nucleic acid sequence for the introduction of the transcribable nucleic acid sequence is a multiple cloning site.

[0080] In certain specific embodiments, the nucleic acid molecule of the application further comprises one or more selected from the group consisting of: (i) a reporter gene; (ii) a selection marker; and (iii) an origin of replication.

[0081] In certain specific embodiments, the nucleic acid molecule of the application is suitable, in particular after linearization, for the in vitro transcription of RNA, in particular mRNA.

[0082] In certain specific embodiments, the method of obtaining RNA or the method of obtaining a peptide or a protein further comprises cleaving the nucleic acid molecule prior to transcribing the nucleic acid molecule.

[0083] In certain specific embodiments, the use further comprises: for the transient expression of a gene, for the preparation of an RNA vaccine (such as an anti-tumor mRNA vaccine), for in vitro transfection into cells or direct in vivo administration, for the in vivo transient expression of a functional recombinant protein, for a therapeutic tool for enzyme replacement therapy (ERT) or protein replacement therapy (PRT), such as: as a medicament.

[0084] In some more specific embodiments, the mRNA molecule can replace, increase or facilitate the expression of a protein or a biologically active fragment thereof in vivo.

[0085] In some more specific embodiments, the nucleic acid molecule or mRNA can be particularly used for transfecting antigen presenting cells and as a tool for presenting an antigen, the antigen to be presented corresponding to the peptide or protein expressed by the mRNA; the antigen presenting cells can be used for stimulating T cells, in particular CD4 + and / or CD8 + T cells in vivo or in vitro.

[0086] In certain embodiments, the gene coding region is replaceable, which encodes a pathogen antigen or a functional fragment thereof, a tumor neoantigen, a tumor-associated antigen, a tumor-specific antigen, a universal tumor mutation site antigen, but not limited to.

[0087] In certain specific embodiments, the RNA comprises or consists of a pharmaceutically active RNA. The "pharmaceutically active RNA" can be an RNA encoding a pharmaceutically active peptide or protein.

[0088] In certain specific embodiments, the "pharmaceutically active peptide or protein" includes peptides and proteins that are antigens, i.e. the peptide or protein elicits an immune response in a subject, which can be prophylactic or therapeutic or partially or completely protective.

[0089] In certain specific embodiments, RNA, in particular mRNA, encoding an antigen, e.g. a disease-associated antigen, is administered to a mammal, in particular if it is desired to treat a mammal suffering from a disease involving the antigen. The RNA, in particular mRNA, is taken up into an antigen-presenting cell (monocyte, macrophage, dendritic cell, thymic cortical epithelial cell or other cell) of the mammal. An antigenic translation product of the RNA, in particular mRNA, is formed and the product is displayed on the cell surface for recognition by a T cell.

[0090] In certain specific embodiments, the antigen is displayed on the cell surface for recognition by a CAR-engineered T cell directed against the antigen. In one embodiment, the antigen or a product produced by optional processing thereof is displayed on the cell surface in the context of an MHC molecule for recognition by a T cell through the T cell receptor of the T cell.

[0091] In certain specific embodiments, RNA, in particular mRNA, expressing an antigen is introduced into an antigen-presenting cell ex vivo (e.g. an antigen-presenting cell taken from a patient) and the antigen-presenting cell, optionally an ex vivo clonally propagated antigen-presenting cell, is transplanted back into the same patient. The transfected cell can be re-introduced into the patient in sterile form using any method known in the art, preferably by intravenous, intracavitary, intraperitoneal or intratumoral administration.

[0092] In certain specific embodiments, the methods of the application can involve antigen presenting cells for expressing RNA, in particular mRNA, encoding an antigen. To this end, the methods of the application can involve introducing RNA, in particular mRNA, encoding an antigen into an antigen presenting cell, e.g. a dendritic cell (DC cell). For transfection of antigen presenting cells, e.g. dendritic cells, a pharmaceutical composition comprising RNA, in particular mRNA, encoding an antigen can be used. The delivery vehicle targeting the RNA, in particular mRNA, to dendritic cells or other antigen presenting cells can be administered to the patient, the transfection being accomplished in vivo. BRIEF DESCRIPTION OF DRAWINGS

[0093] Figure 1 : d2EGFP mean fluorescence intensity MFI experimental results.

[0094] Figure 2: His protein expression positive rate and mean fluorescence intensity MFI experimental results.

[0095] Figure 3: His protein expression flow cytometry plot.

