Artificial polynucleotides for expression of proteins

Abstract

The present invention provides a polynucleotide comprising a 5'untranslated region (5 '-UTR) and an open reading framework (ORF) in the 5'to 3' direction wherein the 5 '-UTR comprises at least two tandem repeat sequences 5'-GCCNCC-3 'operatively linked to the ORF, and wherein N is any nucleotide. The invention also provides a composition and a pharmaceutical composition comprising the lipid nanoparticles and the polynucleotide, and their use in medicine, in particular as a vaccine or for gene therapy.

Description

[0001] This application claims priority to European patent application EP22382946.6 filed on October 7, 2022. Technical Field

[0002] The present invention belongs to the field of polynucleotides, and in particular relates to artificial polynucleotides encoding polypeptides. The polynucleotides of the present invention are particularly suitable for genetic vaccination. Background Art

[0003] Genetic vaccination and gene therapy are two of the most promising and rapidly developing therapeutic areas in modern medicine. They are both based on delivering polynucleotides such as DNA or RNA molecules into the cells or tissues of patients to produce polypeptides with therapeutic effects.

[0004] The use of RNA in gene therapy and vaccination is generally considered safer than the use of DNA because there is no risk of RNA stably integrating into the genome of the transfected cell. In addition, RNA is more easily degraded in the body, making its half-life relatively short compared to DNA. Therefore, compared with DNA-based therapies, RNA-based therapies have a lower risk of the host producing undesirable anti-RNA antibodies that can reduce the effectiveness of the therapy and produce serious side effects. Therefore, in many cases, RNA is considered the molecule of choice for genetic medicine therapy.

[0005] One of the major limitations of RNA-based therapeutics is their limited protein production efficiency. Due to the relatively short half-life of RNA, it is critical that they provide extremely high translation rates to produce sufficient amounts of the desired protein before RNA degradation. There have been attempts to optimize 5' untranslated regions (UTRs) to improve protein production through rational design of UTRs; however, progress to date has been extremely limited.

[0006] Therefore, despite the efforts that have been made in recent years, there is still a need for polynucleotides, particularly RNA, with high translation efficiency that are suitable for genetic vaccination and gene therapy. Summary of the invention

[0007] The inventors have developed a novel nucleic acid sequence that, when inserted into the 3'-end of a 5'UTR, significantly enhances the translation efficiency of mRNA transcripts. This novel sequence can be used to construct artificial transcripts with extremely high protein yields, suitable for a variety of applications, including nucleic acid therapy or industrial recombinant protein production.

[0008] As shown in the following examples, the present inventors found that inserting only one repeat of the sequence 5'-GCCACC-3' at the 3'-end of the 5'-UTR of the transcript did not affect its protein production ability in vitro or in vivo (see Figure 1 and 2, R2 vs. R1). However, when two tandem repeats of the sequence were inserted, Figure 1 , R3 vs. R1) and in vivo (see Figure 2 , R3 compared with R1), which was completely unexpected as no effect was observed when only one repeat sequence was present.

[0009] Notably, the presence of two tandem repeats provided a synergistic effect resulting in an increase in protein expression of approximately 400% after 24 hours compared to transcripts not containing the two tandem repeats ( Figure 2 , R3 vs. R1 or R2), and also outperformed transcripts containing commercial RNA regulatory elements ( Figure 2 , R3 compared with R4 or R6) or previously published RNA transcripts ( Figure 2 , R3 compared with R5).

[0010] Therefore, as can be seen from the data provided below, the polynucleotides provided by the present invention represent an important new tool to overcome the limitations of current artificial RNA, especially their low protein production efficiency. In addition, the higher translation efficiency of the polynucleotides of the present invention can reduce the RNA treatment dose required for genetic vaccination, thereby helping to reduce related secondary effects.

[0011] In a first aspect, the present invention provides an artificial polynucleotide comprising a 5' untranslated region (5'-UTR) and an open reading frame (ORF) in the 5' to 3' direction, wherein the 5'-UTR comprises at its 3'-end at least two tandem repeats of the sequence 5'-GCCNCC-3' operably linked to the ORF, wherein N is any nucleotide.

[0012] In a second aspect, the present invention provides a DNA construct comprising a promoter operably linked to a polynucleotide sequence encoding the polynucleotide sequence defined in the first aspect.

[0013] In a third aspect, the present invention provides an expression vector comprising the DNA construct as defined in the second aspect.

[0014] In a fourth aspect, the present invention provides a cell comprising the polynucleotide as defined in the first aspect, the DNA construct as defined in the second aspect, or the expression vector as defined in the third aspect.

[0015] In a fifth aspect, the present invention provides a composition comprising lipid nanoparticles and the polynucleotide defined in the first aspect, the DNA construct defined in the second aspect, or the expression vector defined in the third aspect.

[0016] In a sixth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the polynucleotide according to the first aspect, the DNA construct according to the second aspect, the expression vector according to the third aspect or the composition according to the fifth aspect, and at least one pharmaceutically acceptable excipient and / or carrier.

[0017] When the polynucleotide of the present invention includes an ORF encoding a protein associated with a disease, the above aspects can be used as a medicine. Therefore, in the seventh aspect, the present invention provides a polynucleotide according to the first aspect, a DNA construct according to the second aspect, an expression vector according to the third aspect, a cell according to the fourth aspect, a composition according to the fifth aspect, or a pharmaceutical composition according to the sixth aspect for use in medicine.

[0018] In an eighth aspect, the present invention provides a method for inducing an immune response in a subject using the polynucleotide according to the first aspect, the DNA construct according to the second aspect, the expression vector according to the third aspect, the cell according to the fourth aspect, the composition according to the fifth aspect or the pharmaceutical composition according to the sixth aspect.

[0019] This aspect can also be expressed as the use of a polynucleotide, DNA construct, expression vector, composition, cell or pharmaceutical composition for the preparation of a medicament for inducing an immune response in a subject. This aspect can also be expressed as a method of inducing an immune response in a subject, the method comprising administering a therapeutically effective amount of a polynucleotide, DNA construct, expression vector, composition, cell or pharmaceutical composition of the present invention to a subject in need thereof.

[0020] In the ninth aspect, the present invention provides the polynucleotide according to the first aspect, the DNA construct according to the second aspect, the expression vector according to the third aspect, the cell according to the fourth aspect, the composition according to the fifth aspect or the pharmaceutical composition according to the sixth aspect for use in a method for therapeutic immunization of a subject.

[0021] This aspect can also be expressed as the use of a polynucleotide, DNA construct, expression vector, composition, cell or pharmaceutical composition for the preparation of a medicament for therapeutic immunization of a subject. This aspect can also be a method for therapeutic immunization of a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polynucleotide, DNA construct, expression vector, composition, cell or pharmaceutical composition of the invention.

[0022] In the tenth aspect, the present invention provides the polynucleotide according to the first aspect, the DNA construct according to the second aspect, the expression vector according to the third aspect, the cell according to the fourth aspect, the composition according to the fifth aspect or the pharmaceutical composition according to the sixth aspect for use as a vaccine or for gene therapy.

[0023] In the eleventh aspect, the present invention provides an in vitro method for producing a polypeptide in a cell, the method comprising contacting the cell with the polynucleotide defined in the first aspect, the DNA construct defined in the second aspect, the expression vector defined in the third aspect, the composition defined in the fifth aspect, or the pharmaceutical composition defined in the sixth aspect.

