WO2024109866A1 - 提高蛋白表达量的utr分子 - Google Patents

提高蛋白表达量的utr分子 Download PDF

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WO2024109866A1
WO2024109866A1 PCT/CN2023/133522 CN2023133522W WO2024109866A1 WO 2024109866 A1 WO2024109866 A1 WO 2024109866A1 CN 2023133522 W CN2023133522 W CN 2023133522W WO 2024109866 A1 WO2024109866 A1 WO 2024109866A1
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utr
nucleic acid
seq
acid sequence
sequence corresponding
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PCT/CN2023/133522
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English (en)
French (fr)
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覃千山
原蕾
周逆水
许珺
郜鹏
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苏州艾博生物科技有限公司
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Publication of WO2024109866A1 publication Critical patent/WO2024109866A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present disclosure relates to UTR molecules and applications thereof. Specifically, the present disclosure relates to mRNA molecules comprising 5'UTR and 3'UTR, wherein the UTR molecules can significantly increase the protein expression of the mRNA molecules.
  • RNA vaccines are a very promising drug.
  • mRNA is unstable and easily degraded by ubiquitous RNases.
  • 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.
  • nucleic acid molecules 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' polyA 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).
  • 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). It is known that the 3'UTR of ⁇ -globin mRNA plays an important role in the stability of ⁇ -globin mRNA (Rodgers ND, Wang Z, Kiledjian M. RNA. 2002; 8(12): 1526. doi: 10.1017/s1355838202029035). The 3'UTR of ⁇ -globin mRNA is involved in the formation of a specific nucleoprotein-complex ( ⁇ -complex), which is closely related to the mRNA.
  • ⁇ -complex specific nucleoprotein-complex
  • 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).
  • 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 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.
  • 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):
  • (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, or a homologue, fragment or variant thereof, wherein the homologue, fragment or variant has the same or better function of improving translation efficiency as the 5'UTR of an 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;
  • RNA sequence represented by any one of SEQ ID NOs: 4, 3, 12, 17 or 19-22
  • the corresponding RNA sequence constitutes the 5'UTR;
  • 3'UTR is selected from the following (1)-(5):
  • (1) comprising a 3'UTR of an RNA sequence corresponding to any one of the nucleic acid sequences shown in SEQ ID NOs: 23-36, a homologue, a fragment or a variant thereof, wherein the homologue, the fragment or the variant has the same or better function of improving translation efficiency as the 3'UTR of an RNA sequence corresponding to any one of the nucleic acid sequences shown in 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 any one of the nucleic acid sequences shown in SEQ ID NOs: 23-36;
  • 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.
  • the 5'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:4, 3, 12, 17, 19, 20, 21 or 22.
  • 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 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.
  • 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.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:17
  • the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:23, 24 or 28.
  • 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 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.
  • 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.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:19
  • the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:24, 25, 28 or 29.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:3
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:23.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:3
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:24.
  • the 5'UTR is a nucleic acid as shown in SEQ ID NO:3
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:25.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:3
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:28.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:3
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:29.
  • 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.
  • 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.
  • 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.
  • 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.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:12
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:23.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:12
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:24.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 12
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:12
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:29.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:17
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:23.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:17
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:24.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:17
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:25.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:17
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:28.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:17
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:29.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:19
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:23.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:19
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:24.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:19
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:25.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:19
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:28.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:19
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:29.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:20
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:23.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:20
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:28.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:20
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:29.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:21
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:23.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:21
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:24.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:21
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:28.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:22
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:24.
  • the 5'UTR is the nucleotide sequence of SEQ ID NO: 22.
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:25.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 22
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 28.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:22
  • the 3'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:29.
  • the mRNA molecule further comprises a nucleic acid sequence encoding a polypeptide of interest.
  • the mRNA molecule further comprises polyA.
  • 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.
  • the present disclosure provides 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, which is used to increase the protein expression amount or translation efficiency of the mRNA.
  • the present disclosure provides use of a combination of a 5'UTR and a 3'UTR in increasing the protein expression amount or translation efficiency of an mRNA, wherein the 5'UTR and the 3'UTR are as defined above.
  • the present disclosure provides a DNA encoding the above-mentioned mRNA molecule.
  • the present disclosure provides a vector comprising the above-mentioned DNA.
  • the present disclosure provides a host cell comprising the above-mentioned vector.
  • the present disclosure provides a lipid nanoparticle comprising the above-mentioned mRNA molecule.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above mRNA molecule, the above DNA, the above vector, the above host cell or the above lipid nanoparticle. particles, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method for 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 to perform gene therapy, gene vaccination, protein replacement therapy, antisense therapy or treatment by interfering RNA.
  • 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.
  • 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.
  • FIG1 shows the effect of different 5'UTRs on GFP expression.
  • FIG2 shows the effect of different 3'UTRs on GFP expression.
  • nucleic acid molecule or nucleic acid sequence refers to nucleic acid, which is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • nucleic acid comprises genomic DNA, cDNA, mRNA, recombinantly prepared molecules and chemically synthesized molecules.
  • nucleic acid can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.
  • RNA refers to a molecule comprising, and preferably consisting entirely or substantially of, ribonucleotide residues.
  • ribonucleotide refers to a nucleotide having a hydroxyl group at the 2' position of the ⁇ -D-ribofuranosyl group.
  • 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 by the addition, deletion, substitution, and/or change of one or more nucleotides.
  • RNA changes may include the addition of non-nucleotide material, such as to the ends of the RNA or internally, such as at one or more nucleotides of the RNA.
  • Nucleotides in RNA molecules Non-standard nucleotides may also be included, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides.
  • the RNA of these changes may be referred to as analogs, particularly analogs of naturally occurring RNA.
  • RNA includes mRNA.
  • mRNA means "messenger RNA” and refers to a transcript produced by using a DNA template and encoding a peptide or protein.
  • 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 the skilled person. For example, a variety of in vitro transcription kits are commercially available. According to the present invention, in addition to the modification according to the present invention, mRNA can also be modified by further stabilization modification and capping.
  • nucleic acid 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.
  • “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.
  • a fragment replaces said nucleic acid sequence in an RNA molecule, it retains RNA stability and/or translation efficiency.
  • 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 said nucleic acid sequence.
  • variants according to the invention with respect to, for example, 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.
  • nucleic acid variants comprise single or multiple nucleotide deletions, additions, mutations and/or insertions compared to a reference nucleic acid.
  • Deletions include removal of one or more nucleotides from a reference nucleic acid.
  • Addition variants comprise 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, wherein at least one nucleotide in the sequence is removed and Insertion of another nucleotide in its place (e.g., transversion and transition); abasic site; crosslinking site; and chemically altered or modified bases. Insertion includes the addition of at least one nucleotide to a reference nucleic acid.
  • variants include 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.
  • 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 for 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 for the entire length of the reference nucleic acid sequence.
  • Sequence similarity refers to the percentage of amino acids that are identical or that represent conservative amino acid substitutions.
  • Sequence identity refers to the percentage of amino acids or nucleotides that are identical between these sequences.
  • % 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 contain additions or deletions to obtain an optimal alignment between the two sequences compared to the reference sequence.
  • the comparison of two sequences is usually performed by comparing the sequences after optimal alignment for a segment or "comparison window" to identify local regions of corresponding sequences.
  • the optimal alignment for comparison may be performed manually, or by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol.
  • the percent identity is obtained by determining the number of identical positions corresponding to the sequences being compared, dividing that number by the number of positions being compared, and multiplying the result by 100.
  • the BLAST program "BLAST 2 sequences" available at the website http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi can be used.
  • the complementarity degree of the present invention is 100%.
  • 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, such as a nucleic acid sequence that exhibits properties that are the same or similar to those of the specific nucleic acid sequence.
  • promoter refers to a DNA sequence upstream (5') of the coding sequence of a gene, which 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. Inducible promoters are expressed only to a small extent or not at all if there is no inducer. In the presence of an inducer, a gene is "turned on” or the level of transcription increases. This is usually mediated by the binding of a specific transcription factor.