[0096] Figure 4: mRNA intracellular stability experimental results. DETAILED DESCRIPTION

[0097] DEFINITIONS

[0098] Unless otherwise indicated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs. For the purposes of the present application, the following terms have the meanings indicated: Amino acid residues The abbreviations for the amino acid residues are those used in the art to designate one of the 20 commonly occurring L-amino acids.

[0099] For numerical values, the application shows numerical ranges and parameters approximations in broad ranges, or in specific embodiments, in as precise numerical values as possible. However, any numerical value inherently involves some error, resulting from the standard deviation found in their respective measurements. Also, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be considered to include any and all subranges between (and including) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, and ending with a maximum value of 10 or less, e.g., 1 to 6.1, or, 5.5 to 10. Additionally, any reference to introduction of an item (such as a nucleic acid or polypeptide) into a cell should be understood to include reference to introduction of that item into a progeny of the cell, e.g., a progeny cell, a progeny organism, etc.

[0100] The term "template sequence of the 3'-untranslated region" refers to the nucleic acid sequence on the template strand which is transcribed into the 3'-untranslated region. Preferably, the nucleic acid sequence is the sequence on the template strand which comprises the same nucleic acid sequence as the 3'-untranslated region of the RNA transcript produced (but with thymine T instead of uracil U). Thus, according to the present application, the "template sequence of the 3'-untranslated region" comprises in one embodiment the sequence of the 3'-untranslated region as described herein (but with thymine T instead of uracil U).

[0101] The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The term "nucleic acid molecule" encompasses genomic DNA, cDNA, RNA (such as mRNA). The nucleic acid molecule can be a recombinantly produced molecule or a chemically synthesized molecule. According to the present application, the nucleic acid molecule can be in the form of a single- or double-stranded, linear or covalently closed circular molecule. The term "nucleic acid molecule" according to the present application also includes chemical derivatization of the nucleic acid on the nucleotide bases, on the sugar, or on the phosphate, as well as nucleic acid molecules containing non-natural nucleotides and nucleotide analogs.

[0102] The term "mRNA" means "messenger RNA" and relates to a transcript which is produced by using a DNA template and which encodes a peptide or protein. Typically, mRNA comprises a 5'UTR, a protein coding region, a 3'UTR and a poly(A) sequence. mRNA can be produced from a DNA template by in vitro transcription. Methods of in vitro transcription are known to the skilled person. For example, various in vitro transcription kits are commercially available. According to the present application, the mRNA can be modified by further stabilizing modifications and capping in addition to the modifications according to the present application.

[0103] The term "variant" includes any variant, in particular mutants, splice variants, conformers, isomers, allelic variants, species variants and species homologues, etc. Allelic variants relate to changes in the normal sequence of a gene, and complete gene sequencing often identifies a large number of allelic variants of a given gene. Species homologues are nucleic acid or amino acid sequences of different species origin with a given nucleic acid or amino acid sequence.

[0104] According to the present application, the nucleic acid variant comprises a single or multiple nucleotide deletions, additions, mutations and / or insertions compared to the reference nucleic acid. Deletions include the removal of one or more nucleotides from the reference nucleic acid. Addition variants comprise a fusion of one or more nucleotides (e.g. 1, 2, 3, 5, 10, 20, 30, 50 or more nucleotides) at the 5' and / or 3' end. Mutations can include, but are not limited to, substitutions, wherein at least one nucleotide in the sequence is removed and another nucleotide is inserted in its place (e.g. transversions and transitions); abasic sites; cross-linking sites; and chemically altered or modified bases. Insertions include the addition of at least one nucleotide in the reference nucleic acid.

[0105] For nucleic acid molecules, the term "variant" further includes degenerate nucleic acid sequences, wherein a degenerate nucleic acid sequence according to the application is a nucleic acid which differs from the reference nucleic acid in the sequence of codons due to the degeneracy of the genetic code.

[0106] The term "% identity" refers in particular to the percentage of nucleotides which are identical in the optimal alignment between the two sequences to be compared, and the differences between the two sequences to be compared can be distributed randomly over the full length of the sequences, and the sequence to be compared can comprise additions or deletions compared to the reference sequence to obtain the optimal alignment between the two sequences. The comparison of two sequences is usually carried out by comparing the sequences after optimal alignment for a segment or "comparison window" to identify local regions of correspondence between the sequences. The optimal alignment for comparison can be carried out manually or with the aid of computer programs using the algorithms (GAP, BESTFIT, FASTA, BLASTP, BLASTN and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0107] The term "3'-untranslated region" relates to a region located at the 3' end of a gene, downstream of the stop codon of a protein-coding region, and which is transcribed but not translated into an amino acid sequence, or to the corresponding region in an RNA molecule.

