UTR molecule for improving protein expression quantity

Abstract

The present disclosure relates to UTR molecules and uses thereof. In particular, the present disclosure relates to mRNA molecules comprising 5 'UTR and 3' UTR, said UTR molecules being capable of significantly increasing the expression level of mRNA.

Description

UTR molecules that increase protein expression

[0001] This application claims priority to PCT application No. PCT / CN2022 / 134038 filed on November 24, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present disclosure relates to UTR molecules and applications thereof. Specifically, the present disclosure relates to mRNA molecules comprising a 5' UTR and a 3' UTR, wherein the UTR molecules can significantly increase the protein expression level of the mRNA molecules. Background Art

[0003] mRNA vaccines are a promising approach to gene therapy. However, mRNA is unstable and easily degraded by ubiquitous RNA enzymes. In vivo, RNA degradation helps regulate RNA half-life and can fine-tune the regulation of eukaryotic gene expression (Friedel CC, L, Ruzsics Z, Koszinowski UH, Zimmer R. Nucleic Acids Res. 2009;37(17):e115-e115.doi:10.1093 / NAR / GKP542). Stable RNA is very necessary for the expression of RNA drugs. There are many methods to regulate the stability of nucleic acid molecules, including adjusting the GC content of nucleic acids (WO2002098443A2), adding UTR (untranslated region) sequences, 5' caps (Galloway A, Cowling VH. Biochim Biophys Acta-Gene Regul Mech. 2019; 1862(3): 270-279. doi: 10.1016 / j.bbagrm.2018.09.011), and 3' poly A tails (Tudek A, Lloret-Llinares M, Heick Jensen T. Philos Trans R Soc B Biol Sci. 2018; 373(1762). doi: 10.1098 / rstb.2018.0169).

[0004] The UTR is a key factor in translation efficiency (Jackson RJ, Hellen CUT, Pestova T V. Nat Rev Mol Cell Biol 2010 112. 2010; 11(2): 113-127. doi: 10.1038 / nrm2838). The 3' UTR of α-globin mRNA is known to play an important role in α-globin mRNA stability (Rodgers ND, Wang Z, Kiledjian M. RNA. 2002; 8(12): 1526. doi: 10.1017 / s1355838202029035). The 3'UTR of α-globin mRNA participates in the formation of a specific nucleoprotein-complex (α-complex) and is associated with the in vitro stability of mRNA (Wang Z, Day N, Trifillis P, Kiledjian M. Mol Cell Biol. 1999; 19(7): 4552. doi: 10.1128 / MCB.19.7.4552). Moderna constructed a neural network based on the experimental data of 280,000 random 5'UTRs and designed a universal 5'UTR that increases expression (Sample PJ, Wang B, Reid DW, et al. Nat Biotechnol. 2019; 37(7): 803-809. doi: 10.1038 / s41587-019-0164-5; US10881730B2). BioNtech screened out 5UTR and 3UTR that can improve expression through a fragment library (US 2022 / 0273820A1; Orlandini von Niessen AG, Poleganov MA, Rechner C, et al. Mol Ther. 2019; 27(4): 824-836. doi: 10.1016 / j.ymthe.2018.12.011).

[0005] Despite the above progress, there is still an urgent need for UTR molecular combinations that can stabilize mRNA molecules and increase their protein expression.

[0006] Summary of the Invention

[0007] The inventors of the present application unexpectedly discovered that the specific 5'UTR and 3'UTR combination described herein can significantly increase the protein expression level of mRNA molecules, and the mRNA can thus be advantageously used in gene therapy, gene vaccination, protein replacement therapy, antisense therapy or treatment by interfering RNA.

[0008] In one aspect, the present disclosure provides an mRNA molecule comprising a 5'UTR and a 3'UTR, wherein the 5'UTR is selected from the following (1)-(5):

[0009] (1) comprising a 5'UTR of an RNA sequence corresponding to any one of the nucleic acid sequences shown in SEQ ID NOs: 1-22, a homologue, a fragment or a variant thereof, wherein the homologue, fragment or variant has the same or better function of improving translation efficiency as the 5'UTR of the RNA sequence corresponding to any one of the nucleic acid sequences shown in SEQ ID NOs: 1-22; preferably, the nucleic acid sequence of the homologue has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA sequence corresponding to any one of the nucleic acid sequences shown in SEQ ID NOs: 1-22;

[0010] (2) a 5'UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in any one of SEQ ID NOs: 1-22;

[0011] (3) a 5'UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in any one of SEQ ID NOs: 4, 3, 12, 17, or 19-22;

[0012] (4) A 5'UTR obtained by concatenating two or more identical 5'UTRs in (1) to (3) above; or

[0013] (5) 5'-UTR obtained by concatenating two or more different 5'UTRs in (1)-(3) above,

[0014] And wherein the 3'UTR is selected from the following (1)-(5):

[0015] (1) comprising a 3'UTR of an RNA sequence corresponding to the nucleic acid sequence of any one of SEQ ID NOs: 23-36, a homologue, a fragment or a variant thereof, wherein the homologue, fragment or variant has the same or better function of improving translation efficiency as the 3'UTR of the RNA sequence corresponding to the nucleic acid sequence of any one of SEQ ID NOs: 23-36; preferably, the nucleic acid sequence of the homologue has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA sequence corresponding to the nucleic acid sequence of any one of SEQ ID NOs: 23-36;

[0016] (2) a 3'UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in any one of SEQ ID NOs: 23-36;

[0017] (3) a 3'UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 28 or 29;

[0018] (4) A 3'UTR obtained by concatenating two or more identical 3'UTRs in (1) to (3) above; or

[0019] (5) 3′-UTR obtained by concatenating two or more different 3′UTRs in (1)-(3) above;

[0020] And the combination of 5'UTR and 3'UTR does not include RNA sequences corresponding to the following nucleic acid sequences: SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 20 and SEQ ID NO: 24, and SEQ ID NO: 21 and SEQ ID NO: 25.