[0024] In the twelfth aspect, the present invention provides an in vitro method for increasing the translation rate of a polynucleotide, wherein the polynucleotide comprises a 5' untranslated region (5'-UTR) and an open reading frame (ORF) in the 5' to 3' direction, the method comprising the step of inserting at least two tandem repeats of the sequence 5'-GCCNCC-3' at the 3'-end of the 5'-UTR, wherein N is any nucleotide.

[0025] In the thirteenth aspect, the present invention provides a vaccination kit comprising (a) a polynucleotide as defined in the first aspect, a DNA construct as defined in the second aspect, a DNA vector as defined in the third aspect, an expression vector as defined in the fourth aspect, or a composition as defined in the fifth aspect; (b) a pharmaceutically acceptable excipient and / or carrier; (c) an optional adjuvant; and (d) optional instructions for its use.

[0026] In a fourteenth aspect, the present invention provides a method for preparing a pharmaceutical composition according to the sixth aspect, comprising the step of mixing the polynucleotide, DNA construct, expression vector or composition of the present invention with at least one pharmaceutically acceptable excipient and / or carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 , related to Example 1, are bar graphs showing the levels of luciferase production in (A) HeLa cells and (B) HEK293T cells after transfection with the indicated mRNAs. The structures of mRNAs R1, R2, R3, R4, R5, and R6 are detailed in Table 2 below. "RLU" stands for relative light units.

[0028] Figure 2 , related to Example 2, are bar graphs showing the level of luciferase production in mouse muscle at (A) 4 hours, (B) 7 hours, and (C) 24 hours after injection of lipid nanoparticles containing the indicated mRNAs. The structures of mRNAs R1, R2, R3, R4, R5, and R6 are detailed in Table 2 below.

[0029] Figure 3 , related to Example 3, are bar graphs showing the levels of luciferase production in (A) HeLa cells and (B) HEK293T cells after transfection with the indicated mRNAs. The structures of mRNAs R4, R5, R7, and R8 are detailed in Table 3 below. "RLU" stands for relative light units.

[0030] Figure 4 , related to Example 4, are bar graphs showing the levels of luciferase production in (A) HeLa cells and (B) HEK293T cells after transfection of the indicated mRNAs. The structures of mRNAs R4, R5, R9, and R10 are detailed in Table 4 below. "RLU" stands for relative light units. Specific embodiments

[0031] Unless otherwise specified, all terms used in this application are understood to have common meanings known in the art. Other more specific definitions of certain terms used in this application are described below and are intended to be uniformly applied throughout the specification and claims, unless otherwise expressly defined definitions provide a broader definition.

[0032] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more." Definite articles such as "the" as used herein also include plural forms of nouns unless otherwise stated.

[0033] The term "polynucleotide" is used interchangeably with "nucleic acid" and refers to a polymer composed of nucleotides, including ribonucleotides or deoxyribonucleotides. A polynucleotide formed by ribonucleotides may be referred to as an "RNA polynucleotide", "ribonucleic acid" or simply "RNA"; a polynucleotide formed by deoxyribonucleotides may be referred to as a "DNA polynucleotide", "deoxyribonucleic acid" or simply "DNA". A polynucleotide may be single-stranded or double-stranded and may optionally contain synthetic, non-natural or modified nucleotides that can be incorporated into DNA or RNA. An "artificial polynucleotide" refers to a polynucleotide whose sequence does not exist in nature or has been altered by human intervention. A "purine nucleotide" refers to a nucleotide containing a purine as a nitrogenous base, particularly adenine or guanine. When polynucleotide sequences are represented, they refer to the information molecule and only one strand is shown. Nevertheless, it will be understood by those skilled in the art that it can be in the form of a single-stranded or double-stranded nucleic acid polymer; for example, mRNA is single-stranded; while a DNA construct is double-stranded.

[0034] As used herein, the term "messenger RNA" or "mRNA" or "transcript" refers to any RNA polynucleotide encoding a target polypeptide and can be translated in vitro, in vivo or ex vivo to generate the encoded target polypeptide. Typically, mRNA is single-stranded and comprises a 5'-cap structure, a 5'UTR, an ORF, a 3'UTR and a 3' tail sequence.

[0035] As used herein, the "untranslated region" or "UTR" of a polynucleotide refers to a region that is located upstream (i.e., the 5' end) or downstream (i.e., the 3' end) of an open reading frame (ORF) and is not translated by the ribosome. Accordingly, the "5' untranslated region" or "5'-UTR" refers to a region that is located upstream (i.e., the 5' end) of the start codon of the ORF, which is transcribed but not translated into an amino acid sequence. The 5'-UTR generally corresponds to a sequence that extends from the nucleotide immediately adjacent to the 3' end of the 5'-cap structure to the nucleotide immediately adjacent to the 5' end of the start codon of the ORF. The "3' untranslated region" or "3'UTR" refers to a region that is located downstream of the stop codon of the ORF, which is transcribed but not translated into an amino acid sequence. The 3'-UTR generally corresponds to a sequence that extends from the nucleotide immediately adjacent to the 3' end of the stop codon of the ORF to the nucleotide immediately adjacent to the 5' end of the poly (A) sequence.

[0036] As used herein, "open reading frame" or "ORF" refers to a plurality of nucleotide triplet sequences encoding a polypeptide, i.e., a sequence that can be translated into a polypeptide. An open reading frame typically contains a start codon, i.e., a combination of three nucleotides (e.g., ATG or AUG) that typically encodes the amino acid methionine at its 5' end, and a subsequent region, the length of which is typically a multiple of 3 nucleotides. An ORF is typically terminated by a stop codon (e.g., TAA, TAG, TGA). An open reading frame may also be referred to as a "protein coding region."

[0037] "5'-cap structure" refers to an entity, usually a modified nucleotide, which generally closes the 5' end of the mature mRNA. The 5'-cap can generally be formed by modified nucleotides, in particular by derivatives of guanine nucleotides. Preferably, the 5'-cap is connected to the 5'-end via a 5'-5'-triphosphate bond. The 5'-cap can be methylated, for example m7GpppN, where N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap, usually the 5'-end of RNA. 5'-cap structures include but are not limited to "cap-0", which refers to a methylated guanosine (located on the seventh nitrogen, N) added to the mRNA at the 5'-to-5' junction. 7 ); "cap-1" refers to the nucleotide at the 5' end of the strand based on cap-0 (N 1 ) adds a methyl group to the 2'-carbon of the ribose sugar; "cap-2" refers to the addition of a methyl group to the next nucleotide (N 2) and then add a 2'-methyl group.

[0038] The "3' tail sequence" refers to a sequence located at the 3'-end of an mRNA, which enhances the stability of the mRNA, is usually rich in adenine nucleotides and is at least 80 nucleotides in length.

[0039] As used herein, "effectively linked" or "operably linked" refers to a sequence connection mode that achieves expression of the coding sequence under conditions compatible with the control sequence. Operably linked sequences include expression control sequences adjacent to the target coding sequence, as well as expression control sequences that control the target coding sequence in trans or remotely. For example, at least two tandem repeats of the sequence 5'-GCCNCC-3' at the 3'-end of the 5'-UTR are an expression control sequence that is effectively linked to the downstream ORF because it enhances the translation of the ORF. In a specific embodiment, an "effectively linked sequence" refers to a directly linked sequence.

[0040] As used herein, the term "tandem repeat sequence" refers to a sequence that appears consecutively in order. For example, two tandem repeat sequences of the sequence 5'-GCCNCC-3' refer to a sequence comprising two consecutive 5'-GCCNCC-3' units (ie, 5'-GCCNCCGCCNCC-3').