  • 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. 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 by which a strand of messenger RNA directs the assembly of an amino acid sequence to produce a peptide or protein.
  • the term “transcription” relates to a process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into protein.
  • the term “transcription” includes “in vitro transcription”, wherein the term “in vitro transcription” relates to a process in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system.
  • a cloning vector is applied for the production of transcripts.
  • These cloning vectors are usually named transcription vectors and are encompassed in the term “vector” according to the present invention.
  • the RNA is preferably an 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.
  • the cDNA can be obtained by reverse transcription of RNA.
  • untranslated region or "UTR” as used according to the 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.
  • 3'-untranslated region 3'UTR:
  • 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 a protein.
  • 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 polyadenylation sequence of the mRNA.
  • ORF open reading frame
  • CDS coding sequence
  • 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 polyadenylation 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-mature mRNA including optional introns.
  • the pre-mature 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-mature mRNA to excise the mRNA, and then cleaving the mRNA.
  • the 3'-UTR corresponds to the sequence of the mature mRNA located between the stop codon of the protein coding region, preferably the 3' end immediately following the stop codon of the protein coding region, and the polyadenylic acid sequence of the mRNA.
  • the term "corresponding to" means that the 3'-UTR sequence can be an RNA sequence in the mRNA sequence as used to define the 3'-UTR sequence, or a DNA sequence corresponding to this RNA sequence.
  • 3'-UTR of a gene such as “3'-UTR of a ribosomal protein gene” is a sequence corresponding to the 3'UTR of a mature mRNA derived from the gene, i.e., an mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA.
  • the term "3'UTR of a gene” includes the DNA sequence and RNA sequence (both the sense strand and the antisense strand and both mature and immature) of the 3'UTR.
  • 5'-untranslated region 5'UTR is typically understood as a specific part of messenger RNA (mRNA). It is located at the 5' side of the mRNA open reading frame. Typically, 5'UTR starts with a transcription start site and ends one nucleotide before the start codon of the open reading frame. 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. 5'UTR may be post-transcriptionally modified, for example, by adding a 5'-cap. Within the scope of the present invention, 5'UTR corresponds to a mature mRNA sequence located between the 5' cap and the start codon.
  • mRNA messenger RNA
  • 5'UTR corresponds to a sequence extending from a nucleotide located at the 3' side of the 5'-cap, preferably from a nucleotide located at the 3' side of the 5'-cap immediately adjacent to the 5'-cap, to a nucleotide located at the 5' side of the start codon of the protein coding region, preferably to a nucleotide located at the 5' side of the start codon of the protein coding region.
  • the nucleotide at the 3' side of the mature mRNA 5'-cap typically corresponds to the transcription start site.
  • the term "corresponding to” means that the 5'UTR sequence can be an RNA sequence as in the mRNA sequence used to define the 5'UTR sequence, or a DNA sequence corresponding to this RNA sequence.
  • the term "5'UTR of a gene” is a sequence corresponding to the 5'UTR of a mature mRNA derived from the gene, i.e., an mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA.
  • the term "5'UTR of a gene” includes both the DNA sequence and the RNA sequence of the 5'UTR.
  • the term "gene” refers to a gene or genes responsible for producing one or more cell products.
  • the term refers to a specific nucleic acid sequence that encodes a specific protein or a functional or structural RNA molecule.
  • Polyadenylation is the addition of a poly(A) sequence (PolyA) or a poly(A) tail to the primary transcript RNA.
  • the poly(A) sequence consists of multiple adenosine monophosphates. In other words, it is a stretch of RNA with only adenine bases.
  • polyadenylation is part of the process of producing mature messenger RNA (mRNA) for translation. It therefore forms part of the larger process of gene expression.
  • the process of polyadenylation begins at the end or termination of transcription of a gene. 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.
  • poly (A) sequence refers to a sequence of adenylic acid residues usually 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 thymidylic acid 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.
  • 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 especially up to 150 A nucleotides, preferably consecutive A nucleotides, and especially about 120 A nucleotides.
  • a nucleotide or "A” refers to an adenylic acid residue.
  • nucleic acids described herein may be recombinant molecules and/or isolated molecules.
  • isolated molecule is intended to refer to a molecule that is substantially free of other molecules, such as other cellular material.
  • isolated nucleic acid means, according to the present invention, that the nucleic acid has been: (i) amplified in vitro, such as by polymerase chain reaction (PCR); (ii) produced by cloning recombination; (iii) purified, such as by cleavage and gel electrophoresis fractionation; or (iv) synthesized, such as by chemical synthesis.
  • An isolated nucleic acid is one that can be used for recombinant nucleic acid. DNA technology manipulates nucleic acids.
  • recombinant in the context of the present invention means “produced by genetic engineering.”
  • recombinant material such as a recombinant cell is not naturally occurring.
  • naturally occurring refers to the fact that a substance can be found in nature.
  • a peptide or nucleic acid that exists in an organism (including a virus) and can be isolated from a natural source and has not been intentionally modified by man in an experiment is naturally occurring.
  • the term “host cell” refers to any cell that can be transformed or transfected with exogenous nucleic acid.
  • 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 be derived from a variety of 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.
  • host cells are antigen presenting cells, particularly dendritic cells, monocytes, or macrophages.
  • 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.
  • 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 up to preferably 50, preferably 100 or preferably 150 consecutive amino acids linked to each other by peptide bonds.
  • protein refers to large peptides, preferably peptides with at least 151 amino acids, but in general the terms "peptide” and “protein” are used as synonyms herein.
  • peptide and protein 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 bonds, thioether bonds or disulfide bonds.
  • a nucleic acid such as RNA may encode a peptide or protein.
  • a transcribable nucleic acid sequence or its transcript may contain an open reading frame encoding a peptide or protein.
  • the nucleic acid may express an encoded peptide or protein.
  • the nucleic acid may be a nucleic acid that encodes and expresses an antigen or a pharmaceutically active peptide or protein such as an immunologically active compound (which is preferably not an antigen).
  • 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 during translation to produce a peptide or protein.
  • the RNA according to the present invention is capable of interacting with the cell translation machinery to allow the peptide or protein to be translated.
  • the inventors of the present application screened out 5'UTR and 3'UTR combinations that can significantly increase the expression 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 of mRNA.
  • the combination of the 5'UTR and 3'UTR can significantly improve the translation efficiency of the target gene coding sequence, thereby significantly improving its expression level.
  • 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.
  • the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 23, 24, 25, 28 or 29.
  • 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.
  • 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.
  • 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 SEQ ID NO: 23, 24 or The RNA sequence corresponding to the nucleic acid sequence shown in 28.
  • 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 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.
  • 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.
  • the 5'UTR is an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:19
  • the 3'UTR is selected from an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:24, 25, 28 or 29.
  • 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.
  • the mRNA molecule further comprises a nucleic acid sequence encoding a polypeptide of interest.
  • the mRNA molecule further comprises polyA.
  • the mRNA can be advantageously used in gene therapy, genetic vaccination, protein replacement therapy, antisense therapy, or therapy by interfering RNA.
  • the present disclosure provides use of a combination of a 5'UTR and a 3'UTR in increasing the protein expression amount or translation efficiency of an mRNA, wherein the 5'UTR and the 3'UTR are as defined above.
  • T7 promoter sequence as shown in SEQ ID NO:37
  • GGATCC BamHI restriction site
  • 5UTR + KOZAK GCCACC
  • CDS EcoRI restriction site
  • GAATTC 3UTR + SpeI restriction site
  • RNA capping After the uncapped RNA is pre-denatured at 65°C, GTP, capping reaction solution, S-adenosylmethionine, vaccinia capping enzyme, RNase inhibitor, 2'-o-methyltransferase, etc. are mixed in a certain proportion and reacted in a thermostat at 37°C for a certain period of time to complete the RNA capping. Purification is done by lithium chloride precipitation.