[0021] In some embodiments, the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4, 3, 12, 17, 19, 20, 21 or 22.

[0022] In some embodiments, the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 25, 28 or 29.

[0023] In some embodiments, the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19 or 22, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 25, 28 or 29.

[0024] In some embodiments, the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4, 3 or 12, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 28 or 29.

[0025] In some embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24 or 28.

[0026] In some embodiments, the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 20 or 21, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 28 or 28.

[0027] In some embodiments, the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 3, 19 or 22, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 25, 28 or 29.

[0028] In some embodiments, the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4, 12, 17, 20 or 21, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 28 or 29.

[0029] In some embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24, 25, 28 or 29.

[0030] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:23.

[0031] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:24.

[0032] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:25.

[0033] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:28.

[0034] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:29.

[0035] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:23.

[0036] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:24.

[0037] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:28.

[0038] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:29.

[0039] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:23.

[0040] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24.

[0041] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28.

[0042] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:29.

[0043] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23.

[0044] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24.

[0045] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 25.

[0046] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28.

[0047] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 29.

[0048] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23.

[0049] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24.

[0050] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 25.

[0051] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28.

[0052] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 29.

[0053] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 20, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23.

[0054] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 20, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28.

[0055] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:20, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:29.

[0056] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:21, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:23.

[0057] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:21, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:24.

[0058] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:21, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:28.

[0059] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24.

[0060] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:25.

[0061] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28.

[0062] In a specific embodiment, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:29.

[0063] In some embodiments, the mRNA molecule further comprises a nucleic acid sequence encoding a polypeptide of interest.

[0064] In some embodiments, the mRNA molecule further comprises polyA.

[0065] In one aspect, the present disclosure provides a method for increasing the protein expression amount or translation efficiency of mRNA using a combination of a 5'UTR and a 3'UTR as defined above or an mRNA molecule comprising a 5'UTR and a 3'UTR.

[0066] In one aspect, the present disclosure provides an mRNA molecule comprising a combination of a 5'UTR and a 3'UTR as defined above or comprising a 5'UTR and a 3'UTR, which is used to increase the protein expression amount or translation efficiency of the mRNA.

[0067] In one aspect, the present disclosure provides use of a combination of a 5'UTR and a 3'UTR in increasing protein expression or translation efficiency of an mRNA, wherein the 5'UTR and the 3'UTR are as defined above.

[0068] In one aspect, the present disclosure provides a DNA encoding the above-mentioned mRNA molecule.

[0069] In one aspect, the present disclosure provides a vector comprising the above-mentioned DNA.

[0070] In one aspect, the present disclosure provides a host cell comprising the above-mentioned vector.

[0071] In one aspect, the present disclosure provides a lipid nanoparticle comprising the above-mentioned mRNA molecule.

[0072] In one aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned mRNA molecule, the aforementioned DNA, the aforementioned vector, the aforementioned host cell or the aforementioned lipid nanoparticle, and a pharmaceutically acceptable carrier.

[0073] In one aspect, the present disclosure provides a method for performing gene therapy, gene vaccination, protein replacement therapy, antisense therapy, or treatment by interfering RNA using the above-mentioned mRNA molecule, the above-mentioned DNA, the above-mentioned vector, the above-mentioned host cell, the above-mentioned lipid nanoparticle, or the above-mentioned pharmaceutical composition.

[0074] In one aspect, the present disclosure provides the above-mentioned mRNA molecule, the above-mentioned DNA, the above-mentioned vector, the above-mentioned host cell, the above-mentioned lipid nanoparticle or the above-mentioned pharmaceutical composition for use in gene therapy, gene vaccination, protein replacement therapy, antisense therapy or treatment by interfering RNA.

[0075] In one aspect, the present disclosure provides the use of the above-mentioned mRNA molecule, the above-mentioned DNA, the above-mentioned vector, the above-mentioned host cell, the above-mentioned lipid nanoparticle or the above-mentioned pharmaceutical composition in the preparation of a drug, wherein the drug is used for gene therapy, gene vaccination, protein replacement therapy, antisense therapy or treatment by interfering RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 shows the effects of different 5'UTRs on GFP expression.

[0077] FIG2 shows the effects of different 3'UTRs on GFP expression.