[0041] As used herein, "Kozak sequence" refers to a sequence located in mRNA that assists the ribosomal translation machinery in recognizing the translation start site of the transcript. The Kozak sequence has a consensus sequence CCRCCAUGG, where R is a purine nucleotide and AUG is the start codon of the ORF. As used herein, a "consensus Kozak sequence" or "strong Kozak sequence" refers to a sequence that is highly matched to the above consensus sequence. A "non-consensus Kozak sequence" or "weak Kozak sequence" refers to a sequence that is not highly matched to the consensus sequence, particularly at the +4 and -3 positions relative to the adenosine (+1) at the 5' end of the start codon.

[0042] The term "heterologous" refers to a combination of elements that do not occur in nature. For example, a 5'-UTR that is heterologous to an ORF means that the combination of the 5'-UTR and the ORF is not found in a natural sequence.

[0043] In the present invention, the term "identical" or "identity" refers to the percentage of identical sites in two sequences when the sequences are optimally aligned. If, in the optimal alignment, a site in the first sequence is occupied by the same nucleotide as the corresponding site in the second sequence, the sequence exhibits identity at that site. The identity percentage determines the number of identical nucleotides within a specific length in a particular alignment. Therefore, the level of identity between two sequences or ("sequence identity percentage") is determined by calculating the ratio of the number of identical sites to the number of aligned sites (i.e., sequence identity percentage = (number of identical sites / total number of aligned sites) × 100). Gaps, i.e., sites where a nucleotide is present in one sequence but not in the other sequence in the alignment, are considered sites of non-identical nucleotides and are counted as aligned sites.

[0044] A variety of mathematical algorithms for rapidly obtaining optimal alignments between two or more sequences and calculating their identity are known and have been incorporated into a variety of existing software programs. For the purposes of the present invention, the sequence identity between two nucleic acid sequences is preferably determined by an algorithm based on a global alignment, such as the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), preferably implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277); or the BLAST global alignment tool (Altschul et al., "Basic local alignment search tool", 1990, J. Mol. Biol, v. 215, pages 403-410), using default settings. When the lengths of the sequences being aligned are substantially the same, local alignments may also be used.

[0045] As used herein, "DNA construct" refers to an artificial polynucleotide comprising a target sequence operably linked to an expression promoter, which controls the expression of the target sequence. "Expression vector" refers to a vector used to introduce a specific nucleic acid, usually a DNA construct, into a target cell to express the nucleic acid by the cell. Examples of suitable expression promoters and expression vectors include those routinely used in molecular biology and well known to those skilled in the art.

[0046] The term 'polypeptide' refers to any peptide or protein formed by two or more amino acids linked by peptide bonds or modified peptide bonds (ie, peptide isosteres). "Polypeptide" refers to short chains, usually called peptides, oligopeptides or oligomers, and also to longer chains, usually called proteins.

[0047] As used herein, the expression "therapeutically effective amount" refers to a dose that, when a compound is administered, is sufficient to prevent the development of the disease being treated or to alleviate to some extent one or more symptoms of the disease being treated. The specific dose of the compound administered according to the present invention will of course be determined according to the specific application, including the compound administered, the route of administration, the specific condition being treated, and similar factors. The term "drug" also includes the concept of "veterinary composition". Therefore, they relate to compositions that are therapeutically effective when administered to any animal, including humans, by any desired or applicable route.

[0048] According to the present invention, the term "antigen" refers to a compound that can be recognized by the immunoglobulin receptor of a B cell, or by a T cell receptor when in complex with MHC. Preferably, the "antigen" is a polypeptide.

[0049] As used herein, the term "nanoparticle" refers to particles in which at least two dimensions are at nanoscale, particularly all three dimensions are at nanoscale, wherein the nanoscale is in the range of about 1 nm to about 300 nm. In particular, when the nanoparticle is substantially rod-shaped and has a substantially circular cross-section, such as a nanowire or a nanotube, "nanoparticle" refers to particles in which at least two dimensions are at nanoscale, and these two dimensions are the cross-sections of the nanoparticles. As used herein, the term "lipid nanoparticle" refers to nanoparticles whose outer envelope is composed entirely or partially of lipids. Examples of lipid nanoparticles suitable for the compositions of the present invention are described in Hassett, KJ et al., "Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines", 2019, Mol. Ther. Nucleic Acid, vol. 15, pp. 1-11.

[0050] As described above, the present invention provides in a first aspect an artificial polynucleotide comprising a 5' untranslated region (5'-UTR) and an open reading frame (ORF) in the 5' to 3' direction, wherein the 5'-UTR comprises at its 3'-end at least two tandem repeats of the sequence 5'-GCCNCC-3' operably linked to the ORF, wherein N is any nucleotide.

[0051] In a specific embodiment of the first aspect, optionally in combination with any of the above or below embodiments, the at least two tandem repeat sequences are the sequence 5'-GCCRCC-3', wherein R is a purine nucleotide.

[0052] In an embodiment of the first aspect, the artificial polynucleotide comprises a 5' untranslated region (5'-UTR) and an open reading frame (ORF) in the 5' to 3' direction, wherein the 5'-UTR comprises at its 3'-end at least two tandem repeat sequences of a tandem repeat sequence sequence 5'-GCCRCC-3' operably linked to the ORF, wherein R is a purine nucleotide.

[0053] In one embodiment of the first aspect, the polynucleotide comprises a 5' untranslated region (5'-UTR) and an open reading frame (ORF) consecutively and in a 5' to 3' direction, wherein the 5'-UTR comprises at least two tandem repeats of the sequence 5'-GCCNCC-3' operatively linked to the ORF at its 3'-end, and wherein N is any nucleotide. In a more specific embodiment, the two tandem repeats are the sequence 5'-GCCRCC-3', wherein R is a purine nucleotide.

[0054] In one embodiment of the first aspect, the polynucleotide comprises a 5' untranslated region (5'-UTR) and an open reading frame (ORF) in the 5' to 3' direction, wherein the 5'-UTR comprises at its 3'-end the sequence 5'-GCCRCC-3', wherein R is an adenine nucleotide or a guanine nucleotide, operably linked to at least two tandem repeat sequences of the ORF.

[0055] In one embodiment of the first aspect, the at least two tandem repeats of the sequence 5'-GCCNCC-3' or 5'-GCCRCC-3' are directly linked to the ORF (ie, the at least two tandem repeats are located immediately upstream of the first codon of the ORF).

[0056] In one embodiment of the first aspect, the 5'-UTR comprises at its 3'-end two tandem repeats of the sequence 5'-GCCNCC-3' operatively linked to the ORF. In one embodiment of the first aspect, the 5'-UTR comprises at its 3'-end three tandem repeats of the sequence 5'-GCCNCC-3' operatively linked to the ORF. In another embodiment, the 5'-UTR comprises at its 3'-end two or three tandem repeats of the sequence 5'-GCCRCC-3' operatively linked to the ORF.

[0057] In one embodiment of the first aspect, the at least two tandem repeats of the sequence 5'-GCCNCC-3' enhance the translation efficiency of the ORF.

[0058] In one embodiment of the first aspect, the 5'-UTR comprises at its 3'-end a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. In a more specific embodiment, the 5'-UTR comprises at its 3'-end SEQ ID NO: 3.

[0059] In one embodiment of the first aspect, the 5'-UTR comprises at its 3'-end a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or a sequence that is identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. NO:6 is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or at least 99.9% identical variants, particularly wherein the variants substantially retain or improve upon SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0060] Polynucleotide sequence variants are well known to those skilled in the art and may include sequence modifications such as deletions, insertions or changes of nucleotides.