[0108] The terms "polyadenosine sequence", "poly(A) sequence", "poly(A)" or "poly(A) tail" are used interchangeably and refer to a sequence of adenosine residues, usually located at the 3' end of an RNA molecule. The present application allows such a sequence to be attached during RNA transcription based on repeated thymidine acid residues in the strand complementary to the coding strand by a DNA template, whereas the sequence is not normally encoded in DNA, but is attached to the free 3' end of the RNA after transcription in the nucleus by a template-independent RNA polymerase. According to the present application, in one embodiment, the poly(A) sequence has at least 20, preferably at least 40, preferably at least 80, preferably at least 100 and preferably up to 500, preferably up to 400, preferably up to 300, preferably up to 200 and in particular up to 150 A nucleotides, preferably consecutive A nucleotides, and in particular about 120 A nucleotides. The term "A nucleotide" or "A" refers to an adenosine residue.

[0109] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, which are all conventional products that can be obtained by purchase.

[0110] Example 1: In vitro synthesis of mRNA

[0111] (1) The candidate 3'UTR template sequences (SEQ ID NO: 1-3) to be evaluated and the control 3'UTR template sequence (SEQ ID NO: 4) were respectively connected to the downstream of the 3' end of the gene coding region containing 5'UTR, EGFP coding sequence (amino acid sequence as shown in SEQ ID NO: 8, nucleotide sequence as shown in SEQ ID NO: 9) or His (amino acid sequence as shown in SEQ ID NO: 10, nucleotide sequence as shown in SEQ ID NO: 11) by homologous recombination, thereby generating plasmids with different 3'UTR template sequences. The E. coli competent cells were transformed, and after overnight culture, single colonies were picked and shaken, and then the plasmid was extracted using a plasmid extraction kit. The linearized plasmid was purified after BspQI enzyme digestion, and the purified linearized plasmid was used as a template for in vitro transcription of mRNA.

[0112] (2) The linearized plasmid, nucleotide raw material, T7 RNA polymerase and cap analog were added to the reaction buffer system in a certain proportion, mixed and incubated at 37°C for 2h. After the reaction was completed, DNase I was incubated at 37°C for 15min to obtain the crude mRNA product.

[0113] (3) The purified mRNA was obtained by magnetic bead purification and 70% ethanol washing. The prepared mRNA was detected for concentration, purity, capping rate, dsRNA content, and poly(A) proportion, and the qualified mRNA was frozen at -80°C for subsequent cell electroporation.

[0114] Example 2: Culture and electroporation of DC cells

[0115] 2.1 The DC cell culture and maturation promotion were carried out according to the following steps:

[0116] (1) iDC cell (Immature dendritic cell) resuscitation: Take the iDC cells out of the liquid nitrogen tank in a 37°C water bath until the cryopreservation solution is completely melted. Take a certain volume of AIM-V medium (Thermo Fisher) into a centrifuge tube, open the lid of the cryopreservation tube, and take the iDC cell cryopreservation suspension in the cryopreservation tube and add it to the medium. After mixing, transfer the cell suspension to the centrifuge tube, cover the centrifuge tube, mix several times by inverting, and centrifuge at 600xg for 10 min at room temperature.

[0117] (2) Resuspend and count: discard the supernatant, resuspend the cell pellet in DC cell culture medium, mix well by blowing, and then count. According to the test results, the appropriate medium is supplemented to adjust the cell density to 1.00E+06 cells / mL.

[0118] (3) Prepare for sub-packaging and electroporation: After mixing the cell suspension, sub-packaging into culture dishes or culture bottles, labeling the corresponding number, and placing in a 37.0°C, 5.0% CO2 incubator for culture. After 24h of culture, collect the mDC cells (mature dendritic cell), and add fresh culture medium, count, and use for subsequent electroporation.

[0119] 2.2 Electroporation process:

[0120] (1) Four groups of mRNA for electroporation, respectively containing D-3'UTR (corresponding to D-3'UTR template sequence SEQ ID NO:4), 134-3'UTR, 136-3'UTR and 137-3'UTR, wherein D-3'UTR is the control group, which is the 3'UTR sequence used in BIONTECH mRNA vaccine products; experimental groups 134-3'UTR, 136-3'UTR and 137-3'UTR mRNA (SEQ ID NO:5-7) correspond to the 3'UTR template sequences (SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3) designed by the research team. The four groups of mRNA are the same except for the different 3'UTR element sequences. The electroporation dose of each group of mRNA is 100 μg, and the total volume of the electroporation system is 300 μL. The prepared electroporation system is transferred to the electroporation cup, and the cell electroporation instrument is used for electroporation.