[0078] Detailed Description of the Invention

[0079] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terminology used herein is only for the purpose of describing certain specific embodiments and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0080] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA technology, which are explained in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd ed., J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0081] Unless otherwise required by the context, in this specification and the appended claims, the word "comprise / include" and its variations should be understood to mean including the elements, wholes or steps or groups of elements, wholes or steps, without excluding any other elements, wholes or steps or groups of elements, wholes or steps. Unless otherwise noted herein or obviously contradicted by the context, nouns without quantifiers used in the context of describing the present invention (especially in the context of the claims) should be interpreted as representing one / kind and / or more / kind. The recording of the range of values ​​herein is only intended to be used as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise noted herein, each individual value is incorporated into this specification as if it were individually recorded herein. Unless otherwise noted herein or obviously contradicted by the context, all methods described herein can be carried out in any appropriate order. The use of any and all examples or exemplary language (e.g., "such as" or "such as") provided herein is only intended to better illustrate the present invention, without limiting the scope of the invention claimed in other ways. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0082] According to the present invention, nucleic acid molecule or nucleic acid sequence refers to nucleic acid, which is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to the present invention, nucleic acid includes genomic DNA, cDNA, mRNA, recombinantly prepared molecules and chemically synthesized molecules. According to the present invention, nucleic acid can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.

[0083] In the context of the present invention, the term "RNA" refers to a molecule comprising ribonucleotide residues, and preferably consisting of ribonucleotide residues as a whole or substantially. The term "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially or completely purified RNA), substantially pure RNA, synthetic RNA, and recombinantly produced RNA, such as modified RNA, which differs from naturally occurring RNA in that it adds, deletes, replaces, and / or changes one or more nucleotides. Such changes can include adding non-nucleotide materials, such as to the end of the RNA or internally, such as adding non-nucleotide materials at one or more nucleotides of the RNA. The nucleotides in the RNA molecule can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. The RNAs of these changes can be referred to as analogs, particularly analogs of naturally occurring RNA. According to the present invention, RNA includes mRNA.

[0084] The term "mRNA" means "messenger RNA" and refers to a transcript that is produced using a DNA template and encodes a peptide or protein. Typically, an mRNA comprises a 5'UTR, a protein coding region, a 3'UTR, and a poly (A) sequence. mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methods are known to those skilled in the art. For example, a variety of in vitro transcription kits are commercially available. According to the present invention, in addition to the modifications according to the present invention, mRNA can also be modified by further stabilization modifications and capping.

[0085] The term "nucleic acid" according to the present invention also includes chemical derivatizations of nucleic acids on the nucleotide bases, on the sugars or on the phosphates, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs.

[0086] "Fragment" or "fragment of a nucleic acid sequence" relates to a portion of a nucleic acid sequence, i.e. a sequence that exhibits a shortened nucleic acid sequence at the 5' and / or 3' end. Preferably, when the fragment replaces the nucleic acid sequence in an RNA molecule, it retains RNA stability and / or translation efficiency. Preferably, a fragment of a nucleic acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98% or 99% of the nucleotide residues from the nucleic acid sequence.

[0087] The term "variant" according to the present invention, for example, with respect to nucleic acid and amino acid sequences, includes any variant, particularly mutants, splice variants, conformers, isomers, allelic variants, species variants and species homologs, particularly those occurring naturally. Allelic variants relate to changes in the normal sequence of a gene, the importance of which is often unclear. Complete gene sequencing often identifies a large number of allelic variants of a given gene. Species homologs are nucleic acid or amino acid sequences that have a different species origin than a given nucleic acid or amino acid sequence.

[0088] According to the present invention, nucleic acid variants comprise single or multiple nucleotide deletions, additions, mutations and / or insertions compared to a reference nucleic acid. Deletions include removing one or more nucleotides from a reference nucleic acid. Addition variants include 5' and / or 3' fusions of one or more nucleotides (e.g., 1, 2, 3, 5, 10, 20, 30, 50 or more nucleotides). Mutations may include, but are not limited to, substitutions, in which at least one nucleotide in a sequence is removed and another nucleotide (e.g., transversions and conversions) is inserted in its place; abasic sites; crosslinking sites; and chemically altered or modified bases. Insertions include adding at least one nucleotide to a reference nucleic acid.

[0089] With respect to nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, wherein a degenerate nucleic acid sequence according to the present invention is a nucleic acid that differs from a reference nucleic acid in codon sequence due to the degeneracy of the genetic code.

[0090] Preferably, the degree of identity between a given nucleic acid sequence and a nucleic acid sequence that is a variant of the given nucleic acid sequence is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%. The degree of identity is preferably given over a region of at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300 or at least about 400 nucleotides. In some preferred embodiments, the degree of identity is given over the entire length of the reference nucleic acid sequence.

[0091] "Sequence similarity" refers to the percentage of amino acids that are identical or that represent conservative amino acid substitutions. "Sequence identity" between two polypeptide or nucleic acid sequences refers to the percentage of amino acids or nucleotides that are identical between those sequences.

[0092] The term "% identity" is intended to refer to the percentage of identical nucleotides, particularly in an optimal alignment between two sequences to be compared, wherein the percentage is purely statistical, and the differences between the two sequences may be randomly distributed over the full length of the sequence, and the sequence to be compared may comprise additions or deletions to obtain an optimal alignment between the two sequences compared to the reference sequence. The comparison of two sequences is typically performed by comparing the sequences after optimal alignment over a segment or "comparison window" to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually, with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, and with the aid of the search similarity algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or with the aid of computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0093] Percent identity is obtained by determining the number of corresponding identical positions in the sequences being compared, dividing this number by the number of positions being compared, and multiplying the result by 100. For example, the BLAST program "BLAST 2 sequences" available at http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi can be used.

