UTR molecules that increase protein expression

Abstract

The present disclosure relates to UTR molecules and uses thereof. Specifically, the present disclosure relates to mRNA molecules comprising a 5'UTR and a 3'UTR. The UTR molecules can significantly increase mRNA expression levels.

Description

[Technical Field]

[0001] This application claims priority to PCT Application No. PCT / CN2022 / 134038, filed November 24, 2022, which is incorporated herein by reference in its entirety.

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

[0003] As a gene therapy, mRNA vaccines are very promising agents. However, mRNA is unstable and easily degraded by ubiquitous RNAases. In vivo, RNA degradation can help regulate RNA half-life and fine-tune gene expression control in eukaryotes (Friedel CC, Dolken L, Ruzsics Z, Koszinowski UH, Zimmer R. Nucleic Acids Res. 2009;37(17):e115-e115. doi:10.1093 / NAR / GKP542). Stable RNA is required for the expression of RNA medicines. Many methods for regulating the stability of nucleic acid molecules include 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] UTRs are important factors for 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 is involved in the formation of a specific nucleoprotein complex (α complex) and is associated with in vitro mRNA stability (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 experimental data from 280,000 randomized 5'UTRs to design 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 for 5UTRs and 3UTRs that could increase expression through a fragment library (US2022 / 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 aforementioned advances, there is still an urgent need for combinations of UTR molecules that can stabilize mRNA molecules and increase protein expression levels. Summary of the Invention

[0006] The inventors of the present application have unexpectedly discovered that certain 5'UTR and 3'UTR combinations described herein can significantly increase the protein expression level of an mRNA molecule, and therefore, the mRNA can be advantageously used in gene therapy, gene vaccination, protein replacement therapy, antisense therapy, or interfering RNA therapy.

[0007] In one aspect, the present disclosure provides an mRNA molecule comprising a 5'UTR and a 3'UTR, wherein the 5'UTR is one of the following (1) to (5): (1) A 5'UTR comprising an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 1 to 22, or homologs, fragments, or variants thereof, wherein the homologs, fragments, or variants have the same or superior function of improving translation efficiency as a 5'UTR comprising an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 1 to 22, and preferably, the homolog nucleic acid sequence has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 1 to 22; (2) a 5'UTR consisting of an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 1 to 22; (3) a 5'UTR consisting of an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 4, 3, 12, 17, or 19 to 22; (4) a 5'UTR derived from two or more identical 5'UTRs in (1) to (3) linked in tandem; or (5) A 5'UTR obtained from two or more different 5'UTRs among (1) to (3) linked in tandem; and the 3'UTR is selected from the following (1) to (5): (1) A 3'UTR comprising an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 23 to 36, or their homologs, fragments, or variants, wherein the homologs, fragments, or variants have the same or superior function of improving translation efficiency as a 3'UTR comprising an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 23 to 36, and preferably, the homolog nucleic acid sequence has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 23 to 36; (2) a 3'UTR consisting of an RNA sequence corresponding to any one of the nucleic acid sequences set forth in SEQ ID NOs: 23 to 36; (3) a 3'UTR consisting of an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 23, 24, 28, or 29; (4) a 3'UTR derived from two or more identical 3'UTRs in (1) to (3) linked in tandem; or (5) A 3'UTR obtained from two or more different 3'UTRs among (1) to (3) linked in tandem; is selected from Furthermore, 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. The mRNA molecule is provided.

[0008] In some embodiments, the 5'UTR is selected from an RNA sequence corresponding to a nucleic acid sequence set forth in SEQ ID NO: 4, 3, 12, 17, 19, 20, 21, or 22.

[0009] In some embodiments, the 3′UTR is selected from an RNA sequence corresponding to a nucleic acid sequence set forth in SEQ ID NO: 23, 24, 25, 28, or 29.

[0010] In some embodiments, the 5' UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 19 or 22, and the 3' UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24, 25, 28, or 29.

[0011] In some embodiments, the 5'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 4, 3, or 12, and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24, 28, or 29.

[0012] In some embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 17, and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24, or 28.

[0013] In some embodiments, the 5'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 20 or 21, and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 28, or 28.

[0014] In some embodiments, the 5'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 3, 19, or 22, and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24, 25, 28, or 29.

[0015] In some embodiments, the 5'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 4, 12, 17, 20, or 21, and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24, 28, or 29.

[0016] In some embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 19, and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 24, 25, 28, or 29.

[0017] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:23.

[0018] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:24.

[0019] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:25.

[0020] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:28.

[0021] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:29.

[0022] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:23.

[0023] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:24.

[0024] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:28.

[0025] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:29.

[0026] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:23.

[0027] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:24.