  • RNA tailing The capped RNA was tailed using a polyA polymerase kit (Vazyme, DD4111) according to the kit instructions, and purified by lithium chloride precipitation after the reaction.
  • EPO whose CDS sequence is shown in SEQ ID NO: 38 expression detection: 16 hours after cell transfection, the supernatant was collected and ELISA was performed using the Human Erythropoietin ELISA (R&D) kit.
  • R&D Human Erythropoietin ELISA
  • Example 2 Expression of GFP with different 5'UTR in 293T cell line (ELISA reader):
  • Example 3 Expression of GFP with different 3'UTR in 293T cell line (ELISA reader):
  • Example 2-3 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 in vitro transcription, RNA capping and tailing, and mRNA purity quality inspection, mRNA preparation and detection were completed.
  • control combination 2 in 293T cells, the EPO expression of the 38 UTR and 3'UTR combinations of the present invention is significantly higher, up to about 4.1 times that of control combination 2 (combination 10 of the present invention); in Hela cells, the EPO expression of combinations 1-33, 34 and 35 of the present invention is significantly higher, up to about 3.2 times that of control combination 2 (combination 10 of the present invention); in A549 cells, the EPO expression of combinations 1, 2, 4-19, 21-28, 30-35 of the present invention is significantly higher, up to about 3.1 times that of control combination 1 (combination 4 of the present invention).
  • 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.
  • Example 4 In vivo tests were performed on some 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 detection were completed to obtain samples 1-8.
  • 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, thereby removing ethanol and using DPBS instead. Finally, the lipid nanoparticles were filtered through a 0.2 ⁇ m sterile filter.
  • lipid nanoparticles of samples 1-8 containing encapsulated human erythropoietin (hEPO) mRNA were systemically administered to female ICR mice aged 6-8 weeks by tail vein injection (Xipuer-Bikai, Shanghai) at a dose of 0.5 mg/kg (wherein sample 8 is the comparative combination 2 described in Example 4, as a control).
  • sample 8 is the comparative combination 2 described in Example 4, as a control.
  • the blood of mice was collected 6 hours after administration, and the blood samples were 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.
  • the characteristics of the tested lipid nanoparticles are listed in the table below, including the hEPO expression levels ( ⁇ g/ml) measured from the test groups.

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Abstract

本公开涉及UTR分子及其应用。具体而言，本公开涉及包含5'UTR和3'UTR的mRNA分子，所述UTR分子能够显著提高mRNA的表达量。

Description

提高蛋白表达量的UTR分子
本申请要求2022年11月24日提交的PCT申请第PCT/CN2022/134038号的优先权，该案的全部内容以引用的方式并入本文中。
技术领域
本公开涉及UTR分子及其应用。具体而言，本公开涉及包含5'UTR和3'UTR的mRNA分子，所述UTR分子能够显著提高所述mRNA分子的蛋白表达量。
背景技术
mRNA疫苗作为基因疗法，是一种非常有前途的药物。但mRNA不稳定，易被普遍存在的RNA酶降解。在体内，RNA降解有助于调节RNA半衰期，可以微调真核基因表达的调节(Friedel CC,L,Ruzsics Z,Koszinowski UH,Zimmer R.Nucleic Acids Res.2009；37(17):e115-e115.doi:10.1093/NAR/GKP542)。稳定的RNA对于RNA药物的表达量非常有必要，调节核酸分子稳定性的方法有很多，包括调节核酸的GC含量(WO2002098443A2)，添加UTR(untranslated region)序列，5'帽子(Galloway A,Cowling VH.Biochim Biophys Acta-Gene Regul Mech.2019；1862(3):270-279.doi:10.1016/j.bbagrm.2018.09.011)，3'polyA尾巴(Tudek A,Lloret-Llinares M,Heick Jensen T.Philos Trans R Soc B Biol Sci.2018；373(1762).doi:10.1098/rstb.2018.0169)。
而UTR是翻译效率的关键要素(Jackson RJ,Hellen CUT,Pestova T V.Nat Rev Mol Cell Biol 2010 112.2010；11(2):113-127.doi:10.1038/nrm2838)。已知α-珠蛋白mRNA的3'UTR对α-珠蛋白mRNA稳定性起着重要作用(Rodgers ND,Wang Z,Kiledjian M.RNA.2002；8(12):1526.doi:10.1017/s1355838202029035)。α-珠蛋白mRNA的3'UTR参与特定核蛋白-复合物(α-复合物)的形成，与mRNA的 体外稳定性相关(Wang Z,Day N,Trifillis P,Kiledjian M.Mol Cell Biol.1999；19(7):4552.doi:10.1128/MCB.19.7.4552)。Moderna公司通过280000条随机的5'UTR的试验数据结果，构建了神经网络并设计出通用的提高表达量的5'UTR(Sample PJ,Wang B,Reid DW,et al.Nat Biotechnol.2019；37(7):803-809.doi:10.1038/s41587-019-0164-5；US10881730B2)。BioNtech通过片段库的方式筛选出了能提高表达的5UTR和3UTR(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)。
尽管有上述进展，但对于能够稳定mRNA分子且提高其蛋白表达量的UTR分子组合，仍然存在迫切需求。
发明内容
本申请发明人预料不到地发现，本文所述的特定5'UTR和3'UTR组合能够显著提高mRNA分子的蛋白表达量，所述mRNA从而可有利地用于基因治疗、基因疫苗接种、蛋白替代疗法、反义治疗或通过干扰RNA进行的治疗。
在一个方面，本公开提供了一种包含5'UTR和3'UTR的mRNA分子，其中所述5'UTR选自下述(1)-(5)：
(1)包含SEQ ID NO:1-22中任一个所示核酸序列相对应的RNA序列的5'UTR、其同源物、片段或变体，所述同源物、片段或变体具有与SEQ ID NO:1-22中任一个所示核酸序列相对应的RNA序列的5'UTR相同或更优的提高翻译效率的功能；优选地，所述同源物的核酸序列与SEQ ID NO:1-22中任一个所示核酸序列相对应的RNA序列具有至少70％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性；