[0094] In some embodiments, the complementarity degree of the present invention is 100%.

[0095] A fragment or variant of a specific nucleic acid sequence or a nucleic acid sequence having a specific degree of identity with a specific nucleic acid sequence preferably has at least one functional property of the specific sequence and is preferably functionally equivalent to the specific sequence, e.g., a nucleic acid sequence that exhibits properties that are identical or similar to those of the specific nucleic acid sequence.

[0096] The term "promoter" or "promoter region" refers to a DNA sequence upstream (5') of the coding sequence of a gene that controls the expression of the coding sequence by providing recognition and binding sites for RNA polymerase. The promoter region may contain other recognition or binding sites for other factors involved in regulating the transcription of the gene. A promoter may control the transcription of a prokaryotic or eukaryotic gene. A promoter may be "inducible" and initiate transcription in response to an inducer, or it may be "constitutive" if transcription is not controlled by an inducer. If there is no inducer, an inducible promoter is expressed only to a small extent or not at all. In the presence of an inducer, the gene is "turned on" or the transcription level is increased. This is typically mediated by the binding of a specific transcription factor.

[0097] According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA or RNA and protein. It also includes the partial expression of nucleic acids. In addition, expression can be transient or stable. With respect to RNA, the term "expression" or "translation" refers to the process in the ribosomes of a cell that directs the assembly of an amino acid sequence to produce a peptide or protein through its messenger RNA chain.

[0098] In the context of the present invention, the term "transcription" refers to a process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into protein. According to the present invention, the term "transcription" includes "in vitro transcription", wherein the term "in vitro transcription" refers to a process in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system. Preferably, cloning vectors are used for the production of transcripts. These cloning vectors are generally designated as transcription vectors and are encompassed by the term "vector" according to the present invention. According to the present invention, the RNA is preferably in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid (particularly cDNA) and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0099] The term "untranslated region" or "UTR" as used in accordance with the present invention relates to the portion of the mRNA upstream of the start codon and downstream of the stop codon, which is not translated and is therefore referred to as the 5' untranslated region (5'UTR) and the 3' untranslated region (3'UTR), respectively. These regions are transcribed with the coding region and are therefore exonic when present in the mature mRNA.

[0100] 3'-untranslated region (3'UTR): Generally, the term "3'UTR" refers to the portion of an artificial nucleic acid molecule that is located 3' (i.e., "downstream") of the open reading frame and is not translated into protein. Typically, a 3'UTR is the portion of an mRNA that is located between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the poly(A) sequence of the mRNA. In the context of the present invention, the term 3'UTR may also include elements that are not encoded in the template from which the RNA is transcribed, but that are added during post-transcriptional maturation, such as a poly(A) sequence. The 3'-UTR of an mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is typically encoded by a gene that is transcribed into the respective mRNA during gene expression. The genomic sequence is first transcribed into a pre-mRNA containing optional introns. The pre-mRNA is then further processed into a mature mRNA during the maturation process. The maturation process comprises the following steps: 5′ capping, splicing of the pre-mRNA to remove optional introns, and 3′ end modification (e.g., polyadenylation and optional endonuclease / exonuclease cleavage at the 3′ end of the pre-mRNA). Within the scope of the present invention, the 3′-UTR corresponds to the sequence of the mature mRNA located between the stop codon of the protein coding region, preferably immediately 3′ to the stop codon of the protein coding region, and the polyadenylation sequence of the mRNA. The term “corresponding to” means that the 3′-UTR sequence can be an RNA sequence, such as in the mRNA sequence used to define the 3′-UTR sequence, or in a DNA sequence corresponding to this RNA sequence. Within the scope of the present invention, the term “3′-UTR of a gene,” such as “3′-UTR of a ribosomal protein gene,” refers to the sequence corresponding to the 3′UTR of the mature mRNA derived from the gene, i.e., the mRNA obtained by transcription of the gene and maturation of the pre-mRNA. The term "3'UTR of a gene" includes the DNA sequence and RNA sequence (both the sense and antisense strands and both mature and immature) of the 3'UTR.

[0101] 5'-untranslated region (5'UTR): 5'UTR is typically understood as a specific part of messenger RNA (mRNA). It is located on the 5' side of the mRNA open reading frame. Typically, the 5'UTR starts with a transcription start site and ends one nucleotide before the open reading frame start codon. The 5'UTR may contain elements that control gene expression, also referred to as regulatory elements. Such regulatory elements may be, for example, ribosome binding sites. The 5'UTR may be post-transcriptionally modified, for example, by adding a 5'-cap. Within the scope of the present invention, the 5'UTR corresponds to the mature mRNA sequence between the 5'cap and the start codon. Preferably, the 5'UTR corresponds to a sequence extending from the nucleotides located at the 3' side of the 5'-cap, preferably from the 3' side nucleotides immediately adjacent to the 5'-cap, to the nucleotides located at the 5' side of the start codon of the protein coding region, preferably to the nucleotides immediately adjacent to the 5' side of the start codon of the protein coding region. The nucleotides immediately adjacent to the 3' side of the mature mRNA 5'-cap typically correspond to the transcription start site. The term "corresponding to" means that the 5'UTR sequence can be an RNA sequence, such as in the mRNA sequence used to define the 5'UTR sequence, or a DNA sequence corresponding to this RNA sequence. Within the context of the present invention, the term "5'UTR of a gene" refers to a sequence corresponding to the 5'UTR of a mature mRNA derived from that gene, i.e., an mRNA obtained by transcription of the gene and maturation of a pre-mRNA. The term "5'UTR of a gene" encompasses both DNA and RNA sequences of the 5'UTR.