[0061] In a specific embodiment of the first aspect, at least two tandem repeats of the sequence 5'-GCCNCC-3' form a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; in particular SEQ ID NO: 3.

[0062] In one embodiment of the first aspect, the 5'-UTR comprises or consists of a sequence from a 5'-UTR of a gene transcript connected to at least two tandem repeats of the sequence 5'-GCCNCC-3' in the 5' to 3' direction. In one embodiment of the first aspect, the 5'-UTR comprises or consists of a sequence from a 5'-UTR of a gene transcript connected to two tandem repeats of the sequence 5'-GCCNCC-3' in the 5' to 3' direction. In another embodiment, the 5'-UTR comprises or consists of a sequence derived from a 5'-UTR of a gene transcript connected to at least two tandem repeats of the sequence 5'-GCCNCC-3' in the 5' to 3' direction. In another embodiment, the 5'-UTR comprises or consists of a 5'-UTR of a gene transcript directly connected to at least two tandem repeats of the sequence 5'-GCCNCC-3' in the 5' to 3' direction. In a more specific embodiment, the at least two tandem repeats are the sequence 5'-GCCRCC-3'. In a more specific embodiment, the gene is a naturally occurring gene. In a more specific embodiment, the gene is a mammalian gene or a human gene.

[0063] In another specific embodiment of the first aspect, the 5'-UTR comprises or consists of a sequence from a 5'-UTR of a gene transcript connected to at least two tandem repeats of the sequence 5'-GCCNCC-3' in the 5' to 3' direction, wherein the sequence from the 5'-UTR of the gene transcript comprises a Kozak sequence, in particular a non-shared Kozak sequence. Therefore, the Kozak sequence is directly connected to the tandem repeats of the sequence 5'-GCCNCC-3'. In another specific embodiment of the first aspect, the 5'-UTR comprises or consists of a sequence from a 5'-UTR of a gene transcript connected to the sequence 5'-GCCNCC-3' of two tandem repeats in the 5' to 3' direction, wherein the sequence from the 5'-UTR of the gene transcript comprises a Kozak sequence, in particular a non-shared Kozak sequence. Therefore, the polynucleotide may sequentially comprise a Kozak sequence nucleotide located upstream of the start codon, (at least) two tandem repeats of the sequence 5'-GCCNCC-3', and a subsequent start codon in the 5' to 3' direction. In a more specific embodiment, the two tandem repeat sequences are the sequence 5'-GCCRCC-3'.

[0064] In a specific embodiment of the first aspect, the 5'-UTR comprises or consists of a sequence from a 5'-UTR of a gene transcript linked to at least two tandem repeats of the sequence 5'-GCCNCC-3' in the 5' to 3' direction, wherein the gene is selected from apolipoprotein A2 (APOA2), hemoglobin beta subunit (HBB), pre-T cell antigen receptor alpha (PTCRA) and small nuclear ribonucleoprotein D1 polypeptide (SNRPD1). In a more specific embodiment, the gene is a human gene selected from apolipoprotein A2 (APOA2) (NCBI gene ID: 336, updated on August 7, 2022), hemoglobin subunit beta (HBB) (NCBI gene ID: 3043, updated on August 5, 2022), pre-T cell antigen receptor alpha (PTCRA) (gene ID: 171558, updated on August 5, 2022), small nuclear ribonucleoprotein D1 polypeptide (SNRPD1) (gene ID: 6632, updated on September 7, 2023). In a more specific embodiment, the at least two tandem repeat sequences are the sequence 5'-GCCRCC-3'.

[0065] In certain embodiments of the first aspect, the 5'-UTR comprises a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 41, or a sequence thereof with SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 41. NO:41 is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or at least 99.9% identical variant; in particular, wherein the variant substantially retains or improves the NO:12, SEQ ID NO:15 or SEQ ID NO:41.

[0066] In a specific embodiment of the first aspect, the polynucleotide further comprises one or more of the following: a 5'-cap structure; a 3' untranslated region (3'-UTR); and a 3' tail sequence.

[0067] In a specific embodiment of the first aspect, the polynucleotide comprises in the 5' to 3' direction: (i) a 5'-cap structure; (ii) a 5' untranslated region (5'-UTR), (iii) an open reading frame (ORF); (iv) a 3' untranslated region (3'-UTR); and (v) a 3' tail sequence.

[0068] In a particular embodiment of the first aspect, the polynucleotide is an RNA polynucleotide, in particular messenger RNA (mRNA).

[0069] In a specific embodiment of the first aspect, the 5'UTR is heterologous to the ORF and / or the 3'UTR.

[0070] In a specific embodiment of the first aspect, the 3'-UTR comprises or consists of a sequence from a 3'-UTR of a gene transcript, in particular a mammalian gene or a human gene.

[0071] In a specific embodiment of the first aspect, the 3'-UTR comprises or consists of at least two tandem repeats of a sequence from the 3'-UTR of a gene transcript, in particular comprises two tandem repeats of a sequence from the 3'-UTR of a gene transcript. In particular, the 3'-UTR comprises or consists of a sequence from the 3'-UTR of a gene transcript or two tandem repeats thereof, wherein the gene is selected from HBB, PTCRA, APOA2 and SNRPD1.

[0072] In a specific embodiment of the first aspect, the 3'-UTR comprises or consists of a 3'-UTR of a gene transcript, in particular a mammalian gene or a human gene. More particularly, the 3'-UTR comprises or consists of two tandem repeats of a 3'-UTR of a gene transcript, in particular, wherein the gene is selected from the group consisting of HBB, PTCRA, APOA2 and SNRPD1.

[0073] In one embodiment of the first aspect, the 3′-UTR comprises a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26; or a sequence thereof with SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26. NO:26 is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or at least 99.9% identical to a variant thereof.

[0074] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 9 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 18.

[0075] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 9 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 19.

[0076] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 9 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 20.

[0077] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 9 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 21.

[0078] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 9 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 22.

[0079] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 9 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 23.

[0080] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 15 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 18.

[0081] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 15 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 19.

[0082] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 15 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 20.

[0083] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 15 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 21.

[0084] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 15 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 22.

[0085] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 15 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 23.

[0086] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 12 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 18.

[0087] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 12 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 19.

[0088] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 12 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 20.

[0089] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 12 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 21.

[0090] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 12 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 22.

[0091] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 12 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 23.

[0092] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 41 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 18.

[0093] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 41 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 19.

[0094] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 41 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 20.

[0095] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 41 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 21.

[0096] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 41 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 22.

[0097] In one embodiment of the first aspect, the polynucleotide comprises in 5' to 3' direction a 5'-UTR, an ORF and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence SEQ ID NO: 41 and the 3'-UTR comprises or consists of the sequence SEQ ID NO: 23.

[0098] In an embodiment of the first aspect, the ORF encodes a polypeptide, in particular, wherein the polypeptide is an antigen. More particularly, the antigen is selected from a viral protein, a bacterial protein and a tumor-associated antigen. In a specific embodiment of the first aspect, the polypeptide is an antibody or a fragment thereof. In a more specific embodiment, the antibody or fragment thereof is a therapeutic antibody or a fragment thereof.

[0099] In one embodiment of the first aspect, the 5'-cap structure is selected from cap-0, cap-1, cap-2, ARCA, inosine, N1-methylguanosine, 2'-fluoroguanosine, 7-deazaguanosine, 8-oxoguanosine, 2-aminoguanosine, LNAguanosine and 2-azidoguanosine. In a more specific embodiment, the 5'-cap structure is cap-1.