[0028] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:28.

[0029] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:29.

[0030] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:23.

[0031] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:24.

[0032] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:25.

[0033] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:28.

[0034] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:29.

[0035] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:23.

[0036] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:24.

[0037] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:25.

[0038] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:28.

[0039] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:29.

[0040] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:20, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:23.

[0041] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:20, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:28.

[0042] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:20, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:29.

[0043] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:21, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:23.

[0044] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:21, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:24.

[0045] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:21, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:28.

[0046] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:24.

[0047] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:25.

[0048] In certain embodiments, the 5' UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 22 and the 3' UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 28. Or

[0049] In certain embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:29.

[0050] In some embodiments, the mRNA molecule further comprises a nucleic acid sequence encoding a target polypeptide.

[0051] In some embodiments, the mRNA molecule further comprises a polyA.

[0052] In one aspect, the present disclosure provides a method for increasing protein expression levels or translation efficiency of an mRNA using an mRNA molecule comprising a combination of 5'UTR and 3'UTR, or a 5'UTR and a 3'UTR, as defined above.

[0053] In one aspect, the present disclosure provides an mRNA molecule comprising a combination of 5'UTR and 3'UTR, or 5'UTR and 3'UTR, as defined above, for use in increasing protein expression levels or translation efficiency of the mRNA.

[0054] In one aspect, the present disclosure provides the use of a combination of 5'UTR and 3'UTR as defined above to increase protein expression yield or translation efficiency of mRNA.

[0055] In one aspect, the present disclosure provides DNA encoding the aforementioned mRNA molecules.

[0056] In one aspect, the present disclosure provides a vector comprising the aforementioned DNA.

[0057] In one aspect, the present disclosure provides a host cell comprising the aforementioned vector.

[0058] In one aspect, the present disclosure provides lipid nanoparticles comprising the aforementioned mRNA molecules.

[0059] In one aspect, the present disclosure provides a pharmaceutical composition comprising the mRNA molecule, the DNA, the vector, the host cell, or the lipid nanoparticle, and a pharmaceutically acceptable carrier.

[0060] In one aspect, the present disclosure provides a method for performing gene therapy, gene vaccination, protein replacement therapy, antisense therapy, or interfering RNA therapy using the aforementioned mRNA molecule, the aforementioned DNA, the aforementioned vector, the aforementioned host cell, the aforementioned lipid nanoparticle, or the aforementioned pharmaceutical composition.

[0061] 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 interfering RNA therapy.

[0062] In one aspect, the present disclosure provides use of the aforementioned mRNA molecule, the aforementioned DNA, the aforementioned vector, the aforementioned host cell, the aforementioned lipid nanoparticle, or the aforementioned pharmaceutical composition in the preparation of a medicament for gene therapy, gene vaccination, protein replacement therapy, antisense therapy, or interfering RNA therapy. [Brief explanation of the drawings]

[0063] [Figure 1] The effect of different 5'UTRs on GFP expression levels is shown. [Figure 2] The effect of different 3'UTRs on GFP expression levels is shown. DETAILED DESCRIPTION OF THE INVENTION

[0064] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. Furthermore, it should be understood that the terminology used herein is intended only to describe the purpose of some particular embodiments, and does not 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 one of ordinary skill in the art.

[0065] Unless otherwise indicated, the practice of the present invention employs conventional chemical, biochemical, cell biological, immunological, and recombinant DNA techniques as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0066] Unless the context requires otherwise, throughout this specification and the appended claims, the word "comprising" and variations thereof shall be understood to imply the inclusion of the stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps. Unless otherwise indicated herein or clearly contradicted by context, nouns without quantifiers used in the context of describing the invention (particularly in the context of the claims) should be construed to refer to one and / or more than one. The recitation of ranges of values ​​herein is intended merely as shorthand for individual reference to each individual value falling within the range. Unless otherwise stated herein, each individual value is incorporated herein as if individually recorded herein. Unless otherwise indicated herein or clearly contradicted by context, all methods described herein may be performed in any suitable order. Any use of examples or exemplary language (e.g., "for example / such as") provided herein is intended only to better describe the invention and does not limit the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element necessary to the practice of the invention.

[0067] According to the present invention, a nucleic acid molecule or nucleic acid sequence refers to a nucleic acid, preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to the present invention, nucleic acid includes genomic DNA, cDNA, mRNA, recombinant molecules, and chemically synthesized molecules. According to the present invention, a nucleic acid can be in the form of a single-stranded or double-stranded linear or covalently closed circular molecule.