[0102] According to the present invention, the term "gene" refers to a specific nucleic acid sequence responsible for producing one or more cellular products and / or carrying out one or more intercellular or intracellular functions. More specifically, the term relates to a DNA segment comprising a nucleic acid encoding a specific protein or a functional or structural RNA molecule.

[0103] Polyadenylation is the addition of a poly(A) sequence (PolyA) or a poly(A) tail to the primary transcript RNA. The poly(A) sequence is composed of multiple adenosine monophosphates. In other words, it is a section of RNA containing only adenine bases. In eukaryotes, polyadenylation is part of the process of producing mature messenger RNA (mRNA) for translation. Therefore, it forms part of the larger gene expression process. The polyadenylation process begins when transcription of a gene ends or terminates. The most 3' segment of the newly produced pre-mRNA is first cleaved off by a group of proteins; these proteins then synthesize the poly(A) sequence at the 3' end of the RNA. The poly(A) sequence is important for nuclear export, translation, and mRNA stability. This sequence shortens over time, and when it is short enough, the mRNA is enzymatically degraded.

[0104] The term "poly(A) sequence", "poly(A) sequence" or "poly(A) tail" refers to a sequence of adenylate residues typically located at the 3' end of an RNA molecule. The present invention allows such a sequence to be attached during RNA transcription by a DNA template based on repeated thymidylate residues in a strand complementary to the coding strand, whereas the sequence is not normally encoded in DNA but is attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription in the nucleus. According to the present invention, 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 adenylate residue.

[0105] The nucleic acids described herein can be recombinant molecules and / or isolated molecules.

[0106] As used herein, "isolated molecule" is intended to refer to a molecule that is substantially free of other molecules, such as other cellular material. The term "isolated nucleic acid" means, according to the present invention, that the nucleic acid has been: (i) amplified in vitro, for example, by polymerase chain reaction (PCR); (ii) produced recombinantly by cloning; (iii) purified, for example, by cleavage and gel electrophoresis fractionation; or (iv) synthesized, for example, by chemical synthesis. An isolated nucleic acid is a nucleic acid that can be used for manipulation by recombinant DNA techniques.

[0107] The term "recombinant" in the context of the present invention means "produced by genetic engineering." Preferably, in the context of the present invention, "recombinant material" such as recombinant cells is not naturally occurring.

[0108] As used herein, the term "naturally occurring" refers to the fact that a substance can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus) and can be isolated from a source in nature and has not been intentionally modified by man in an experiment is naturally occurring.

[0109] According to the present invention, the term "host cell" refers to any cell that can be transformed or transfected with exogenous nucleic acid. According to the present invention, the term "host cell" includes prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., yeast cells and insect cells). Particularly preferred are mammalian cells, such as cells from humans, mice, hamsters, pigs, goats, primates. Cells can derive from various tissue types and include primary cells and cell lines. Some specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In other embodiments, the host cell is an antigen presenting cell, particularly a dendritic cell, a monocyte, or a macrophage. Nucleic acid can be present in a host cell with a single copy or with several copies, and is expressed in a host cell in one embodiment.

[0110] According to the present invention, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising 2 or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 20 or more, and preferably up to 50, preferably 100 or preferably 150 consecutive amino acids linked to each other by peptide bonds. The term "protein" refers to large peptides, preferably peptides having at least 151 amino acids, but in general the terms "peptide" and "protein" are used synonymously herein.

[0111] The terms "peptide" and "protein" according to the invention encompass substances containing not only amino acid components but also non-amino acid components such as sugar or phosphate structures, and also substances containing bonds such as ester, thioether or disulfide bonds.

[0112] According to the present invention, nucleic acid such as RNA can encode peptides or proteins. Therefore, transcribable nucleic acid sequence or its transcript can comprise an open reading frame (ORF) encoding a peptide or protein. The nucleic acid can express the encoded peptide or protein. For example, the nucleic acid can be a nucleic acid that encodes and expresses an antigen or a pharmaceutically active peptide or protein such as an immunoreactive compound (which is preferably not an antigen).

[0113] According to the present invention, the term "nucleic acid encoding a peptide or protein" means that, if present in a suitable environment, preferably within a cell, the nucleic acid can direct the assembly of amino acids to produce a peptide or protein during translation. Preferably, the RNA according to the present invention is capable of interacting with the cellular translation machinery to allow translation of the peptide or protein.

[0114] In one aspect, the inventors of the present application screened out 5'UTR and 3'UTR combinations that can significantly increase the expression level of mRNA. As demonstrated in the examples of the present application, various 5'UTR and 3'UTR combinations can unexpectedly significantly increase the protein expression level of mRNA.

[0115] More specifically, when the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 3, 4, 12, 17, 19, 20, 21 or 22, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 25, 28 or 29, the combination of the 5'UTR and the 3'UTR can significantly improve the translation efficiency of the target gene coding sequence, thereby significantly improving its expression level.

[0116] Therefore, in some embodiments, the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 3, 4, 12, 17, 19, 20, 21 or 22.

[0117] In some embodiments, the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 25, 28 or 29.