(2)由SEQ ID NO:1-22中任一个所示核酸序列相对应的RNA序列组成的5'UTR；
(3)由SEQ ID NO:4、3、12、17或19-22中任一个所示核酸序列 相对应的RNA序列组成的5'UTR；
(4)上述(1)-(3)中2个或2个以上相同5'UTR经串联获得的5'UTR；或
(5)上述(1)-(3)中2个或2个以上不同5'UTR经串联获得的5'–UTR，
并且其中所述3'UTR选自下述(1)-(5)：
(1)包含SEQ ID NO:23-36中任一个所示核酸序列相对应的RNA序列的3'UTR、其同源物、片段或变体，所述同源物、片段或变体具有与SEQ ID NO:23-36中任一个所示核酸序列相对应的RNA序列的3'UTR相同或更优的提高翻译效率的功能；优选地，所述同源物的核酸序列与SEQ ID NO:23-36中任一个所示核酸序列相对应的RNA序列具有至少70％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性；
(2)由SEQ ID NO:23-36中任一个所示核酸序列相对应的RNA序列组成的3'UTR；
(3)由SEQ ID NO:23、24、28或29所示核酸序列相对应的RNA序列组成的3'UTR；
(4)上述(1)-(3)中2个或2个以上相同3'UTR经串联获得的3'UTR；或
(5)上述(1)-(3)中2个或2个以上不同3'UTR经串联获得的3'–UTR；
并且其中所述5'UTR和3'UTR组合不包括以下所示核酸序列相对应的RNA序列：SEQ ID NO:22和SEQ ID NO:23、SEQ ID NO:20和SEQ ID NO:24以及SEQ ID NO:21和SEQ ID NO:25。
在一些实施方案中，所述5'UTR选自SEQ ID NO:4、3、12、17、19、20、21或22所示核酸序列相对应的RNA序列。
在一些实施方案中，所述3'UTR选自SEQ ID NO:23、24、25、28或29所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR选自SEQ ID NO:19或22所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、25、28或29所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR选自SEQ ID NO:4、3或12所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、28或29所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24或28所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR选自SEQ ID NO:20或21所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、28或28所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR选自SEQ ID NO:3、19或22所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、25、28或29所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR选自SEQ ID NO:4、12、17、20或21所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、28或29所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:24、25、28或29所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:3所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:3所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:3所示核酸 序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:25所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:3所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:3所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:4所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:4所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:4所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:4所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:12所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:12所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:12所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核 酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:12所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:25所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:25所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:20所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:20所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:20所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:21所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:21所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:21所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:22所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:22所示核 酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:25所示核酸序列相对应的RNA序列。
在一个具体实施方案中，所述5'UTR是SEQ ID NO:22所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列。或
在一个具体实施方案中，所述5'UTR是SEQ ID NO:22所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序列。
在一些实施方案中，所述mRNA分子进一步包含编码目的多肽的核酸序列。
在一些实施方案中，所述mRNA分子进一步包含polyA。
在一个方面，本公开提供了一种使用如上文所定义的5'UTR和3'UTR组合或包含5'UTR和3'UTR的mRNA分子来提高mRNA的蛋白表达量或翻译效率的方法。
在一个方面，本公开提供了如上文所定义的5'UTR和3'UTR组合或包含5'UTR和3'UTR的mRNA分子，其用于提高mRNA的蛋白表达量或翻译效率。
在一个方面，本公开提供了5'UTR和3'UTR组合在提高mRNA的蛋白表达量或翻译效率中的用途，其中所述5'UTR和3'UTR如上文所定义。
在一个方面，本公开提供了一种编码上述mRNA分子的DNA。
在一个方面，本公开提供了一种载体，所述载体包含上述DNA。
在一个方面，本公开提供了一种宿主细胞，其包含上述载体。
在一个方面，本公开提供了一种脂质纳米颗粒，其包含上述mRNA分子。
在一个方面，本公开提供了一种药物组合物，其包含上述mRNA分子、上述DNA、上述载体、上述宿主细胞或上述脂质纳米 颗粒，以及药学上可接受的载剂。
在一个方面，本公开提供了一种使用上述mRNA分子、上述DNA、上述载体、上述宿主细胞、上述脂质纳米颗粒或上述药物组合物来进行基因治疗、基因疫苗接种、蛋白替代疗法、反义治疗或通过干扰RNA进行的治疗的方法。
在一个方面，本公开提供了上述mRNA分子、上述DNA、上述载体、上述宿主细胞、上述脂质纳米颗粒或上述药物组合物，用于基因治疗、基因疫苗接种、蛋白替代疗法、反义治疗或通过干扰RNA进行的治疗。
在一个方面，本公开提供了上述mRNA分子、上述DNA、上述载体、上述宿主细胞、上述脂质纳米颗粒或上述药物组合物在制备药物中的用途，所述药物用于基因治疗、基因疫苗接种、蛋白替代疗法、反义治疗或通过干扰RNA进行的治疗。
附图说明
图1显示了不同5'UTR对GFP表达量的影响。
图2显示了不同3'UTR对GFP表达量的影响。
发明详述
尽管下文中详细地描述了本发明，但是应理解，本发明不限于本文中所述的特定方法、方案和试剂，因为这些可改变。还应理解，本文中所使用的术语只是为了描述一些特定实施方案的目的，而不旨在限制将仅受所附权利要求书限制的本发明的范围。除非另外限定，否则本文中所使用的所有技术和科学术语均具有与本领域普通技术人员通常所理解的相同的含义。