[0068] In the context of the present invention, the term "RNA" refers to a molecule containing ribonucleotide residues, preferably consisting entirely or substantially of ribonucleotide residues. 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, e.g., modified RNA that differs from naturally occurring RNA in that one or more nucleotides have been added, deleted, substituted, and / or modified. Such modifications may include, for example, the addition of non-nucleotide material to the end or within the RNA, e.g., the addition of non-nucleotide material to one or more nucleotides of the RNA. The nucleotides of an RNA molecule may also include non-standard nucleotides, e.g., non-naturally occurring nucleotides, or chemically synthesized nucleotides, or deoxynucleotides. These modified RNAs may be referred to as analogs, particularly analogs of naturally occurring RNA. According to the present invention, RNA includes mRNA.

[0069] The term "mRNA" refers to "messenger RNA" and relates to a transcription product produced using a DNA template and encoding a peptide or protein. Generally, mRNA comprises a 5'UTR, a protein-coding region, a 3'UTR, and a poly(A) sequence. mRNA can be produced by transcribing a DNA template in vitro. In vitro transcription methods are known to those skilled in the art. For example, various 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 stabilizing modifications and capping.

[0070] The term "nucleic acid" according to the present invention also includes chemical derivatization of nucleic acids on the nucleotide base, sugar, or phosphate, as well as nucleic acids containing non-naturally occurring nucleotides and nucleotide analogs.

[0071] A "fragment" or "fragment of a nucleic acid sequence" refers to a portion of a nucleic acid sequence, i.e., a sequence that appears as a nucleic acid sequence truncated at the 5' and / or 3' end. Preferably, when the fragment replaces a nucleic acid sequence in an RNA molecule, it retains the stability and / or translation efficiency of the RNA. 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.

[0072] The term "variant" according to the present invention, for example with respect to nucleic acid and amino acid sequences, includes all variants, in particular mutants, splice variants, conformers, isomers, allelic variants, species variants, and species homologs, especially those occurring naturally. Allelic variants involve changes in the normal sequence of a gene, the significance of which is often unknown. Complete gene sequencing often identifies multiple allelic variants of a gene. Species homologs are nucleic acid or amino acid sequences that have a different species origin from the given nucleic acid or amino acid sequence.

[0073] According to the present invention, nucleic acid variants include deletions, additions, mutations, and / or insertions of single or multiple nucleotides compared to a reference nucleic acid. Deletions involve removing one or more nucleotides from the reference nucleic acid. Addition variants include 5' and / or 3' terminal fusions of one or more nucleotides (e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more nucleotides). Mutations include, but are not limited to, substitutions (e.g., translocations and transitions) in which at least one nucleotide in a sequence is removed and another nucleotide is inserted in its place, abasic sites, crosslinked sites, and chemically altered or modified bases. Insertions involve adding at least one nucleotide to the reference nucleic acid.

[0074] In the context of nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, which in the present invention are nucleic acids that differ in codon sequence from a reference nucleic acid due to the degeneracy of the genetic code.

[0075] 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 having 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.

[0076] "Sequence similarity" refers to the percentage of amino acids that are identical or that exhibit 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 the sequences.

[0077] The term "% identity" is intended to refer, inter alia, to the percentage of identical nucleotides in optimal alignment between two sequences being compared, where this percentage is purely statistical and the differences between the two sequences may be randomly distributed over the entire length of the sequences, and the compared sequence may contain additions or deletions relative to the reference sequence to achieve optimal alignment between the two sequences. Comparison of two sequences is typically performed by comparing the sequences after optimal alignment over a portion or "comparison window" to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually, using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, and using the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or using computer programs that employ such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0078] The percentage of identity is obtained by determining the number of identical positions in the sequences being compared, dividing that number by the number of compared positions, and multiplying the result by 100. For example, the BLAST program "BLAST 2 sequences," available on the website (http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi), can be used.

[0079] All consecutive residues of a nucleic acid sequence will hydrogen bond with the same number of consecutive residues of a second nucleic acid sequence. Preferably, the degree of complementarity according to the present invention is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98%, or 99%. Most preferably, the degree of complementarity according to the present invention is 100%.

[0080] A fragment or variant of a particular nucleic acid sequence, or a nucleic acid sequence having a particular identity to a particular nucleic acid sequence, preferably has at least one functional characteristic of the particular sequence, and preferably is functionally equivalent to the particular sequence, e.g., is a nucleic acid sequence that exhibits the same or similar characteristics as the particular nucleic acid sequence.