[0118] In some embodiments, the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19 or 22, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 25, 28 or 29.

[0119] In some embodiments, the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 3, 4 or 12, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 28 or 29.

[0120] In some embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24 or 28.

[0121] In some embodiments, the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 20 or 21, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 28 or 28.

[0122] In some embodiments, the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 3, 19 or 22, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 25, 28 or 29.

[0123] In some embodiments, the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4, 12, 17, 20 or 21, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 28 or 29.

[0124] In some embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24, 25, 28 or 29.

[0125] In some embodiments, the 5'UTR and 3'UTR combination does not include RNA sequences corresponding to the following nucleic acid sequences: SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 20 and SEQ ID NO: 24, and SEQ ID NO: 21 and SEQ ID NO: 25.

[0126] In some embodiments, the mRNA molecule further comprises a nucleic acid sequence encoding a polypeptide of interest.

[0127] In some embodiments, the mRNA molecule further comprises polyA.

[0128] In some embodiments, the mRNA can be advantageously used in gene therapy, genetic vaccination, protein replacement therapy, antisense therapy, or therapy by interfering RNA.

[0129] In one aspect, the present disclosure provides use of a combination of a 5'UTR and a 3'UTR in increasing protein expression or translation efficiency of an mRNA, wherein the 5'UTR and the 3'UTR are as defined above.

[0130] Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" as used herein are intended to include the plural forms as well. In addition, the open-ended expressions "including" and "comprising" are interpreted as also including structural components or method steps that are not mentioned, but it should be noted that such open-ended expressions also cover situations where the composition consists only of the components and method steps (i.e., they cover situations where the closed-ended expression "consists of...").

[0131] In general, the term "about" is used herein to refer to a numerical value above and below the stated value with a modification of 5%.

[0132] The exemplary 5'UTR and 3'UTR sequences of the present application are shown in Table 1.

[0133] Table 1. Exemplary 5'UTR and 3'UTR sequences

[0134] Example 1: Preparation of RNA

[0135] 1) In vitro transcription template preparation: Use PCR to amplify a DNA sequence containing the T7 promoter, 5' UTR, target gene, and 3' UTR for in vitro transcription. The specific construct is: T7 promoter (sequence shown in SEQ ID NO: 37) + BamHI restriction site (GGATCC) + 5' UTR + KOZAK (GCCACC) + CDS + EcoRI restriction site (GAATTC) + 3' UTR + SpeI restriction site (ACTAGT).

[0136] 2) In vitro transcription: Using the PCR product as a template, in the presence of a mixture of rNTPs, DTT, T7 RNA polymerase, RNase inhibitor, and reaction buffer, complete in vitro transcription at 37°C for 16 hours. DNase I was added to the reaction system and incubated at 37°C for 30 minutes to digest the DNA template. The transcription product was purified by lithium chloride precipitation.

[0137] 3) RNA Capping: Uncapped RNA is pre-denatured at 65°C. GTP, capping reaction solution, S-adenosylmethionine, vaccinia capping enzyme, RNase inhibitor, and 2'-O-methyltransferase are mixed in a specific ratio and incubated at 37°C for a specified time to complete RNA capping. Purification is performed by lithium chloride precipitation.

[0138] 4) RNA tailing: The capped RNA was tailed using a polyA polymerase kit (Vazyme, DD4111) according to the kit instructions. After the reaction, the RNA was purified by lithium chloride precipitation.

[0139] In vitro expression detection

[0140] 1) Cell culture and in vitro transfection: The complete culture medium for HEK293T and Hela cells was DMEM high glucose medium (Hyclone) containing 10% FBS (Hyclone), and the complete culture medium for A459 cells was RPMI 1640 medium (Gibco) containing 10% FBS (Hyclone). The day before mRNA transfection, cells were plated at 2*10 4 Cells / well were seeded in 96-well cell culture plates (Corning). The next day, mRNA was transfected using Lipofectamine 3000 (Invitrogen). The transfection method was described in the instructions for Lipofectamine 3000.

[0141] 2) Detection of GFP expression: 16 h after cell transfection, the 96-well plate was placed in a microplate reader (Tecan), excited at a wavelength of 488 nm, and the OD value at a wavelength of 507 nm was detected.

[0142] 3) EPO (its CDS sequence is shown in SEQ ID NO: 38) expression detection: 16 hours after cell transfection, the supernatant was collected and the expression of ELISA was performed using the Human Erythropoietin ELISA (R&D) kit. For detailed procedures, refer to the kit instructions.

[0143] Example 2: Expression of GFP with different 5'UTR in 293T cell lines (microplate reader):

[0144] In order to test the effect of different 5'UTRs on the expression level of GFP (whose CDS sequence is shown in SEQ ID NO: 39), in this example, the 3'UTR was fixed to UTR3-1. The experimental results are shown in Table 2 and Figure 1 below.

[0145] Table 2. Effects of different 5'UTRs on GFP expression

[0146] Example 3: Expression of GFP with different 3'UTRs in 293T cell lines (microplate reader):

[0147] In order to test the effect of different 3'UTRs on GFP expression, in this example, the 5'UTR was fixed at 0.28 M. The experimental results are shown in Table 3 and Figure 2 below.