[0100] In an embodiment of the first aspect, the 3' tail sequence is a poly(A) region, in particular, the length of the poly(A) region is at least 80 nucleotides, at least 90 nucleotides or at least 100 nucleotides. In a more specific embodiment, the 3' tail sequence comprises the sequence of SEQ ID NO: 39, or a sequence thereof with SEQ ID NO: NO: 39 is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or at least 99.9% identical to a variant thereof.

[0101] In one embodiment of the first aspect, the polynucleotide comprises at least one of: a 5' untranslated region (5'-UTR) of sequence SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 41; a 3' untranslated region (3'-UTR) of sequence SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23; and a 3' tail sequence of sequence SEQ ID NO: 39.

[0102] In one embodiment of the first aspect, the polynucleotide comprises at least one of: a 5' untranslated region (5'-UTR) of sequence SEQ ID NO: 9; a 3' untranslated region (3'-UTR) of sequence SEQ ID NO: 18 or 19; and a 3' tail sequence of sequence SEQ ID NO: 39.

[0103] In one embodiment of the first aspect, the polynucleotide comprises: a 5' untranslated region (5'-UTR) of sequence SEQ ID NO:9; a 3' untranslated region (3'-UTR) of sequence SEQ ID NO:18 or 19; and a 3' tail sequence of sequence SEQ ID NO:39.

[0104] In one embodiment of the first aspect, the polynucleotide comprises at least one of: a 5' untranslated region (5'-UTR) of sequence SEQ ID NO:41; a 3' untranslated region (3'-UTR) of sequence SEQ ID NO:18 or 19; and a 3' tail sequence of sequence SEQ ID NO:39.

[0105] In one embodiment of the first aspect, the polynucleotide comprises: a 5' untranslated region (5'-UTR) of sequence SEQ ID NO:41; a 3' untranslated region (3'-UTR) of sequence SEQ ID NO:18 or 19; and a 3' tail sequence of sequence SEQ ID NO:39.

[0106] In one embodiment of the first aspect, the polynucleotide comprises or consists of the following structures in the 5' to 3' direction: (i) a 5'-cap 1 structure; (ii) a 5' untranslated region (5'-UTR), in particular the 5' untranslated region of sequence SEQ ID NO: 9 or 41, (iii) an open reading frame (ORF); in particular the polypeptide encoded thereof; (iv) a 3' untranslated region (3'-UTR), in particular the 3' untranslated region of sequence SEQ ID NO: 18 or 19; and (v) a 3' tail sequence, in particular the 3' tail sequence of sequence SEQ ID NO: 39.

[0107] In one embodiment of the first aspect, the polynucleotide comprises at least one chemical modification. In a specific embodiment, the chemical modification is selected from the following pseudouridine, N1-methyl pseudouridine (also known as 1-methyl pseudouridine or m1Ψ), N6-methyladenosine (also known as m6A), 2-thiouridine (also known as s2U), 4'-thiouridine, 5-methylcytosine (also known as 5mC), 2-thiol-1-methyl-1-deazapseudouridine, 2-thiol-1-methylpseudouridine, 2-thiol-5-azauridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol pseudouridine, 4-methoxypseudouridine, 4-thiol-L-methylpseudouridine, 4-thiol pseudouridine, 5-azauridine, dihydropseudouridine, 5-methyluridine (also known as m5U), 5-methoxyuridine, 2'-O-methyluridine and combinations thereof. In a more specific embodiment, the chemical modification is N1-methylpseudouridine, 5-methoxyuridine or a combination thereof; in particular, the chemical modification is N1-methylpseudouridine.

[0108] In a specific embodiment of the first aspect, the polynucleotide is partially modified with N1-methyluridine. In another specific embodiment, the polynucleotide is partially modified with at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% of N1-methyluridine. In another specific embodiment, the polynucleotide is fully modified with N1-methyluridine, 5-methoxyuridine or a combination thereof. In a more specific embodiment, the polynucleotide is fully modified with N1-methylpseudouridine.

[0109] In a specific embodiment of the first aspect, the polynucleotide is an isolated artificial polynucleotide.

[0110] As described above, the second aspect of the present invention provides a DNA construct comprising a promoter operably linked to a sequence encoding the polynucleotide of the first aspect. That is, this aspect provides a DNA construct that transcribes and generates a polynucleotide as defined in the first aspect.

[0111] All embodiments of the polynucleotide of the first aspect are equally applicable to the DNA construct of the second aspect.

[0112] In one embodiment of the second aspect, the polynucleotide is an RNA polynucleotide, in particular mRNA.

[0113] Those skilled in the art know how to prepare the polynucleotides, DNA constructs or expression vectors of the present invention by conventional methods known in the art, such as by chemical synthesis or by molecular biology techniques, without expending any creative technical effort.

[0114] As described above, the present invention provides a composition in the fifth aspect, comprising lipid nanoparticles and a polynucleotide as defined in the first aspect, a DNA construct as defined in the second aspect, or an expression vector as defined in the third aspect. That is, the composition comprises lipid nanoparticles encapsulating a polynucleotide as defined in the first aspect, a DNA construct as defined in the second aspect, or an expression vector as defined in the third aspect.

[0115] The synthetic method of the composition formed by lipid nanoparticle encapsulation polynucleotide, DNA construct or expression vector is well known to those skilled in the art and has been fully established in the molecular biology experimental procedures. Specific conditions are described in the embodiments. Those skilled in the art can select the lipid nanoparticle suitable for encapsulating the polynucleotide, DNA construct or expression vector according to the intended use of the composition.

[0116] In one embodiment of the fifth aspect, the lipid nanoparticle comprises at least one component selected from PEG-modified lipids, non-cationic lipids, sterols and ionizable cationic lipids. In one embodiment of the fifth aspect, the lipid nanoparticle comprises or consists of PEG-modified lipids, non-cationic lipids, sterols and ionizable cationic lipids. In a more specific embodiment, the lipid nanoparticle comprises at least one of SM-102, distearyl phosphatidylcholine (DSPC), cholesterol and DMG-PEG2000. In a more specific embodiment, the lipid nanoparticle comprises or consists of SM-102, DSPC, cholesterol and DMG-PEG2000.

[0117] In a specific embodiment of the fifth aspect, the lipid nanoparticles are ionizable or charged.

[0118] In a specific embodiment of the fifth aspect, the composition comprises a polynucleotide, DNA construct or expression vector of the invention encapsulated in lipid nanoparticles.

[0119] In a specific embodiment of the fifth aspect, the composition is administered in the form of a pharmaceutical composition.

[0120] As described above, the present invention provides a pharmaceutical composition in a sixth aspect, comprising a therapeutically effective amount of the polynucleotide, DNA construct, expression vector or composition of the present invention, and at least one pharmaceutically acceptable excipient and / or carrier.

[0121] The term "pharmaceutically acceptable excipient and / or carrier" refers to a pharmaceutically acceptable material, composition or vehicle. Each ingredient must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with tissues or organs of humans and non-human animals without excessive toxicity, irritation, allergic reaction, immunogenicity or other problems or complications matched with a reasonable benefit / risk ratio.

[0122] Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents or other liquid carriers, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. Unless any conventional excipient medium is incompatible with the substance or its derivatives, such as producing any undesirable biological effect or interacting in a deleterious manner with any other component of the pharmaceutical composition, its use should be considered to be within the scope of the present invention.

[0123] The relative amounts of active ingredient, pharmaceutically acceptable excipient and / or any other ingredients in the pharmaceutical compositions of the present invention will vary depending on the individual characteristics, body size and / or health of the subject being treated, and also on the route of administration of the composition.