[0081] The term "promoter" or "promoter region" refers to a DNA sequence upstream (5') of a gene's coding sequence that controls expression of the coding sequence by providing recognition and binding sites for RNA polymerase. A promoter region may also contain recognition or binding sites for other factors involved in regulating gene transcription. Promoters can control the transcription of prokaryotic or eukaryotic genes. Promoters can be "inducible," initiating transcription in response to an inducer, or "constitutive," in which transcription is not controlled by an inducer. In the absence of an inducer, an inducible promoter is expressed to a low degree or not at all. In the presence of an inducer, the gene is "turned on," resulting in increased levels of transcription, usually mediated by the binding of specific transcription factors.

[0082] 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 partial expression of nucleic acids. Furthermore, expression can be transient or stable. With respect to RNA, the terms "expression" or "translation" refer to the process by which a chain of messenger RNA directs the assembly of a sequence of amino acids in the ribosomes of a cell to produce a peptide or protein.

[0083] In the context of the present invention, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into proteins. According to the invention, the term "transcription" includes "in vitro transcription," which refers to the process by which RNA, particularly mRNA, is synthesized in vitro in a cell-free system. Preferably, a cloning vector is applied to produce the transcript. These cloning vectors are generally named transcription vectors and, according to the present invention, are referred to by the term "vector." According to the present invention, the RNA is preferably in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for 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.

[0084] The term "untranslated region" or "UTR" as used in accordance with the present invention relates to the portions of the mRNA upstream of the start codon and downstream of the stop codon, which are not translated and are therefore called the 5' untranslated region (5'UTR) and the 3' untranslated region (3'UTR), respectively. These regions are transcribed together with the coding region and are therefore exon-like when present in the mature mRNA.

[0085] 3'-Untranslated Region (3'UTR): Generally, the term "3'UTR" refers to a portion of an artificial nucleic acid molecule located 3' (i.e., "downstream") of an open reading frame and not translated into protein. Typically, the 3'UTR is the portion of an mRNA between the protein-coding region (open reading frame (ORF) or coding sequence (CDS)) and the polyadenylate sequence of the mRNA. In the context of the present invention, the term 3'UTR may also include elements, such as polyadenylate sequences, that are not encoded in the template from which the RNA is transcribed but are added during post-transcriptional maturation. The 3'UTR of an mRNA is not translated into an amino acid sequence. The 3'UTR sequence is usually encoded by the gene, which is transcribed into the respective mRNA during gene expression. The genomic sequence is first transcribed into pre-mRNA, including any introns. This pre-mRNA is then further processed into mature mRNA during maturation. This pre-mRNA is then further processed into mature mRNA during maturation. The maturation process includes the following steps: 5'-capping, splicing of the pre-mRNA to remove any introns, and modification of the 3' end (e.g., polyadenylation of the 3' end of the pre-mRNA and any endonuclease / exonuclease cleavage). Within the scope of the present invention, the 3'-UTR corresponds to the sequence of the mature mRNA between the stop codon of the protein-coding region, preferably the stop codon immediately 3'-end of 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, for example, an RNA sequence in the mRNA sequence used to define the 3'-UTR sequence, or a DNA sequence corresponding to the RNA sequence. Within the scope of the present invention, the term "3'-UTR of a gene," e.g., "3'-UTR of a ribosomal protein gene," refers to a sequence corresponding to the 3'-UTR derived from the mature mRNA of the gene, the mature mRNA being the mRNA obtained by gene transcription and maturation of the pre-mRNA. The term "3'UTR of a gene" includes DNA and RNA sequences (both sense and antisense, and both mature and premature) of the 3'UTR.

[0086] 5'-untranslated region (5'UTR): 5'UTR is generally understood as a specific portion of messenger RNA (mRNA). It is located 5' to the open reading frame of the mRNA. Typically, the 5'UTR begins at the transcription initiation site and ends at the nucleotide preceding the start codon of the open reading frame. The 5'UTR may contain elements, also known as regulatory elements, that control gene expression. Such regulatory elements are, 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 located between the 5'-cap and the start codon. Preferably, the 5'UTR corresponds to the sequence extending from the nucleotide 3' to the 5'-cap, preferably the nucleotide immediately adjacent to the 3' side of the 5'-cap, to the nucleotide 5' to the start codon of the protein-coding region, preferably the nucleotide 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, for example, an RNA sequence in the mRNA sequence used to define the 5'-UTR sequence, or a DNA sequence corresponding to the RNA sequence. Within the scope of the present invention, the term "5'-UTR of a gene" refers to a sequence corresponding to the 5'-UTR derived from the mature mRNA of a gene, which is the mRNA obtained by gene transcription and maturation of a pre-mRNA. The term "5'-UTR" of a gene includes the DNA and RNA sequences of the 5'-UTR.

[0087] According to the present invention, the term "gene" refers to a specific nucleic acid sequence that produces one or more cellular products and / or performs one or more inter- or intracellular functions. More specifically, the term relates to a DNA segment that comprises a nucleic acid that encodes a specific protein or a functional or structural RNA molecule.