[0121] (2) After electroporation, dilute the cell suspension in the electroporation cup with culture medium to adjust the cell density to 1.00E+06 cells / mL. The diluted cell suspension is sub-packaged into culture dishes and placed in a 37.0°C, 5.0% CO2 incubator for culture.

[0122] Example 3: mRNA transfection efficiency and intracellular level detection

[0123] The cells obtained in Example 2 were subjected to mRNA transfection efficiency and intracellular level detection, with the following specific steps:

[0124] (1) Cell washing, collection and resuspension: collect the cell samples of each group at 0h, 4h and 20h after electroporation, centrifuge at 400xg for 5min at room temperature; discard the supernatant, resuspend the cells with 1mL of PBS containing 2% FBS and transfer to a 1.5mL centrifuge tube. Centrifuge at 400xg for 5min at room temperature, discard the supernatant.

[0125] (2) Extract RNA using an RNA extraction kit (QIAGEN GmbH) and perform reverse transcription, then perform qPCR reaction after reverse transcription. Add 7.6μL of the above-mentioned cDNA sample obtained by reverse transcription to each PCR tube, then add 0.4μL of the primers and probes (10μM) of the target gene EGFP (SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14), 0.4μL of the primers and probes (10μM) of the internal reference gene hACTB2 (SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17), 10μL of Perfect start II probe qPCR super MIX (Quanjing Biological Co., Ltd.), to a total volume of 20μL. Set 2 replicates for each group, and set a blank control. Place the above- configured reaction system in a real-time fluorescence quantitative PCR instrument, and perform the reaction according to the reaction program: 94℃ for 30sec; 94℃ for 5sec, 60.3℃ for 30sec, for a total of 40 cycles.

[0126] (3) After the reaction is completed, the CT value is exported for subsequent analysis of the level of target mRNA in each sample.

[0127] Example 4: Intracellular EGFP protein expression detection

[0128] (1) Collect the cell samples of each group at 2h, 4h, 20h, 24h and 48h after electroporation, centrifuge at 600xg for 5min at room temperature;

[0129] (2) Discard the supernatant, add 1mL of FACS buffer to wash the cell precipitate, centrifuge at 600xg for 5min at room temperature; discard the supernatant, resuspend the cell precipitate with 200μL of FACS buffer, mix well, and then load the sample into a flow cytometer for sample loading. Detect the average fluorescence intensity MFI of EGFP expression of each group of samples.

[0130] The results of the study show that: the protein expression determination results at different time points after the mRNA of different mDCs is electrically transferred show (Table 1, FIG. 1) that, compared with D-3'UTR, the mRNA 134-3'UTR, 136-3'UTR and 137-3'UTR using the 3'UTR sequence independently designed by the present study have stronger protein translation ability in DCs, and achieve the highest protein expression level at multiple time points after electrical transfer.

[0131] Example 5: His intracellular staining detection

[0132] The following steps are followed:

[0133] 5.1 Blocking

[0134] (1) Collect the cell samples of each group 6h after electrical transfer, centrifuge at 600xg at room temperature for 5min.

[0135] (2) Discard the supernatant, add 1mL FACS buffer to each group to wash the cell precipitate, mix and count the sample.

[0136] (3) Take out the cell suspension containing 5.00E+05 cells from each tube, centrifuge at 600xg for 5min; discard the supernatant, add 100μL 5% BSA (5% BSA bovine serum albumin: 10mL FACS buffer + 0.5g BSA) and 5μL Human TruStain FcX TM to each tube, and incubate at room temperature for 10min.

[0137] 5.2 Fixation

[0138] (1) After incubation, centrifuge at 600xg for 5min, discard the supernatant, and add 1mL Phosphate Buffered Saline to wash the sample at 600xg for 5min, discard the supernatant; repeat the washing twice.

[0139] (2) Add 200μL flow cytometry fixing solution, and incubate at room temperature in the dark for 20min.

[0140] 5.3 Membrane breaking-staining

[0141] (1) Add 1mL 1x membrane breaking buffer to wash the sample, centrifuge at 600xg for 5min, discard the supernatant; repeat the washing twice.

[0142] (2) Add 100μL 1x membrane breaking buffer containing 0.2μg His-Tag Monoclonal antibody (Proteintech) to each group, and incubate at 4°C in the dark for 30min.