除非另外指出，否则本发明的实施将采用本领域的文献中阐明的常规化学、生物化学、细胞生物学、免疫学和重组DNA技术方法(参见例如，Molecular Cloning：ALaboratory Manual，第二版，J.Sambrook等编辑，Cold Spring Harbor LaboratoryPress，Cold Spring Harbor 1989)。
除非上下文另外要求，否则在本说明书和所附权利要求书通篇，词语“包含/包括”及其变化形式应理解为意指包括所述要素、整体或步骤或者要素、整体或步骤的组，而不排除任何其他要素、整体或步骤或者要素、整体或步骤的组。除非本文中另外指出或者明显与上下文相矛盾，否则在描述本发明的上下文中(尤其在权利要求书的上下文中)使用的没有数量词修饰的名词应解释为表示一个/种和/或更多个/种。本文中值的范围的记载仅旨在用作单独提及落在所述范围内的各个单独值的速记法。除非本文中另外指出，否则各个单独值被并入本说明书中，就像其在本文中被单独记载。除非本文中另外指出或者明显与上下文相矛盾，否则本文中所述所有方法均可以以任何合适的顺序进行。本文中提供的任何和所有实例或示例性语言(例如，“例如/如”)的使用仅旨在更好地举例说明本发明，而不对以其他方式要求保护的本发明范围提出限制。本说明书中的语言均不应被解释为指明对实施本发明必需的任何未要求保护的要素。
根据本发明，核酸分子或核酸序列是指核酸，其优选地是脱氧核糖核酸(DNA)或核糖核酸(RNA)。根据本发明，核酸包含基因组DNA、cDNA、mRNA、重组制备分子和化学合成分子。根据本发明，核酸可以是单链或双链的线性或共价闭合环状分子的形式。
在本发明的上下文中，术语“RNA”是指包含核糖核苷酸残基，并且优选地整体上或基本上由核糖核苷酸残基构成的分子。术语“核糖核苷酸”是指在β-D-呋喃核糖基的2’位具有羟基的核苷酸。术语“RNA”包含双链RNA、单链RN、分离的RNA(例如经部分或完全纯化的RNA)、基本上纯的RNA、合成的RNA和重组产生的RNA，例如经修饰的RNA，其与天然存在RNA的不同之处在于添加、缺失、替换和/或改变一个或更多个核苷酸。这样的改变可以包括添加非核苷酸材料，例如向RNA的末端或在内部，例如在RNA的一个或更多个核苷酸处添加非核苷酸材料。RNA分子中的核苷酸 还可以包含非标准核苷酸，例如非天然存在的核苷酸或化学合成的核苷酸或脱氧核苷酸。这些改变的RNA可以被称为类似物，特别是天然存在RNA的类似物。根据本发明，RNA包括mRNA。
术语“mRNA”意指“信使RNA”，并且涉及通过使用DNA模板产生且编码肽或蛋白质的转录物。通常，mRNA包含5'UTR、蛋白质编码区、3'UTR和多聚(A)序列。mRNA可以由DNA模板通过体外转录产生。体外转录方法是技术人员已知的。例如，多种体外转录试剂盒可商购获得。根据本发明，除根据本发明的修饰之外，还可以通过进一步的稳定修饰和加帽来修饰mRNA。
根据本发明的术语“核酸”还包括对核酸在核苷酸碱基上、在糖上或在磷酸上的化学衍生化，以及含有非天然核苷酸和核苷酸类似物的核酸。
“片段”或“核酸序列的片段”涉及核酸序列的一部分，即展现为在5’和/或3’端缩短的核酸序列的序列。优选地，当片段替代RNA分子中的所述核酸序列时，其保留RNA稳定性和/或翻译效率。优选地，核酸序列的片段包含来自所述核酸序列的至少80％，优选至少90％、95％、96％、97％、98％或99％的核苷酸残基。
根据本发明关于例如核酸和氨基酸序列的术语“变体”包括任何变体，特别是突变体、剪接变体、构象体、异构体、等位基因变体、物种变体和物种同源物，特别是天然存在的那些。等位基因变体涉及基因的正常序列的改变，其重要性常常不清楚。完整基因测序常常鉴定出给定基因的大量等位基因变体。物种同源物是与给定核酸或氨基酸序列具有不同物种来源的核酸或氨基酸序列。
根据本发明，与参考核酸相比，核酸变体包含单一或多个核苷酸缺失、添加、突变和/或插入。缺失包括从参考核酸中移除一个或更多个核苷酸。添加变体包含一个或更多个核苷酸(例如1、2、3、5、10、20、30、50个或更多个核苷酸)的5’和/或3’端融合。突变可以包括但不限于替换，其中序列中的至少一个核苷酸被移除并且 在其位置插入另一个核苷酸(例如颠换和转换)；无碱基位点；交联位点；和化学改变的或经修饰的碱基。插入包括在参考核酸中添加至少一个核苷酸。
对于核酸分子，术语“变体”包括简并性核酸序列，其中根据本发明的简并性核酸序列是由于遗传密码的简并性而在密码子序列中与参考核酸不同的核酸。
优选地，给定核酸序列与作为所述给定核酸序列之变体的核酸序列之间的同一性程度为至少70％，优选至少75％，优选至少80％，更优选至少85％，更优选至少90％或最优选至少95％、96％、97％、98％或99％。同一性程度优选地针对具有至少约30个、至少约50个、至少约70个、至少约90个、至少约100个、至少约150个、至少约200个、至少约250个、至少约300个或至少约400个核苷酸的区域给出。在一些优选实施方案中，同一性程度针对参考核酸序列的整个长度给出。
“序列相似性”表示相同或代表保守氨基酸替换的氨基酸的百分比。两个多肽或核酸序列之间的“序列同一性”表示这些序列之间相同的氨基酸或核苷酸的百分比。
术语“％同一性”旨在指特别地在待比较的两个序列之间的最佳比对中相同的核苷酸的百分比，其中所述百分比是纯统计学的，并且所述两个序列之间的差异可以在序列的全长内随机分布，并且与参考序列相比，待比较序列可以包含添加或缺失以获得两个序列之间的最佳比对。两个序列的比较通常通过针对区段或“比较窗”在最佳比对之后比较所述序列来进行以鉴定对应序列的局部区域。用于比较的最佳比对可以人工进行，或者借助于Smith和Waterman，1981，Ads App.Math.2，482的局部同源性算法、借助于Neddleman和Wunsch，1970，J.Mol.Biol.48，443的局部同源性算法和借助于Pearson和Lipman，1988，Proc.Natl Acad.Sci.USA 85，2444的相似性检索算法，或者借助于使用所述算法的计算机程序 (Wisconsin Genetics Software Package，Genetics Computer Group，575 ScienceDrive，Madison，Wis.中的GAP、BESTFIT、FASTA、BLAST P、BLAST N和TFASTA)来进行。
百分比同一性如下获得：确定待比较序列对应的相同位置的数目，将该数目除以所比较的位置的数目，并将该结果乘以100。例如，可以使用可在网站http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi上获得的BLAST程序“BLAST 2 sequences”。
酸序列的所有连续残基将与第二核酸序列中相同数目的连续残基氢键键合。优选地，根据本发明的互补性程度为至少70％，优选至少75％，优选至少80％，更优选至少85％，甚至更优选至少90％，或者最优选至少95％、96％、97％、98％或99％。最优选地，根据本发明的互补性程度为100％。
特定核酸序列的片段或变体或者与特定核酸序列具有特定同一性程度的核酸序列优选地具有所述特定序列的至少一种功能特性，并且优选地与所述特定序列是功能上等同的，例如展现出与特定核酸序列的特性相同或相似的特性的核酸序列。
术语“启动子”或“启动子区”是指基因的编码序列上游(5’)的DNA序列，其通过提供RNA聚合酶的识别和结合位点来控制所述编码序列的表达。启动子区可以包含参与调节所述基因的转录的另一些因子的另一些识别或结合位点。启动子可以控制原核或真核基因的转录。启动子可以是“诱导型”的并且响应于诱导物而启动转录，或者如果转录不受诱导物控制的话，所述启动子可以是“组成型的”。如果没有诱导物，诱导型启动子只在很小的程度上表达或根本不表达。在存在诱导物的情况下，基因“开启”或转录水平提高。这通常通过特定转录因子的结合来介导。
根据本发明，术语“表达”以其最通常的含义使用，并且包括RNA或者RNA和蛋白质的产生。其还包括核酸的部分表达。此外， 表达可以是瞬时的或稳定的。对于RNA，术语“表达”或“翻译”涉及通过其信使RNA的链指导组装氨基酸序列以产生肽或蛋白质的在细胞的核糖体中的过程。
在本发明的上下文中，术语“转录”涉及其中DNA序列中的遗传密码转录成RNA的过程。随后，RNA可以翻译成蛋白质。根据本发明，术语“转录”包括“体外转录”，其中术语“体外转录”涉及其中RNA、特别地mRNA是在无细胞系统中体外合成的过程。优选地，克隆载体应用于转录物的产生。这些克隆载体通常命名为转录载体，并且根据本发明涵盖在术语“载体”中。根据本发明，RNA优选地是体外转录的RNA(IVT-RNA)，并且可以通过合适DNA模板的体外转录获得。用于控制转录的启动子可以是任何RNA聚合酶的任何启动子。用于体外转录的DNA模板可以通过克隆核酸(特别地cDNA)并将其引入用于体外转录的合适载体中来获得。cDNA可以通过RNA的逆转录获得。
根据本发明使用的术语“非翻译区”或“UTR”涉及起始密码子上游和终止密码子下游的mRNA部分，其不被翻译，因此分别被称为5’非翻译区(5'UTR)和3’非翻译区(3'UTR)。这些区域用编码区转录，因此在成熟mRNA中存在时是外显子的。
3’-非翻译区(3'UTR)：通常，术语“3'UTR”是指人工核酸分子的位于可读框的3’(即“下游”)并且不翻译为蛋白的部分。典型地，3'UTR是mRNA的位于mRNA的蛋白编码区(可读框(ORF)或编码序列(CDS))和聚腺苷酸序列之间的部分。在本发明的情况下，术语3'UTR还可以包含RNA被转录自的模板中不编码的，但在转录后成熟期间添加的元件，例如聚腺苷酸序列。mRNA的3′-UTR不翻译为氨基酸序列。3′-UTR序列通常由在基因表达过程中被转录为各自的mRNA的基因编码。基因组序列首先转录为包含任选内含子的成熟前mRNA。成熟前mRNA随后在成熟过程中进一步被加工为成熟mRNA。该成熟过程包含以下步骤：5′加帽、剪接成熟前mRNA以切 除任选内含子和3′末端修饰(如成熟前mRNA 3′末端的聚腺苷酸化和任选的核酸内切酶/或核酸外切酶切割等)。在本发明范围内，3′-UTR对应于位于蛋白编码区终止密码子，优选紧接蛋白编码区终止密码子的3′端，和mRNA的聚腺苷酸序列之间的成熟mRNA的序列。术语“对应于”意为3′-UTR序列可以是如用于限定3′-UTR序列的mRNA序列中的RNA序列，或对应于此RNA序列的DNA序列中。在本发明范围内，术语“基因的3′-UTR”，如“核糖体蛋白基因的3′-UTR”，为对应于源自该基因的成熟mRNA的3′UTR的序列，所述成熟mRNA即通过基因转录和成熟前mRNA的成熟获得的mRNA。术语“基因的3′UTR”包括3′UTR的DNA序列和RNA序列(正义链和反义链二者和成熟和不成熟的二者)。