[0088] Polyadenylation adds a poly(A) sequence (PolyA) or poly(A) tail to the primary transcript RNA. A poly(A) sequence consists of multiple adenosine monophosphates. In other words, it is a segment of RNA containing only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation. Thus, polyadenylation forms part of the larger gene expression process. Polyadenylation begins at the end, or termination, of gene transcription. The 3'-most segment of the newly produced pre-mRNA is first cleaved by a series of proteins. These proteins then synthesize a poly(A) sequence at the 3' end of the RNA. The poly(A) sequence is important for nuclear transport, translation, and mRNA stability. This sequence shortens over time, and when it becomes short enough, the mRNA is enzymatically degraded.

[0089] The terms "polyadenylate sequence," "poly(A) sequence," or "poly(A) tail" refer to a sequence of adenylate residues typically found at the 3' end of an RNA molecule. The present invention allows such sequences to be added during RNA transcription via a DNA template based on repeated thymidylate residues in the strand complementary to the coding strand. However, such sequences are not typically encoded within DNA, but are added to the free 3' end of RNA by 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, particularly up to 150 A nucleotides, preferably consecutive A nucleotides, particularly about 120 A nucleotides. The term "A nucleotide" or "A" refers to an adenylate residue.

[0090] The nucleic acids described herein may be recombinant and / or isolated molecules.

[0091] As used herein, the term "isolated molecule" is intended to refer to a molecule that is substantially free of other molecules, such as other cellular material. According to the present invention, the term "isolated nucleic acid" refers to a nucleic acid that is (i) amplified in vitro, e.g., amplified in vitro by polymerase chain reaction (PCR); (ii) produced by cloning or recombination; (iii) purified, e.g., purified by cleavage and gel electrophoresis fractionation; or (iv) synthesized, e.g., synthesized by chemical synthesis. An isolated nucleic acid is a nucleic acid that can be manipulated by recombinant DNA techniques.

[0092] 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 a recombinant cell, is not naturally occurring.

[0093] As used herein, the term "naturally occurring" means that a substance exists in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), can be isolated from a natural source, and has not been artificially modified in an experimental setting is naturally occurring.

[0094] 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., E. coli) or eukaryotic cells (e.g., yeast cells and insect cells). Mammalian cells, such as human, mouse, hamster, pig, goat, and primate cells, are particularly preferred. Cells can be derived from a variety of tissue types and include primary cells and cell lines. Specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In yet another embodiment, the host cell is an antigen-presenting cell, particularly a dendritic cell, monocyte, or macrophage. The nucleic acid can be present in a single copy or multiple copies in the host cell and, in one embodiment, is expressed in the host cell.

[0095] According to the present invention, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising two or more, preferably three or more, more preferably four or more, preferably six or more, preferably eight or more, preferably ten or more, preferably thirteen or more, preferably six or more, preferably twenty or more, and preferably up to fifty, preferably one hundred or preferably one hundred and fifty consecutive amino acids linked together by peptide bonds. The term "protein" refers to a macropeptide, preferably a peptide having at least 151 amino acids, although generally the terms "peptide" and "protein" are used synonymously herein.

[0096] According to the present invention, the terms "peptide" and "protein" include substances that contain not only amino acid components but also non-amino acid components such as sugar or phosphate structures, and further include substances that contain bonds such as ester bonds, thioether bonds or disulfide bonds.

[0097] According to the present invention, nucleic acids such as RNA can encode peptides or proteins. Thus, the transcribable nucleic acid sequence or its transcript can contain 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 may encode and express a pharmaceutically active peptide or protein, such as an antigen or an immunologically active compound, but is preferably not an antigen.

[0098] According to the present invention, the term "nucleic acid encoding a peptide or protein" means that the nucleic acid, when present in an appropriate environment, preferably a cell, is capable of directing the assembly of amino acids during translation to produce a peptide or protein. Preferably, the RNA according to the present invention is capable of interacting with the cell's translation machinery to allow translation of the peptide or protein.

[0099] In one aspect, the inventors of the present application screen for combinations of 5'-UTR and 3'-UTR that can significantly increase mRNA expression levels. As confirmed by the embodiments of the present application, various combinations of 5'-UTR and 3'-UTR can unexpectedly significantly increase the protein expression levels of mRNA.

[0100] More specifically, when the 5'UTR is selected from RNA sequences corresponding to the nucleic acid sequences set forth in SEQ ID NO: 3, 4, 12, 17, 19, 20, 21 or 22, and the 3'UTR is selected from RNA sequences corresponding to the nucleic acid sequences set forth in SEQ ID NO: 23, 24, 25, 28 or 29, the combination of the 5'UTR and 3'UTR can significantly improve the translation efficiency of the coding sequence of the target gene, thereby significantly increasing its expression level.