[0143] (3) After incubation, each tube was added with 1 mL FACS buffer to wash the cells, centrifuged at 600xg for 5 min, and the supernatant was discarded; the washing was repeated twice.

[0144] (4) Each tube was added with 200 μL FACS buffer to resuspend the cell precipitate, and the flow detection was performed.

[0145] The experimental results show that the method of membrane breaking staining is used to detect the His-tag protein translated from 6h mRNA in mDC after electroporation, and the results show that the His expression levels of 134-3'UTR, 136-3'UTR and 137-3'UTR are higher than that of D-3'UTR (Table 2, Figure 2, Figure 3).

[0146] Table 2 His protein expression positive rate and average fluorescence intensity MFI

[0147] Example 6: Comparison results of evaluation of intracellular stability of different mRNA

[0148] The mRNA levels of D-3'UTR, 134-3'UTR, 136-3'UTR and 137-3'UTR in the cells after electroporation were analyzed by qRT-PCR, and the stability of different mRNA was evaluated according to the mRNA transfection efficiency in the cells of each group at 0h after electroporation. As shown in Table 3 and Figure 4, the mRNA levels of each group in DC at the same time point after electroporation are basically the same, and the stability of the 3'UTR element obtained in the application is slightly better than that of the control group.

[0149] Table 3 Relative mRNA levels in mDC at different time points after electroporation

[0150] Note: The values in the table are the results after normalization according to the mRNA transfection efficiency in the cells of each group at 0h after electroporation.

[0151] In summary, the 3'UTR obtained in the application can effectively improve the expression of exogenous protein and has strong stability, and can relatively persistently express exogenous mRNA in cells, and the comprehensive effect is significantly better than that of the control group (3'UTR used by BIONTECH).

[0152] The sequences involved in the application are as follows:

Claims

1. A nucleic acid molecule comprising in 5’-3’ transcriptional direction: 1) a promoter; 2) a transcribable nucleic acid sequence or a nucleic acid sequence for introducing a transcribable nucleic acid sequence; and 3) a template sequence of a 3’-untranslated region (3’UTR) which, when transcribed under the control of the promoter, is transcribed into a nucleic acid sequence of a 3’-untranslated region (3’UTR) in the transcript, the 3’-untranslated region (3’UTR) comprising a sequence selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO: 1-3 or having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity with the nucleotide sequence as shown in SEQ ID NO: 1-3 or a fragment thereof, or a variant of said nucleic acid sequence or fragment thereof.

2. The nucleic acid molecule of claim 1, which is suitable for in vitro transcription of RNA.

3. An mRNA having the structure shown in Formula II: Y1-Y2-Y3-Y4-Y5-Y6 (II), in which, Y1 is a 5’ cap element; Y2 is nothing or an internal ribosome entry site sequence (IRES); Y3 is a 5’UTR element; Y4 is a gene coding region; Y5 is a 3’UTR element; Y6 is a poly(A) tail element; the nucleotide sequence of the 3’UTR element is a nucleic acid sequence as shown in SEQ ID NO: 5-7 or having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity with the nucleotide sequence as shown in SEQ ID NO: 5-7 or a fragment thereof, or a variant of said nucleic acid sequence or fragment thereof.

4. A 3’UTR element for use in constructing an mRNA transcript, improving the translation efficiency of a coding region in the mRNA transcript, the nucleotide sequence of the 3’UTR element being a nucleic acid sequence as shown in SEQ ID NO: 1-3 or having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity with the nucleotide sequence as shown in SEQ ID NO: 1-3 or a fragment thereof, or a variant of said nucleic acid sequence or fragment thereof.

5. Use of the nucleic acid molecule of any one of claims 1-2, the mRNA of claim 3 or the 3’UTR element of claim 4 for the manufacture of a medicament for transfecting a host cell.

6. The use of claim 5, wherein the host cell is an antigen presenting cell, comprising: monocytes, dendritic cells, B cells, Langerhans cells, preferably dendritic cells.

7. The use of claim 5, the medicament being an anti-tumor mRNA vaccine.

8. A method of preparing an RNA, comprising: (i) providing the nucleic acid molecule of any one of claims 1 to 2, and (ii) transcribing into RNA using the nucleic acid molecule as a template.

9. A method of obtaining a peptide or protein, comprising: (i) obtaining RNA encoding said peptide or protein according to the method of claim 8, and (ii) translating said RNA.

10. RNA obtained by the method of claim 8 or a peptide or protein obtained by the method of claim 9.

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