[0148] Table 3. Effects of different 3'UTRs on GFP expression

[0149] Example 4: Experiments on combinations of different 5'UTRs and different 3'UTRs

[0150] The sequences with better effects in Example 2-3 (UTR5-32, UTR5-30, UTR5-83, UTR5-59, UTR5-92, UTR5-91, ABOC-028M; UTR3_32, UTR3_34, UTR3_1, UTR3_7, UTR3_5) were combined. After template preparation, in vitro transcription, RNA capping and tailing, and mRNA purity quality inspection, mRNA preparation and detection were completed. The qualified mRNA was transfected into HEK293T, Hela and A549 cells respectively, and the cell supernatant was removed 16 hours after transfection. ELISA was performed using the Human Erythropoietin ELISA (R&D) kit to measure EPO (erythropoietin) expression in various cell lines (unit: mIU / ml). This example tested a total of 38 UTR and 3'UTR combinations of the present invention. The effects of various 5'UTR and 3'UTR combinations on EPO expression in HEK293T, HeLa, and A549 cell lines are shown in Tables 4, 5, and 6, respectively.

[0151] Table 4. Effects of various combinations on EPO expression in HEK293T cell lines

[0152] Table 5. Effects of various combinations on EPO expression in Hela cell lines

[0153] Table 6. Effects of various combinations on EPO expression in A549 cell lines

[0154] The above examples demonstrate that, relative to control combination 1, in 293T cells, the EPO expression of combinations 1-19, 21, 23-28, 30-32, and 34 of the present invention is significantly higher, with the highest being approximately 2.3 times that of control combination 1 (combination 10 of the present invention); in Hela cells, the EPO expression of combinations 1-28, 30-32, and 34 of the present invention is significantly higher, with the highest being approximately 2.8 times that of control combination 1 (combination 10 of the present invention); in A549 cells, the EPO expression of combinations 2, 4-11, 13, 15, 16, 18, 19, 23, 26-28, and 30 of the present invention is significantly higher, with the highest being approximately 1.8 times that of control combination 1 (combination 4 of the present invention).

[0155] Based on these data, it can be concluded that when the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19 or 22, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 25, 28 or 29; when the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 3, 4 or 12, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 28 or 29; when the 5'UTR is the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24 or 28; or when the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 20 or 21, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 28 or 28; the combination of the 5'UTR and the 3'UTR can significantly improve the translation efficiency of the target gene coding sequence, thereby significantly improving its expression level.

[0156] The above examples also confirmed that, compared with the control combination 2, in 293T cells, the EPO expression of the 38 UTR and 3'UTR combinations of the present invention was significantly higher, up to about 4.1 times that of the control combination 2 (combination 10 of the present invention); in Hela cells, the EPO expression of the combinations 1-33, 34 and 35 of the present invention was significantly higher, up to about 3.2 times that of the control combination 2 (combination 10 of the present invention); in A549 cells, the EPO expression of the combinations 1, 2, 4-19, 21-28, 30-35 of the present invention was significantly higher, up to about 3.1 times that of the control combination 1 (combination 4 of the present invention).

[0157] Based on these data, it can be concluded that when the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 3, 19 or 22, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 25, 28 or 29; when the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4, 12, 17, 20 or 21, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 28 or 29; or when the 5'UTR is the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24, 25, 28 or 29; the combination of the 5'UTR and the 3'UTR can significantly improve the translation efficiency of the target gene coding sequence, thereby significantly improving its expression level.

[0158] Example 5: In vivo experiments

[0159] In vivo testing was performed on some of the combinations in Example 4 (as shown in Table 7). The CDS sequence is shown in SEQ ID NO: 38. After template preparation, in vitro transcription, RNA capping and tailing, and mRNA purity quality inspection, mRNA preparation and testing were completed to obtain samples 1-8.

[0160] Preparation and detection of nanolipid particles

[0161] Cationic lipids, DSPC, cholesterol and PEG-lipids were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5, and mRNA was diluted in 10 to 50 mM citrate buffer (pH = 4). Liposomes were prepared by mixing ethanol lipid solution and mRNA aqueous solution in a volume ratio of 1:3 using a microfluidic device. The total dialysis flow rate was 9-30 mL / min, whereby ethanol was removed and replaced with DPBS. Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter.

[0162] Animal studies

[0163] Lipid nanoparticles containing samples 1-8 of encapsulated human erythropoietin (hEPO) mRNA were systemically administered to 6-8 week old female ICR mice via tail vein injection (Xipuer-Bikai, Shanghai) at a dose of 0.5 mg / kg (wherein sample 8 was the comparative combination 2 described in Example 4, as a control). Blood was collected from the mice 6 hours after administration, and the blood samples were snap-frozen and stored at -80°C for analysis. ELSA analysis was performed using a commercially available kit (DEP00, R&D Systems) according to the manufacturer's instructions.

[0164] The following table lists the characteristics of the lipid nanoparticles tested, including the hEPO expression levels (μg / ml) measured from the test groups.

[0165] Table 7

[0166] The results of the above in vivo experiments showed that, consistent with the results of the in vivo experiments, the combination of the 5'UTR and 3'UTR can also significantly improve the translation efficiency of the target gene coding sequence in vivo, thereby significantly increasing its expression level.

[0167] Although various embodiments of the present invention have been described above, it should be understood that they are provided by way of example only and not limitation. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications will fall within the scope of the present invention as claimed. The scope of the present invention as claimed is defined by the appended claims and their equivalents.