[0101] Thus, in some embodiments, the 5'UTR is selected from an RNA sequence corresponding to a nucleic acid sequence set forth in SEQ ID NO: 3, 4, 12, 17, 19, 20, 21 or 22.

[0102] In some embodiments, the 3′UTR is selected from an RNA sequence corresponding to a nucleic acid sequence set forth in SEQ ID NO: 23, 24, 25, 28, or 29.

[0103] In some embodiments, the 5' UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 19 or 22, and the 3' UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24, 25, 28, or 29.

[0104] In some embodiments, the 5'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 3, 4 or 12, and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24, 28 or 29.

[0105] In some embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 17, and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24, or 28.

[0106] In some embodiments, the 5'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 20 or 21, and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 28, or 28.

[0107] In some embodiments, the 5'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 3, 19, or 22, and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24, 25, 28, or 29.

[0108] In some embodiments, the 5'UTR is selected from an RNA sequence corresponding to a nucleic acid sequence set forth in SEQ ID NO: 4, 12, 17, 20, or 21, and the 3'UTR is selected from an RNA sequence corresponding to a nucleic acid sequence set forth in SEQ ID NO: 23, 24, 28, or 29.

[0109] In some embodiments, the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 19, and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 24, 25, 28, or 29.

[0110] In some embodiments, 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.

[0111] In some embodiments, the mRNA molecule further comprises a nucleic acid sequence encoding a target polypeptide.

[0112] In some embodiments, the mRNA molecule further comprises a polyA.

[0113] In some embodiments, mRNA may be advantageously used in gene therapy, genetic vaccination, protein replacement therapy, antisense therapy, or interfering RNA therapy.

[0114] In one aspect, the present disclosure provides the use of a combination of 5'UTR and 3'UTR as defined above to increase protein expression yield or translation efficiency of mRNA.

[0115] As used herein, the singular terms "a," "an," and "the" are intended to include their plural references unless the context clearly dictates otherwise. Additionally, it is important to note that while the open-ended terms "including" and "comprising" may be interpreted to include structural components or methodological steps, the open-ended terms also cover situations consisting only of the recited components and methodological steps (i.e., also cover the closed-ended context of "consisting of").

[0116] Generally, the term "about" as used herein refers to a numerical value that varies within 5% above and below the stated value.

[0117] Exemplary 5'UTR and 3'UTR sequences of the present application are shown in Table 1.

[0118] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Example]

[0119] Example 1: RNA preparation 1) Preparation of in vitro transcription template: A DNA sequence containing a T7 promoter, 5'UTR, target gene, and 3'UTR was amplified using PCR for in vitro transcription. The specific construction was T7 promoter (the sequence of which is set forth in SEQ ID NO: 37) + BamHI restriction enzyme cleavage site (GGATCC) + 5'UTR + KOZAK (GCCACC) + CDS + EcoRI restriction enzyme cleavage site (GAATTC) + 3'UTR + SpeI restriction enzyme cleavage site (ACTAGT).

[0120] 2) In vitro transcription: Using the PCR product as a template, a reaction system containing rNTP mix, DTT, T7 RNA polymerase, RNAase inhibitor, and reaction buffer was incubated in a 37°C incubator for 16 hours to complete the in vitro transcription. DNase I was added to the reaction system, and the reaction system was incubated in a 37°C incubator for 30 minutes to digest the DNA template. The transcription product was purified by lithium chloride precipitation.

[0121] 3) RNA capping: Uncapped RNA was pre-denatured at 65°C, and then GTP, capping reaction solution, S-adenosylmethionine, vaccinia capping enzyme, RNA enzyme inhibitor, and 2'-o-methyltransferase were mixed in a specific ratio and reacted in a constant temperature bath at 37°C for a specific time to complete the capping of the RNA. Purification was performed by lithium chloride precipitation.

[0122] 4) RNA tailing: The capped RNA was tailed using a polyA polymerase kit (Vazyme, DD4111) according to the kit's instructions, and purified by lithium chloride precipitation after the reaction was completed.

[0123] In vitro expression assay 1) Cell culture and in vitro transfection: The complete medium for HEK293T and HeLa cells was DMEM high glucose medium (Hyclone) containing 10% FBS (Hyclone), and the complete medium for A459 cells was RPMI 1640 medium (Gibco) containing 10% FBS (Hyclone). The day before mRNA transfection, cells were plated in a 96-well cell culture plate (Corning) at 2 × 10 4 Cells were seeded at 1000 x g / well. The next day, mRNA transfection was performed using Lipofectamine 3000 (Invitrogen). For transfection procedures, please refer to the Lipofectamine 3000 manual.