5′-非翻译区(5′UTR)：5'UTR典型地理解为信使RNA(mRNA)的特定部分。其位于mRNA可读框的5′侧。典型地，5'UTR以转录起始位点开始并在可读框起始密码子前一个核苷酸结束。5'UTR可以包含控制基因表达的元件，也称为调节元件。此种调节元件可以是，例如，核糖体结合位点。5'UTR可以例如通过添加5′-帽而被转录后修饰。在本发明范围内，5'UTR对应于位于5′帽和起始密码子之间的成熟mRNA序列。优选地，5'UTR对应于从位于5′-帽的3′侧的核苷酸，优选从紧邻5′-帽的3′侧核苷酸，向位于蛋白编码区起始密码子5′侧的核苷酸，优选向紧接蛋白编码区起始密码子5′侧的核苷酸延伸的序列。紧接成熟mRNA5′-帽的3′侧的核苷酸典型地对应于转录起始位点。术语“对应于”意为5'UTR序列可以是如用于限定5'UTR序列的mRNA序列中的RNA序列，或与此RNA序列对应的DNA序列。在本发明范围内，术语“基因的5'UTR”是对应于源自该基因的成熟mRNA的5'UTR的序列，所述成熟mRNA即通过基因转录和成熟前mRNA的成熟获得的mRNA。术语“基因的5'UTR”包括5'UTR的DNA序列和RNA序列。
根据本发明，术语“基因”是指负责产生一种或更多种细胞产 物和/或实现一种或更多种细胞间或细胞内功能的特定核酸序列。更具体地，所述术语涉及包含编码特定蛋白质或者功能性或结构性RNA分子的核酸的DNA区段。
多聚腺苷酸化是将多聚(A)序列(PolyA)或多聚(A)尾添加至初级转录物RNA。多聚(A)序列由多个腺苷单磷酸组成。换言之，其是一段只具有腺嘌呤碱基的RNA。在真核生物中，多聚腺苷酸化是产生用于翻译的成熟信使RNA(mRNA)的过程的一部分。因此，其构成更大的基因表达过程的一部分。多聚腺苷酸化的过程开始于基因的转录结束或终止时。新产生的前mRNA的最3’区段首先被一组蛋白质切掉；接着这些蛋白质在RNA 3’端合成多聚(A)序列。多聚(A)序列对于核输出、翻译和mRNA稳定性十分重要。该序列随着时间的推移而缩短，并且当其足够短时，mRNA会被酶促降解。
术语“多聚腺苷酸序列”、“多聚(A)序列”或“多聚(A)尾”是指通常位于RNA分子的3’端的腺苷酸残基序列。本发明允许这样的序列通过DNA模板基于在与编码链互补的链中的重复胸苷酸残基来在RNA转录期间附着，然而所述序列在正常情况下不在DNA中编码，而是在细胞核中转录后通过模板独立性RNA聚合酶附着至RNA的游离3’端。根据本发明，在一个实施方案中，多聚(A)序列具有至少20，优选至少40，优选至少80，优选至少100且优选多至500，优选多至400，优选多至300，优选多至200并且特别地多至150个A核苷酸，优选连续A核苷酸，并且特别地约120个A核苷酸。术语“A核苷酸”或“A”是指腺苷酸残基。
本文所述的核酸可以是重组分子和/或分离的分子。
本文所用的“分离的分子”旨在是指基本上不含其他分子例如其他细胞物质的分子。术语“分离的核酸”根据本发明意指，核酸已经：(i)在体外扩增，例如通过聚合酶链反应(PCR)体外扩增；(ii)通过克隆重组产生；(iii)纯化，例如通过切割和凝胶电泳分级纯化；或(iv)合成，例如通过化学合成而合成。分离的核酸是可用于通过重组 DNA技术操作的核酸。
术语“重组的”在本发明的上下中意指“通过基因工程产生的”。优选地，在本发明的上下文中，“重组物质”例如重组细胞是非天然存在的。
本文所用的术语“天然存在的”是指物质可见于自然界中的事实。例如，存在于生物体(包括病毒)中和可从自然界来源中分离的并且未在实验中人工有意修饰的肽或核酸是天然存在的。
根据本发明，术语“宿主细胞”是指可以用外源核酸转化或转染的任何细胞。根据本发明，术语“宿主细胞”包含原核细胞(例如大肠杆菌)或真核细胞(例如酵母细胞和昆虫细胞)。特别地优选哺乳动物细胞，例如来自人、小鼠、仓鼠、猪、山羊、灵长类的细胞。细胞可以来源于多种组织类型，并且包含初级细胞和细胞系。一些具体实例包括角质形成细胞、外周血白细胞、骨髓干细胞和胚胎干细胞。在另一些实施方案中，宿主细胞是抗原呈递细胞，特别地树突细胞、单核细胞或巨噬细胞。核酸可以以单拷贝或以数个拷贝存在于宿主细胞中，并且在一个实施方案中在宿主细胞中表达。
根据本发明，术语“肽”包含寡肽和多肽，并且是指包含通过肽键彼此连接的2个或更多个，优选3个或更多个，优选4个或更多个，优选6个或更多个，优选8个或更多个，优选10个或更多个，优选13个或更多个，优选16个或更多个，优选20个或更多个，并且多至优选50个，优选100个或优选150个连续氨基酸的物质。术语“蛋白质”是指大肽，优选具有至少151个氨基酸的肽，但通常术语“肽”和“蛋白质”在本文中作为同义词使用。
术语“肽”和“蛋白质”根据本发明包含不仅含有氨基酸组分而且还含有非氨基酸组分例如糖或磷酸结构的物质，并且还包含含有例如酯键、硫醚键或二硫键的键的物质。
根据本发明，核酸例如RNA可以编码肽或蛋白质。因此，可转录核酸序列或其转录物可以包含编码肽或蛋白质的开放阅读框(open  reading frame，ORF)。所述核酸可以表达编码的肽或蛋白质。例如，所述核酸可以是编码并表达抗原或者药物活性肽或蛋白质例如免疫活性化合物(其优选地不是抗原)的核酸。
根据本发明，术语“编码肽或蛋白质的核酸”意指，如果存在于合适的环境中，优选地在细胞内，核酸可以在翻译过程中指导氨基酸的组装以产生肽或蛋白质。优选地，根据本发明的RNA能够与细胞翻译机构相互作用从而允许肽或蛋白质翻译。
在一个方面，本申请发明人筛选出能够显著提高mRNA的表达量的5'UTR和3'UTR组合。如本申请实施例证实的，多种5'UTR和3'UTR组合预料不到地能够显著提高mRNA的蛋白表达量。
更具体而言，当所述5'UTR选自SEQ ID NO:3、4、12、17、19、20、21或22所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、25、28或29所示核酸序列相对应的RNA序列时，所述5'UTR和3'UTR组合能够显著提高目的基因编码序列的翻译效率，从而显著提高其表达量。
因此，在一些实施方案中，所述5'UTR选自SEQ ID NO:3、4、12、17、19、20、21或22所示核酸序列相对应的RNA序列。
在一些实施方案中，所述3'UTR选自SEQ ID NO:23、24、25、28或29所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR选自SEQ ID NO:19或22所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、25、28或29所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR选自SEQ ID NO:3、4或12所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、28或29所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24或 28所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR选自SEQ ID NO:20或21所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、28或28所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR选自SEQ ID NO:3、19或22所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、25、28或29所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR选自SEQ ID NO:4、12、17、20或21所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、28或29所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:24、25、28或29所示核酸序列相对应的RNA序列。
在一些实施方案中，所述5'UTR和3'UTR组合不包括以下所示核酸序列相对应的RNA序列：SEQ ID NO:22和SEQ ID NO:23、SEQ ID NO:20和SEQ ID NO:24以及SEQ ID NO:21和SEQ ID NO:25
在一些实施方案中，所述mRNA分子进一步包含编码目的多肽的核酸序列。
在一些实施方案中，所述mRNA分子进一步包含polyA。
在一些实施方案中，所述mRNA可有利地用于基因治疗、基因疫苗接种、蛋白替代疗法、反义治疗或通过干扰RNA进行的治疗。
在一个方面，本公开提供了5'UTR和3'UTR组合在提高mRNA的蛋白表达量或翻译效率中的用途，其中所述5'UTR和3'UTR如上文所定义。
除非上下文另有明确指示，否则本文所用的单数形式“一”、“一个”和“所述”也意图包括复数形式。此外，开放式的表述“包括”和“包含”解释为还可以含有没有述及的结构组成部分或 方法步骤，但需要注意的是，该开放式的表述也涵盖仅由所述的组分和方法步骤组成的情形(即涵盖了封闭式表述“由……组成”的情形)。
一般而言，术语“约”在本文中用于指所述值之上和之下的数值修改5％的变化。
本申请的示例性5'UTR和3'UTR序列如表1所示。
表1.示例性5'UTR和3'UTR序列



实施例1：RNA的制备
1)体外转录模板制备：用PCR的方法扩增包含T7启动子，5'UTR,目的基因，3'UTR的DNA序列，用于体外转录。具体构建体为：T7启动子(序列如SEQ ID NO:37所示)+BamHI酶切位点(GGATCC)+5UTR+KOZAK(GCCACC)+CDS+EcoRI酶切位点(GAATTC)+3UTR+SpeI酶切位点(ACTAGT)。
2)体外转录：以PCR产物为模板，在rNTPs混合液、DTT、T7 RNA聚合酶、RNA酶抑制剂、反应Buffer的参与下，在37℃恒温器中反应16h完成体外转录，在反应体系中加入DNase I在37℃恒温器中反应30min以消化DNA模板。转录产物用氯化锂沉淀的方式纯化。