[0124] Pharmaceutically acceptable excipients for preparing pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients such as colorants, coating agents, sweeteners and flavoring agents may be included in the composition at the discretion of the formulator.

[0125] The polynucleotides or compositions described herein can be used in vaccine therapy to enhance the efficacy of conventional vaccines and / or as a new vaccine format for use against infectious pathogens such as viruses, bacteria, fungi, protozoa, prions and parasitic helminths (worms); or for the treatment of diseases such as cancer.

[0126] In one embodiment of the sixth aspect, the pharmaceutical composition is a vaccine. In a more specific embodiment, the pharmaceutical composition is a vaccine and further comprises an adjuvant. Those skilled in the art, based on their common knowledge, can determine which excipients, carriers and adjuvants to include in the vaccine according to the intended use.

[0127] In another embodiment, the polynucleotides, DNA constructs, expression vectors, cells, compositions or pharmaceutical compositions of the present invention are used to prevent COVID-19.

[0128] In embodiments of the eighth, ninth and tenth aspects, the polynucleotide, DNA construct, expression vector, composition or pharmaceutical composition is administered orally, intranasally, intravenously, intraperitoneally, intramuscularly, intradermally, subcutaneously, topically or intraarticularly.

[0129] As mentioned above, the present invention also provides an in vitro method for producing a polypeptide in a cell in the eleventh aspect, the method comprising contacting the cell with a polynucleotide, DNA construct, expression vector, composition or pharmaceutical composition of the present invention; particularly under suitable conditions. Those skilled in the art will be able to determine the effective and optimal conditions for producing the polypeptide by routine experimentation.

[0130] The present invention also provides a method for increasing the translation rate of a polynucleotide in a twelfth aspect. A person skilled in the art knows how to insert a tandem repeat sequence into a polynucleotide sequence using conventional molecular biology techniques.

[0131] In a specific embodiment of the twelfth aspect, the method comprises the step of inserting at least two tandem repeats of the sequence 5'-GCCRCC-3' at the 3'-end of the 5'-UTR, wherein R is a purine nucleotide. In particular, the at least two tandem repeats of the sequence 5'-GCCRCC-3' are located immediately upstream of the start codon of the ORF.

[0132] In a specific embodiment of the twelfth aspect, the method comprises the step of inserting two tandem repeats of the sequence 5'-GCCRCC-3' at the 3'-end of the 5'-UTR, particularly inserting two tandem repeats of the sequence 5'-GCCACC-3' at the 3'-end of the 5'-UTR.

[0133] All embodiments regarding the polynucleotide sequence in the first aspect are also applicable to this twelfth aspect.

[0134] Throughout the specification and claims, the term "comprising" and its variants are not intended to exclude other technical features, additives, components or steps. In addition, the term "comprising" covers the situation of "consisting of...". Other objects, advantages and features of the present invention will become apparent to those skilled in the art upon reading the specification or through the implementation of the present invention. The following embodiments and drawings are for illustrative purposes only and are not intended to limit the present invention. In addition, the present invention covers all possible combinations of the specific embodiments and preferred embodiments described herein. In addition, the present invention covers all possible combinations of the specific embodiments and preferred embodiments described herein.

[0135] The sequence of the present invention is shown in Table 1 below:

[0136] Table 1

[0137]

[0138]

[0139]

[0140] The following examples are for illustrative purposes only and are not intended to limit the present invention. In addition, the present invention encompasses all possible combinations of the specific embodiments and preferred embodiments described herein.

[0141] Example

[0142] Example 1: Protein expression in cells transfected with RNA of the present invention

[0143] In vitro transcription of RNA

[0144] The plasmids used in the experiment are listed in Table 2 below:

[0145] Table 2

[0146]

[0147] All plasmids encode transcripts containing the firefly luciferase coding sequence regulated by the different UTRs shown in Table 2. Plasmid P2 was generated by inserting the sequence 5'-GCCACC-3' at the 3'-end of the 5'-UTR of plasmid P1, while plasmid P3 was generated by inserting two tandem repeats of the sequence 5'-GCCACC-3' at the 3'-end of the 5'-UTR of plasmid P1. Plasmids P4-P6 encode transcripts with regulatory elements (i.e., UTRs) of commercially available RNAs and were used together with P1 and P2 for comparison purposes.

[0148] 1 μg of each RNase-free plasmid was digested with BspQI restriction endonuclease for P1, P2, P3, and P5 plasmids, and NotI restriction endonuclease for P4 and P6 plasmids. The restriction endonuclease cuts the plasmid just behind the fragment to be transcribed. The plasmid linearization reaction was performed according to the manufacturer's instructions. Purification was performed using the SV Gel and PCR Purification Kit (Promega A7270) protocol. In all cases, the recovery of plasmid linearization was at least 50%.

[0149] The purified linear DNA was then used for in vitro transcription to generate RNA using T7 RNA polymerase according to the manufacturer's instructions. Briefly, the transcription reaction was performed at 37°C for 3 hours using the following reagents:

[0150] Template DNA (50 μg / mL)

[0151] T7 polymerase (5.000 U / mL; ON-004)

[0152] · RNase inhibitor (1.000U / mL, ON-039)

[0153] Inorganic pyrophosphatase (2U / mL, ON-025)

[0154] vATP (5 μg / mL, R1331)

[0155] GTP (5 μg / mL, R2331)

[0156] CTP (5 μg / mL, R3331)

[0157] N1-methylpseudouridine (5 μg / mL, R5-027)

[0158] · AG (4 μg / mL, N-7113-10)

[0159] Following the manufacturer's instructions, DNase I ( The transcripts generated were purified by incubation with 4% 4% 4-amino-2-nitropropene (M0303L), followed by LiCl precipitation and 75% ethanol washing. The concentration of RNA redissolved in sodium citrate buffer was determined by measuring the optical density at 260 nm. The RNA yield was at least 80 μg RNA per 1 μg of linearized plasmid DNA.

[0160] All RNA samples were analyzed by denaturing agarose gel electrophoresis to ensure quality. RNA was stored at −80°C in aliquots until use. Each RNA type was synthesized in at least 2 independent transcription experiments, and all experiments were performed using at least 2 different batches of mRNA.

[0161] As shown in Table 2 above, in vitro transcription of P1 generates transcript R1; P2 generates transcript R2; P3 generates transcript R3; P4 generates transcript R4; P5 generates transcript R5; and P6 generates transcript R6.

[0162] Cell transfection

[0163] One day before transfection, HeLa (ACC57) or HEK293T (CRL-3216) cells were cultured at 1×10 4The cells were seeded into 96-well plates at a density of 10 cells / well. The culture medium used for both cells was DMEM high glucose (Merk D6429) supplemented with 10% fetal bovine serum (Sigma F7524), 1% antibiotic antimycotic solution (Sigma A5955) and 2 mM Glutamax (Fisher 35050038).

[0164] For transfection, the corresponding culture medium was replaced with 90 μL of fresh culture medium. Then, 10 μL of a mixture of mRNA R1-R6 (0.1 μg / well) prepared above and LipofectamineTM MessengerMAX reagent (Invitrogen 15397974; 0.2 μL / well) pre-incubated in OptiMEM medium was added to the cell culture. The cells containing the mRNA-Lipofectamine MessengerMAX mixture were incubated at 37°C, 5% CO 2 Incubate for 24 hours in the environment for subsequent analysis.