[0124] 2) GFP expression detection: After the cells were transfected for 16 hours, 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.

[0125] 3) Expression detection of EPO (its CDS sequence is shown in SEQ ID NO: 38): After the cells were transfected for 16 hours, the supernatant was collected and subjected to ELISA using the Quantikine® IVD® Human Erythropoietin ELISA (R&D) kit. For detailed procedures, please refer to the kit's instruction manual.

[0126] Example 2: GFP expression levels of different 5'UTRs in 293T cell line (microplate reader): To examine the effect of different 5'UTRs on the expression level of GFP (whose CDS sequence is shown in SEQ ID NO: 39), the 3'UTR was fixed to UTR3-1 in this example. The experimental results are shown in Table 2 below and Figure 1.

[0127] [Table 2]

[0128] Example 3: GFP expression levels of different 3'UTRs in 293T cell line (microplate reader): To examine the effect of different 3'UTRs on the expression level of GFP, in this example the 5'UTR was fixed at 028M. The experimental results are shown in Table 3 below and Figure 2.

[0129] [Table 3]

[0130] Example 4: Experiments on combinations of different 5'UTRs and different 3'UTRs The sequences that showed good results in Examples 2 and 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 testing, mRNA preparation and detection were completed. Qualified mRNA was transfected into HEK293T, HeLa, and A549 cells, respectively. Cell supernatants were removed 16 hours after transfection, and ELISA was performed using the Quantikine® IVD® Human Erythropoietin ELISA (R&D) kit to examine the EPO (erythropoietin) expression levels (unit: mlU / ml) in different cells. In this example, a total of 38 combinations of UTRs and 3'UTRs of the present invention were tested. The effects of various combinations of 5'UTRs and 3'UTRs on EPO expression levels in HEK293T, Hela, and A549 cell lines are shown in Tables 4, 5, and 6, respectively.

[0131] [Table 4]

[0132] [Table 5]

[0133] [Table 6]

[0134] The above examples show that, compared to Comparative Combination 1, in 293T cells, Combinations 1 to 19, 21, 23 to 28, 30 to 32 and 34 of the present invention have significantly higher EPO expression, up to about 2.3 times that of Comparative Combination 1 (Combination 10 of the present invention); in Hela cells, Combinations 1 to 28, 30 to 32 and 34 of the present invention have significantly higher EPO expression, up to about 2.8 times that of Comparative Combination 1 (Combination 10 of the present invention); and in A549 cells, Combinations 2, 4 to 11, 13, 15, 16, 18, 19, 23, 26 to 28 and 30 of the present invention have significantly higher EPO expression, up to about 1.8 times that of Comparative Combination 1 (Combination 4 of the present invention).

[0135] Based on these data, it can be concluded that the combination of 5'UTR and 3'UTR can significantly improve the translation efficiency of the coding sequence of a target gene, thereby significantly increasing its expression level, when the 5'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 19 or 22 and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24, 25, 28 or 29; when the 5'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 3, 4 or 12 and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24, 28 or 29; when the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 17 and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24 or 28; or when the 5'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 20 or 21 and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 28 or 28.

[0136] The foregoing examples further show that, compared to Comparative Combination 2, in 293T cells, combinations of 38 UTRs and 3'UTRs of the present invention have significantly higher EPO expression, up to about 4.1 times that of Comparative Combination 2 (Combination 10 of the present invention); in HeLa cells, Combinations 1 to 33, 34 and 35 of the present invention have significantly higher EPO expression, up to about 3.2 times that of Comparative Combination 2 (Combination 10 of the present invention); and in A549 cells, Combinations 1, 2, 4 to 19, 21 to 28, 30 to 35 of the present invention have significantly higher EPO expression, up to about 3.1 times that of Comparative Combination 1 (Combination 4 of the present invention).

[0137] Based on these data, it can be concluded that when the 5'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 3, 19 or 22 and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24, 25, 28 or 29; when the 5'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 4, 12, 17, 20 or 12 and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, 24, 28 or 29; or when the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 19 and the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 24, 25, 28 or 29, the combination of the 5'UTR and 3'UTR can significantly improve the translation efficiency of the coding sequence of the target gene, thereby significantly increasing its expression level.

[0138] Example 5: In vivo assay Several combinations of Example 4 (shown in Table 7) were tested in vivo. The CDS sequences are as set forth in SEQ ID NO: 38. mRNA preparation and detection were completed through template preparation, in vitro transcription, RNA capping and tailing, and mRNA purity testing, yielding Samples 1 to 8.