3)RNA加帽：未加帽的RNA用65℃预变性后，在GTP、加帽反应液、S-腺苷蛋氨酸、牛痘加帽酶、RNA酶抑制剂、2’-o-甲基转移酶等按照一定比例混合后，在37℃恒温器中反应一定时间后完成RNA的加帽。用氯化锂沉淀的方式纯化。
4)RNA加尾：加帽后的RNA使用polyA聚合酶试剂盒(Vazyme,DD4111)参照试剂盒说明书进行加尾反应，反应结束后用氯化锂沉淀的方式纯化。
体外表达检测
1)细胞培养和体外转染：HEK293T和Hela细胞的完全培养基为 含10％FBS(Hyclone)DMEM高糖培养基(Hyclone)，A459细胞的完全培养基为含10％FBS(Hyclne)的RPMI 1640培养基(Gibco)。在转染mRNA的前一天，将细胞以2*104细胞/孔种在96孔细胞培养板(Corning)中,第二天用Lipofectamine 3000(Invitrogen)转染mRNA，转染方法参见Lipofectamine 3000说明书。
2)GFP表达检测：细胞转染16h后，把96孔板放入酶标仪(Tecan)中，以波长488nm激发，检测波长507nm处OD值。
3)EPO(其CDS序列如SEQ ID NO:38所示)表达检测：细胞转染16h后，收集上清，用Human Erythropoietin ELISA(R&D)试剂盒做ELISA，具体操作参见试剂盒说明书。
实施例2：不同5'UTR的GFP在293T细胞系的表达量(酶标仪)：
为了测试不同5'UTR对GFP(其CDS序列如SEQ ID NO:39所示)表达量的影响，在本实施例中，3'UTR固定为UTR3-1。实验结果如下表2和图1所示。
表2.不同5'UTR对GFP表达量的影响

实施例3：不同3'UTR的GFP在293T细胞系的表达量(酶标仪)：
为了测试不同3'UTR对GFP表达量的影响，在本实施例中，5'UTR固定为028M。实验结果如下表3和图2所示。
表3.不同3'UTR对GFP表达量的影响
实施例4：不同5'UTR和不同3'UTR组合实验
对实施例2-3中效果较好的序列(UTR5-32、UTR5-30、UTR5-83、UTR5-59、UTR5-92、UTR5-91、ABOC-028M；UTR3_32、UTR3_34、UTR3_1、UTR3_7、UTR3_5)进行组合。经过模板制备、 体外转录、RNA加帽加尾、mRNA纯度质检后完成mRNA的制备和检测。检测合格后的mRNA分别转染HEK293T、Hela和A549细胞，转染后16h分别去细胞上清，用Human Erythropoietin ELISA(R&D)试剂盒进行ELISA，测试EPO(促红细胞生成素)在不同细胞中的表达量(单位为mIU/ml)。本实施例总共测试了本发明的38种UTR和3'UTR组合。各种组合的5'UTR和3'UTR在HEK293T、Hela和A549细胞系中对EPO表达量的影响分别如表4、表5和表6所示。
表4.各种组合在HEK293T细胞系中对EPO表达量的影响

表5.各种组合在Hela细胞系中对EPO表达量的影响

表6.各种组合在A549细胞系中对EPO表达量的影响

以上实施例证实，相对于对比组合1，在293T细胞中，本发明的组合1-19、21、23-28、30-32和34的EPO表达显著更高，最高为对比组合1的约2.3倍(本发明的组合10)；在Hela细胞中，本发明的组合1-28、30-32和34的EPO表达显著更高，最高为对比组合1的约2.8倍(本发明的组合10)；在A549细胞中，本发明的组合2、4-11、13、15、16、18、19、23、26-28、30的EPO表达显著更高，最高为对比组合1的约1.8倍(本发明的组合4)。
基于这些数据可得出，当所述5'UTR选自SEQ ID NO:19或22所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、25、28或29所示核酸序列相对应的RNA序列时；当所述5'UTR选自SEQ ID NO:3、4或12所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、28或29所示核酸序列 相对应的RNA序列；当所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24或28所示核酸序列相对应的RNA序列时；或者当所述5'UTR选自SEQ ID NO:20或21所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、28或28所示核酸序列相对应的RNA序列时；所述5'UTR和3'UTR组合能够显著提高目的基因编码序列的翻译效率，从而显著提高其表达量。
以上实施例还证实，相对于对比组合2，在293T细胞中，本发明的38种UTR和3'UTR组合的EPO表达显著更高，最高为对比组合2的约4.1倍(本发明的组合10)；在Hela细胞中，本发明的组合1-33、34和35的EPO表达显著更高，最高为对比组合2的约3.2倍(本发明的组合10)；在A549细胞中，本发明的组合1、2、4-19、21-28、30-35的EPO表达显著更高，最高为对比组合1的约3.1倍(本发明的组合4)。
基于这些数据可得出，当所述5'UTR选自SEQ ID NO:3、19或22所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、25、28或29所示核酸序列相对应的RNA序列时；当所述5'UTR选自SEQ ID NO:4、12、17、20或21所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、28或29所示核酸序列相对应的RNA序列时；或者当所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:24、25、28或29所示核酸序列相对应的RNA序列时；所述5'UTR和3'UTR组合能够显著提高目的基因编码序列的翻译效率，从而显著提高其表达量。
实施例5：体内实验
对实施例4中部分组合(如表7所示)进行体内测试。其CDS序列如SEQ ID NO:38所示，经过模板制备、体外转录、RNA加帽和加尾、mRNA纯度质检后完成mRNA的制备和检测，得到样品1-8。
纳米脂质颗粒的制备和检测
将阳离子脂质、DSPC、胆固醇和PEG-脂质以50：10：38.5：1.5的摩尔比溶于乙醇，并将mRNA在10至50mM柠檬酸盐缓冲液(pH＝4)中稀释。通过使用微流控装置以1：3的体积比混合乙醇脂质溶液和mRNA水溶液制备脂质体。总透析流速在9-30mL/分钟，由此除去乙醇并使用DPBS代替。最后，脂质纳米颗粒通过0.2μm无菌过滤器过滤。
动物研究
以0.5mg/kg的剂量对6-8周龄雌性ICR小鼠通过尾静脉注射(Xipuer-Bikai,Shanghai)系统性地施用包含封装人促红细胞生成素(hEPO)mRNA的样品1-8的脂质纳米颗粒(其中样品8为实施例4所述的对比组合2，作为对照)。并在给药后6小时采集小鼠血液，血液样品速冻并在-80℃下储存以进行分析。根据制造商的说明，使用市售试剂盒(DEP00，R&D系统)进行ELSA分析。
下表中列出了测试脂质纳米颗粒的特征，包括从测试组测得的hEPO表达水平(μg/ml)。
表7
上述体内实验的结果表明，与体内实验结果相一致，所述5'UTR和3'UTR组合也能够显著提高目的基因编码序列在体内的翻译效率，从而显著提高其表达量。
虽然上文已描述了本发明的各种实施方案，但是应理解的是，其仅以示例的方式提供，而并非限制。在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都将落入要求保护的本发明范围内。本发明要求保护的范围由所附的权利要求书及其等效物界定。

Claims (16)

  1. 一种包含5'UTR和3'UTR的mRNA分子，其中所述5'UTR选自下述(1)-(5)：
    (1)包含SEQ ID NO:1-22中任一个所示核酸序列相对应的RNA序列的5'UTR、其同源物、片段或变体，所述同源物、片段或变体具有与SEQ ID NO:1-22中任一个所示核酸序列相对应的RNA序列的5'UTR相同或更优的提高翻译效率的功能；优选地，所述同源物的核酸序列与SEQ ID NO:1-22中任一个所示核酸序列相对应的RNA序列具有至少70％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性；
    (2)由SEQ ID NO:1-22中任一个所示核酸序列相对应的RNA序列组成的5'UTR；
    (3)由SEQ ID NO:4、3、12、17或19-22中任一个所示核酸序列相对应的RNA序列组成的5'UTR；
    (4)上述(1)-(3)中2个或2个以上相同5'UTR经串联获得的5'UTR；或
    (5)上述(1)-(3)中2个或2个以上不同5'UTR经串联获得的5'–UTR，
    并且其中所述3'UTR选自下述(1)-(5)：
    (1)包含SEQ ID NO:23-36中任一个所示核酸序列相对应的RNA序列的3'UTR、其同源物、片段或变体，所述同源物、片段或变体具有与SEQ ID NO:23-36中任一个所示核酸序列相对应的RNA序列的3'UTR相同或更优的提高翻译效率的功能；优选地，所述同源物的核酸序列与SEQ ID NO:23-36中任一个所示核酸序列相对应的RNA序列具有至少70％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性；
    (2)由SEQ ID NO:23-36中任一个所示核酸序列相对应的RNA 序列组成的3'UTR；
    (3)由SEQ ID NO:23、24、28或29所示核酸序列相对应的RNA序列组成的3'UTR；
    (4)上述(1)-(3)中2个或2个以上相同3'UTR经串联获得的3'UTR；或
    (5)上述(1)-(3)中2个或2个以上不同3'UTR经串联获得的3'–UTR；
    并且其中所述5'UTR和3'UTR组合不包括以下所示核酸序列相对应的RNA序列：SEQ ID NO:22和SEQ ID NO:23、SEQ ID NO:20和SEQ ID NO:24以及SEQ ID NO:21和SEQ ID NO:25。