Claims

1. An mRNA molecule comprising a 5'UTR and a 3'UTR, wherein the 5'UTR is selected from the following (1)-(5): (1) comprising a 5'UTR of an RNA sequence corresponding to any one of the nucleic acid sequences shown in SEQ ID NOs: 1-22, a homologue, a fragment or a variant thereof, wherein the homologue, fragment or variant has the same or better function of improving translation efficiency as the 5'UTR of the RNA sequence corresponding to any one of the nucleic acid sequences shown in SEQ ID NOs: 1-22; preferably, the nucleic acid sequence of the homologue has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA sequence corresponding to any one of the nucleic acid sequences shown in SEQ ID NOs: 1-22; (2) a 5'UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in any one of SEQ ID NOs: 1-22; (3) a 5'UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in any one of SEQ ID NOs: 4, 3, 12, 17, or 19-22; (4) A 5'UTR obtained by concatenating two or more identical 5'UTRs in (1) to (3) above; or (5) 5'-UTR obtained by concatenating two or more different 5'UTRs in (1)-(3) above, And wherein the 3'UTR is selected from the following (1)-(5): (1) comprising a 3'UTR of an RNA sequence corresponding to the nucleic acid sequence of any one of SEQ ID NOs: 23-36, a homologue, a fragment or a variant thereof, wherein the homologue, fragment or variant has the same or better function of improving translation efficiency as the 3'UTR of the RNA sequence corresponding to the nucleic acid sequence of any one of SEQ ID NOs: 23-36; preferably, the nucleic acid sequence of the homologue has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA sequence corresponding to the nucleic acid sequence of any one of SEQ ID NOs: 23-36; (2) RNA corresponding to the nucleic acid sequence shown in any one of SEQ ID NOs: 23-36 The 3'UTR composed of the sequence; (3) a 3'UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 28 or 29; (4) A 3'UTR obtained by concatenating two or more identical 3'UTRs in (1) to (3) above; or (5) 3′–UTR obtained by concatenating two or more different 3′UTRs in (1)-(3) above; And the combination of 5'UTR and 3'UTR does not include RNA sequences corresponding to the following nucleic acid sequences: SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 20 and SEQ ID NO: 24, and SEQ ID NO: 21 and SEQ ID NO:

25.

2. The mRNA molecule according to claim 1, wherein the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4, 3, 12, 17, 19, 20, 21 or 22.

3. The mRNA molecule according to claim 1, wherein the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 25, 28 or 29.

4. The mRNA molecule according to any one of claims 1 to 3, wherein (1) The 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4, 3 or 12, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 28 or 29; (2) the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19 or 22, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 25, 28 or 29; (3) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24 or 28; (4) The 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 20 or 21, and the 3'UTR is selected from the nucleic acid sequence shown in SEQ ID NO: 23, 28 or 29. The RNA sequence corresponding to the acid sequence; (5) the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 3, 19 or 22, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 25, 28 or 29; (6) the 5'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4, 12, 17, 20 or 21, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 28 or 29; or (7) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is selected from the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24, 25, 28 or 29.

5. The mRNA molecule according to any one of claims 1 to 3, wherein (1) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23; (2) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23; (3) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24; (4) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 25; (5) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28; (6) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:

29. List; (7) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24; (8) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28; (9) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 29; (10) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23; (11) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24; (12) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28; (13) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 29; (14) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23; (15) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24; (16) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 25; (17) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28; (18) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 29; (19) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23; (20) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24; (21) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 25; (22) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28; (23) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 29; (24) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 20, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23; (25) The 5'UTR is the RNA corresponding to the nucleic acid sequence shown in SEQ ID NO: 20 sequence, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28; (26) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 20, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 29; (27) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 21, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23; (28) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 21, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24; (29) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 21, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28; (30) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 24; (31) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 25; (32) the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28; or (33) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:

29.

6. The mRNA molecule according to any one of claims 1 to 3, wherein the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:

23.

7. The mRNA molecule according to any one of claims 1 to 3, wherein the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:

28.

8. The mRNA molecule according to any one of claims 1 to 3, further comprising a nucleic acid sequence encoding a polypeptide of interest.

9. The mRNA molecule according to any one of claims 1 to 3, further comprising polyA.

10. Use of a combination of a 5'UTR and a 3'UTR for increasing protein expression or translation efficiency of mRNA, wherein the 5'UTR and the 3'UTR are as defined in any one of claims 1 to 7.

11. A DNA encoding the mRNA molecule according to any one of claims 1 to 9. A vector comprising the DNA according to claim 11 . A host cell comprising the vector according to claim 12 .

14. A lipid nanoparticle comprising the mRNA molecule of any one of claims 1-9.

15. A pharmaceutical composition comprising the mRNA molecule according to any one of claims 1 to 9, the DNA according to claim 11, the vector according to claim 12, the host cell according to claim 13 or the lipid nanoparticle according to claim 14, and a pharmaceutically acceptable carrier.

16. Use of the mRNA molecule according to any one of claims 1 to 9, the DNA according to claim 11, the vector according to claim 12, the host cell according to claim 13, the lipid nanoparticle according to claim 14, or the pharmaceutical composition according to claim 15 in the preparation of a medicament for gene therapy, gene vaccination, protein replacement therapy, antisense therapy, or treatment by interfering RNA.

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