[0165] Quantification of firefly luciferase activity

[0166] The transfected cells were lysed in 100 μL 0.1% PBS-Triton and 98 μL of the lysate was transferred to an opaque 96-well white plate. Then, 102 μL of buffered d-luciferin (GoldBio LUCK-100) (100 mM Tris-HCl pH 7.8, 5 mM MgCl 2 , 250 μM CoA, 150 μM ATP) were added to a white 96-well plate at a final concentration of 150 μg / mL. After incubation at room temperature for 5 minutes according to the manufacturer's instructions, the luminescence intensity was measured using a FLUOstar Omega microplate reader.

[0167] like Figure 1 As shown in A and B, different mRNAs produce luciferase at different rates depending on the combination of 5'-UTR and 3'-UTR sequences on both sides of the coding region. Insertion of one repeat of the sequence 5'-GCCACC-3' just before the ORF in R1 (i.e., R2) did not increase protein production. However, insertion of two 5'-GCCACC-3' sequences in tandem just before the ORF in R1 (i.e., R3) significantly increased protein production in HeLa and HEK293T cells, reaching the highest production level among all tested mRNAs.

[0168] These results clearly indicate that the presence of at least two 5′-GCCACC-3′ sequences in tandem at the 3′-end of the 5′-UTR can synergistically enhance the translation rate of mRNA, thereby achieving higher protein production levels.

[0169] Example 2: Protein expression in mice injected intramuscularly with RNA of the present invention

[0170] RNA encapsulated in lipid nanoparticles

[0171] For in vivo administration, the mRNA R1-R6 prepared above was encapsulated in lipid nanoparticles (LNPs), referring to the description of Hassett, KJ et al. "Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines", 2019, Mol. Ther. Nucleic Acid, vol. 15, pp. 1-11. In short, the purified capped mRNA was first diluted in sodium citrate buffer at pH = 4 to a final concentration of 266 μg / mL. In addition, lipid SM-102 (BOCSI 2089251-47-6), DSPC (Merk 850365P), cholesterol (Sigma C3045) and DMG-PEG2000 (Cayman 33945-1) were dissolved in ethanol at a molar ratio of 50: 10: 38.5: 1.5, and the N: P ratio was 5.5: 1.

[0172] The aqueous solution was then carefully added on top of the ethanol solution and the resulting solution was homogenized by pipetting up and down 4-5 times. The resulting LNPs were immediately diluted 1:1 with Tris buffer and dialyzed overnight against Tris buffer containing 15% sucrose. The resulting LNP solution was then collected and used according to the manufacturer's instructions. Encapsulated mRNA was assessed by Ribogreen (Invitrogen R11490).

[0173] The LNP solution was then adjusted to a final mRNA concentration of 100 μg / ml. Particle size distribution, polydispersity index, and Z-potential were measured by dynamic light scattering (DLS). Typical values ​​for these parameters ranged from: particle size distribution, 90-120 nm; polydispersity index, 0.08-1.5; Z-potential, -10-+10 mV. RNA encapsulation was assessed by Quant-Ribogreen according to the manufacturer's instructions, and the encapsulation efficiency of all mRNAs was generally about 80%-95%.

[0174] Finally, the LNP solution was filtered through a 0.22 mm filter and the LNPs were stored at -80 °C until use.

[0175] mRNA (LNP) was administered to mice

[0176] Female BALB / c mice (Charles River Laboratories), 8-10 weeks old, weighing 18-23 g, were acclimatized to the new environment for 3-7 days after arriving at the experimental facility. The housing conditions were room temperature 20-24°C, humidity 50-70%, light intensity 60 lux, and light-dark cycle 12 hours.

[0177] To measure firefly luciferase activity in mice, LNPs generated as described above containing 5 μg of the indicated mRNA in a final volume of 50 μl were injected intramuscularly.

[0178] 4 to 72 hours after RNA-LNP inoculation, mice were anesthetized by inhalation of 4% isoflurane using a nebulizer. Anesthesia was maintained under 1.5% isoflurane. Subsequently, D-luciferin (Quimigen, Cat. No.: 12507) was injected intraperitoneally at a dose of 150 mg / kg, and for 20 g mice, about 200 μL of PBS stock solution with a concentration of 15 mg / mL was usually used. According to the manufacturer's instructions, luciferase images were collected using the IVIS LuminaXRMS imaging system 10 minutes after luciferin inoculation.

[0179] like Figure 2 As shown, LNPs containing two tandem 5'-GCCACC-3' sequences (R3) significantly increased in vivo protein production compared to mRNAs containing only one repeat sequence or no repeat sequence.

[0180] These results suggest that the presence of two tandem 5'-GCCACC-3' sequences in a polynucleotide used to produce a protein greatly enhances its expression capacity, which may improve the therapeutic efficiency of known and future protein expression vectors.

[0181] Example 3: Protein expression in cells transfected with RNA of the present invention

[0182] The protein expression experiment was carried out according to the method described in Example 1 using the plasmids listed in Table 3 below:

[0183] Table 3

[0184]

[0185]

[0186] like Figure 3 As shown in A and B, different mRNAs produce luciferase at different rates depending on the combination of 5'-UTR and 3'-UTR sequences on either side of the coding region. Adding two tandem 5'-GCCACC-3' sequences before the ORF of R7 (i.e., R8) significantly increased protein production in HeLa and HEK293T cells.

[0187] These results demonstrate that the presence of at least two tandem 5′-GCCACC-3′ sequences at the 3′-end of the 5′-UTR can consistently enhance the translation rate of mRNAs with different 3′-UTRs.

[0188] Example 4: Protein expression in cells transfected with RNA of the present invention

[0189] The protein expression experiment was carried out according to the method described in Example 1, except that the amount of mRNA added to the cell culture during cell transfection was 0.05 μg / well instead of 0.1 μg / well. The plasmids and mRNA used are listed in Table 4 below:

[0190] Table 4

[0191]

[0192] like Figure 4 Different mRNAs produce luciferase at different rates depending on the combination of 5'-UTR and 3'-UTR sequences on either side of the coding region, as shown in A and B. Two 5'-GCCACC-3' sequences added in tandem before the ORF of R9 (i.e., R10) significantly increased protein production in HeLa and HEK293T cells.

[0193] These results demonstrate that the presence of at least two tandem 5'-GCCACC-3' sequences at the 3'-end of different 5'-UTRs can consistently increase the translation rate of mRNA, thereby achieving higher levels of protein production.

[0194] The present invention includes the following embodiments:

[0195] 1. An artificial polynucleotide comprising, in the 5' to 3' direction:

[0196] -5' untranslated region (5'-UTR), and

[0197] - Open reading frame (ORF),

[0198] Wherein, the 5'-UTR comprises at its 3'-end at least two tandem repeats of the sequence 5'-GCCNCC-3' operably linked to the ORF, and wherein N is any arbitrary nucleotide.

[0199] 2. The polynucleotide according to embodiment 1, wherein the at least two tandem repeat sequences are the sequence 5'-GCCRCC-3', wherein R is any purine nucleotide.

[0200] 3. The polynucleotide according to any one of embodiments 1-2, wherein:

[0201] - said 5'-UTR comprises at its 3'-end two tandem repeats of the sequence 5'-GCCRCC-3', which are operably linked to the ORF; or alternatively,

[0202] - the 5'-UTR comprises at its 3'-end a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, which is operably linked to the ORF.

[0203] 4. A polynucleotide according to any one of embodiments 1-3, wherein the 5'-UTR comprises or consists of a sequence from a 5'-UTR of a gene transcript linked to at least two tandem repeats of the sequence 5'-GCCNCC-3' in the 5' to 3' direction; in particular, wherein the at least two tandem repeats are the sequence 5'-GCCRCC-3'.