  2. 根据权利要求1所述的mRNA分子，其中所述5'UTR选自SEQ ID NO:4、3、12、17、19、20、21或22所示核酸序列相对应的RNA序列。
  3. 根据权利要求1所述的mRNA分子，其中所述3'UTR选自SEQ ID NO:23、24、25、28或29所示核酸序列相对应的RNA序列。
  4. 根据权利要求1-3中任一项所述的mRNA分子，其中
    (1)所述5'UTR选自SEQ ID NO:4、3或12所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、28或29所示核酸序列相对应的RNA序列；
    (2)所述5'UTR选自SEQ ID NO:19或22所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、25、28或29所示核酸序列相对应的RNA序列；
    (3)所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24或28所示核酸序列相对应的RNA序列；
    (4)所述5'UTR选自SEQ ID NO:20或21所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、28或28所示核 酸序列相对应的RNA序列；
    (5)所述5'UTR选自SEQ ID NO:3、19或22所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、25、28或29所示核酸序列相对应的RNA序列；
    (6)所述5'UTR选自SEQ ID NO:4、12、17、20或21所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:23、24、28或29所示核酸序列相对应的RNA序列；或
    (7)所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR选自SEQ ID NO:24、25、28或29所示核酸序列相对应的RNA序列。
  5. 根据权利要求1-3中任一项所述的mRNA分子，其中
    (1)所述5'UTR是SEQ ID NO:4所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列；
    (2)所述5'UTR是SEQ ID NO:3所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列；
    (3)所述5'UTR是SEQ ID NO:3所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列；
    (4)所述5'UTR是SEQ ID NO:3所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:25所示核酸序列相对应的RNA序列；
    (5)所述5'UTR是SEQ ID NO:3所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列；
    (6)所述5'UTR是SEQ ID NO:3所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序 列；
    (7)所述5'UTR是SEQ ID NO:4所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列；
    (8)所述5'UTR是SEQ ID NO:4所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列；
    (9)所述5'UTR是SEQ ID NO:4所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序列；
    (10)所述5'UTR是SEQ ID NO:12所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列；
    (11)所述5'UTR是SEQ ID NO:12所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列；
    (12)所述5'UTR是SEQ ID NO:12所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列；
    (13)所述5'UTR是SEQ ID NO:12所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序列；
    (14)所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列；
    (15)所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列；
    (16)所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:25所示核酸序列相对应的RNA序列；
    (17)所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列；
    (18)所述5'UTR是SEQ ID NO:17所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序列；
    (19)所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列；
    (20)所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列；
    (21)所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:25所示核酸序列相对应的RNA序列；
    (22)所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列；
    (23)所述5'UTR是SEQ ID NO:19所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序列；
    (24)所述5'UTR是SEQ ID NO:20所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列；
    (25)所述5'UTR是SEQ ID NO:20所示核酸序列相对应的RNA 序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列；
    (26)所述5'UTR是SEQ ID NO:20所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序列；
    (27)所述5'UTR是SEQ ID NO:21所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列；
    (28)所述5'UTR是SEQ ID NO:21所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列；
    (29)所述5'UTR是SEQ ID NO:21所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列；
    (30)所述5'UTR是SEQ ID NO:22所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:24所示核酸序列相对应的RNA序列；
    (31)所述5'UTR是SEQ ID NO:22所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:25所示核酸序列相对应的RNA序列；
    (32)所述5'UTR是SEQ ID NO:22所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列；或
    (33)所述5'UTR是SEQ ID NO:22所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:29所示核酸序列相对应的RNA序列。
  6. 根据权利要求1-3中任一项所述的mRNA分子，其中所述5'UTR是SEQ ID NO:4所示核酸序列相对应的RNA序列，而所述3' UTR是SEQ ID NO:23所示核酸序列相对应的RNA序列。
  7. 根据权利要求1-3中任一项所述的mRNA分子，其中所述5'UTR是SEQ ID NO:22所示核酸序列相对应的RNA序列，而所述3'UTR是SEQ ID NO:28所示核酸序列相对应的RNA序列。
  8. 根据权利要求1-3中任一项所述的mRNA分子，其进一步包含编码目的多肽的核酸序列。
  9. 根据权利要求1-3中任一项所述的mRNA分子，其进一步包含polyA。
  10. 5'UTR和3'UTR组合在提高mRNA的蛋白表达量或翻译效率中的用途，其中所述5'UTR和3'UTR如权利要求1-7中任一项所定义。
  11. 编码权利要求1-9中任一项所述的mRNA分子的DNA。
  12. 一种载体，所述载体包含权利要求11所述的DNA。
  13. 一种宿主细胞，其包含权利要求12所述的载体。
  14. 一种脂质纳米颗粒，其包含权利要求1-9中任一项所述的mRNA分子。
  15. 一种药物组合物，其包含权利要求1-9中任一项所述的mRNA分子、权利要求11所述的DNA、权利要求12所述的载体、权利要求13所述的宿主细胞或权利要求14所述的脂质纳米颗粒，以及药学上可接受的载剂。
  16. 权利要求1-9中任一项所述的mRNA分子、权利要求11所述的DNA、权利要求12所述的载体、权利要求13所述的宿主细胞、权利要求14所述的脂质纳米颗粒或权利要求15所述的药物组合物在制备药物中的用途，所述药物用于基因治疗、基因疫苗接种、蛋白替代疗法、反义治疗或通过干扰RNA进行的治疗。
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