[0139] Preparation and detection of lipid nanoparticles 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 to 10–50 mM with citrate buffer (pH = 4). Liposomes were prepared by mixing the ethanolic lipid solution with the aqueous mRNA solution at a volume ratio of 1:3 using a microfluidic splitter. The total dialysis flow rate was 9–30 mL / min, and the ethanol was removed and replaced with DPBS. Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter.

[0140] Animal experiments Lipid nanoparticles containing samples 1 to 8 encapsulating human erythropoietin (hEPO) mRNA were administered systemically to 6- to 8-week-old female ICR mice via tail vein injection (Xipuer-Bikai, Shanghai) at a dose of 0.5 mg / kg (Sample 8 was the control, Comparative Formulation 2 described in Example 4). Six hours after administration, blood was collected from the mice, and the blood samples were flash-frozen and stored at -80°C for analysis. ELISA analysis was performed using a commercially available kit (DEP00, R&D system) according to the manufacturer's instructions.

[0141] The table below lists the characteristics of the lipid nanoparticles tested, including the hEPO expression levels (μg / ml) measured from the test groups.

[0142] [Table 7]

[0143] According to the results of the in vivo assay described above, the combination of the 5'UTR and 3'UTR can significantly improve the translation efficiency of the coding sequence of the target gene in vivo, thereby significantly improving its expression level.

[0144] While 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. The present invention may be subject to various changes and modifications without departing from the spirit and scope of the present invention, which remain within the scope of the invention as claimed. The scope of protection claimed by the present invention 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 one of the following (1) to (5): (1) A 5'UTR comprising an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 1 to 22, or homologs, fragments, or variants thereof, wherein the homologs, fragments, or variants have the same or superior function of improving translation efficiency as a 5'UTR comprising an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 1 to 22, and preferably, the homolog nucleic acid sequence has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 1 to 22; (2) a 5'UTR consisting of an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 1 to 22; (3) a 5'UTR consisting of an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 4, 3, 12, 17, or 19-22; (4) a 5'UTR derived from two or more identical 5'UTRs in (1) to (3) linked in tandem; or (5) A 5'UTR obtained from two or more different 5'UTRs in (1) to (3) linked in tandem; and the 3'UTR is selected from the following (1) to (5): (1) A 3'UTR comprising an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 23 to 36, or any of its homologs, fragments, or variants, wherein the homologs, fragments, or variants have the same or superior function of improving translation efficiency as a 3'UTR comprising an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 23 to 36, and preferably, the homolog nucleic acid sequence has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 23 to 36; (2) a 3'UTR consisting of an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 23 to 36; (3) a 3′UTR consisting of an RNA sequence corresponding to any of the nucleic acid sequences set forth in SEQ ID NOs: 23, 24, 28, or 29; (4) a 3'UTR derived from two or more identical 3'UTRs in (1) to (3) linked in tandem; or (5) A 3'UTR obtained from two or more different 3'UTRs in (1) to (3) above linked in tandem; is selected from, 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; The mRNA molecule.

2. 2. The mRNA molecule of claim 1, wherein the 5'UTR is selected from RNA sequences corresponding to the nucleic acid sequences set forth in SEQ ID NOs: 4, 3, 12, 17, 19, 20, 21 or 22.

3. 2. The mRNA molecule of claim 1, wherein the 3'UTR is selected from RNA sequences corresponding to the nucleic acid sequences set forth in SEQ ID NOs: 23, 24, 25, 28 or 29.

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

5. (1) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, or (2) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, or (3) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 24, or (4) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 25, or (5) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 28, or (6) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 3, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 29, or (7) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 24, or (8) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 28, or (9) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 4, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 29, or (10) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, or (11) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 24, or (12) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 28, or (13) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 12, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 29, or (14) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, or (15) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 24, or (16) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 25, or (17) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 28, or (18) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 17, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 29, or (19) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, or (20) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 24, or (21) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 25, or (22) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 28, or (23) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 19, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 29, or (24) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 20, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, or (25) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 20, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 28, or (26) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 20, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 29, or (27) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 21, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 23, or (28) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 21, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 24, or (29) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 21, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 28, or (30) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 24, or (31) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 25, or (32) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 28, or (33) The 5'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 22, and the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO:

29. An mRNA molecule according to any one of claims 1 to 3.

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

23.

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

28.

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

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

10. Use of a combination of the 5'UTR and 3'UTR according to any one of claims 1 to 7 in increasing the protein expression level or translation efficiency of mRNA.

11. A DNA encoding the mRNA molecule according to any one of claims 1 to 9.

12. A vector comprising the DNA of claim 11.

13. A host cell comprising the vector of claim 12.

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

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

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

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