[0204] 5. The polynucleotide according to embodiment 4, wherein the sequence from the 5'-UTR of the gene transcript comprises a Kozak sequence, in particular a non-consensus Kozak sequence.

[0205] 6. The polynucleotide of any one of embodiments 4-5, wherein the sequence from the 5'-UTR of the gene transcript is from a gene selected from the group consisting of apolipoprotein A2 (APOA2), hemoglobin subunit β (HBB), pre-T cell antigen receptor alpha (PTCRA), and small nuclear ribonucleoprotein D1 polypeptide (SNRPD1).

[0206] 7. The polynucleotide according to any one of embodiments 1-6, wherein at least two tandem repeats of the sequence 5'-GCCNCC-3' form a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0207] 8. The polynucleotide according to any one of embodiments 1-7, wherein the 5'-UTR comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 41.

[0208] 9. The polynucleotide according to any one of embodiments 1-8, further comprising one or more of the following:

[0209] -5'-cap structure;

[0210] -3' untranslated region (3'-UTR); and

[0211] -3' tail sequence.

[0212] 10. The polynucleotide according to any one of embodiments 1-9, which comprises or consists of the following structure in the 5' to 3' direction:

[0213] (i) 5'-cap structure;

[0214] (ii) 5' untranslated region (5'-UTR),

[0215] (iii) open reading frame (ORF),

[0216] (iv) 3' untranslated region (3'-UTR); and

[0217] (v) 3' tailing sequence.

[0218] 11. The polynucleotide according to any one of embodiments 1-10, which is an RNA polynucleotide, in particular messenger RNA (mRNA).

[0219] 12. The polynucleotide of any one of embodiments 1-11, wherein the 5'UTR is heterologous to the ORF and / or 3'UTR.

[0220] 13. The polynucleotide according to any one of embodiments 9 to 12, wherein the 3'-UTR comprises or consists of a sequence from a 3'-UTR of a gene transcript, in particular a 3'-UTR sequence of a mammalian gene.

[0221] 14. The polynucleotide according to any one of embodiments 9 to 13, wherein the 3'-UTR comprises or consists of at least two tandem repeats of a sequence from a 3'-UTR of a gene transcript, in particular two tandem repeats of a sequence from a 3'-UTR of a gene transcript.

[0222] 15. The polynucleotide of any one of embodiments 9-14, wherein the 3'-UTR comprises or consists of a sequence from a 3'-UTR of a gene transcript selected from the group consisting of: apolipoprotein A2 (APOA2), hemoglobin β subunit (HBB), pre-T cell antigen receptor alpha (PTCRA), and small nuclear ribonucleoprotein D1 polypeptide (SNRPD1).

[0223] 16. A polynucleotide according to any one of embodiments 9-15, wherein the 3'-UTR comprises or consists of a sequence selected from SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.

[0224] 17. The polynucleotide according to any one of embodiments 1-16, wherein the ORF encodes a polypeptide, in particular an antigen.

[0225] 18. The polynucleotide of embodiment 17, wherein the antigen is a SARS-CoV-2 antigen, in particular a SARS-CoV-2 spike antigen.

[0226] 19. The polynucleotide according to any one of embodiments 9-18, wherein the 5'-cap structure is selected from cap-0, cap-1, cap-2, ARCA, inosine, N1-methylguanosine, 2'-fluoroguanosine, 7-deazaguanosine, 8-oxoguanosine, 2-aminoguanosine, LNA-guanosine and 2-azidoguanosine; in particular, the 5'-cap structure is cap-1.

[0227] 20. A polynucleotide according to any one of embodiments 9-19, wherein the 3' tail sequence is a poly-A region, in particular the sequence SEQ ID NO:39.

[0228] 21. The polynucleotide according to any one of embodiments 1-20, comprising at least one of the following:

[0229] - 5' untranslated region (5'-UTR) of sequence SEQ ID NO: 9 or 41;

[0230] - the 3' untranslated region (3'-UTR) of sequence SEQ ID NO: 18 or 19; and

[0231] - a 3' tail sequence of SEQ ID NO:39.

[0232] 22. The polynucleotide according to any one of embodiments 1-21, which comprises or consists of the following structure in the 5' to 3' direction:

[0233] (i) 5'-cap 1 structure;

[0234] (ii) the 5' untranslated region (5'-UTR) of SEQ ID NO: 9 or 41;

[0235] (iii) an open reading frame (ORF) encoding a polypeptide;

[0236] (iv) the 3' untranslated region (3'-UTR) of SEQ ID NO: 18 or 19; and

[0237] (v) The 3' tail sequence of SEQ ID NO:39.

[0238] 23. The polynucleotide according to any one of embodiments 1 to 22, wherein the polynucleotide comprises at least one chemical modification, in particular the chemical modification is selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deazapseudouridine, 2-thiol-1-methylpseudouridine, 2-thiol-5-azauridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxypseudouridine, 4-thiol-1-methylpseudouridine, 4-thiol-pseudouridine, 5-azauridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2'-O-methyluridine, N6-methyladenosine and combinations thereof.

[0239] 24. A polynucleotide according to embodiment 23, wherein the chemical modification is N1-methylpseudouridine, 5-methoxyuridine or a combination thereof; in particular wherein the chemical modification is N1-methylpseudouridine.

[0240] 25. The polynucleotide according to any one of embodiments 23-24, wherein the polynucleotide is fully modified with N1-methylpseudouridine, 5-methoxyuridine or a combination thereof; in particular, the polynucleotide is fully modified with N1-methylpseudouridine.

[0241] 26. A DNA construct comprising a promoter operably linked to a sequence encoding a polynucleotide as defined in any one of embodiments 1 to 25.

[0242] 27. An expression vector comprising the DNA construct defined in embodiment 26.

[0243] 28. A cell comprising a polynucleotide as defined in any one of embodiments 1 to 25, a DNA construct as defined in embodiment 26 or an expression vector as defined in embodiment 27.

[0244] 29. A composition comprising a lipid nanoparticle and a polynucleotide as defined in any one of embodiments 1-25, a DNA construct as defined in embodiment 26, or an expression vector as defined in embodiment 27.

[0245] 30. A composition according to embodiment 29, wherein the lipid nanoparticles comprise at least one selected from PEG-modified lipids, non-cationic lipids, sterols and ionizable cationic lipids.

[0246] 31. A pharmaceutical composition comprising a therapeutically effective amount of a polynucleotide as defined in any one of embodiments 1-25, a DNA construct as defined in embodiment 26, an expression vector as defined in embodiment 27, or a composition as defined in any one of embodiments 29 to 30, and at least one pharmaceutically acceptable excipient and / or carrier.

[0247] 32. The pharmaceutical composition according to embodiment 31, which is a vaccine, optionally further comprising an adjuvant.

[0248] 33. The polynucleotide according to any one of embodiments 1-25, the DNA construct according to embodiment 26, the expression vector according to embodiment 27, the cell according to embodiment 28, the composition according to any one of embodiments 29-30 or the pharmaceutical composition according to any one of embodiments 31-32 for use in medicine, in a method for inducing an immune response in a subject, in a method for therapeutic immunization of a subject, or as a vaccine or for gene therapy.

[0249] 34. The polynucleotide, DNA construct, expression vector, composition or pharmaceutical composition according to embodiment 33, which is used to prevent and / or treat COVID-19.

[0250] 35. An in vitro method for producing a polypeptide in a cell, the method comprising contacting the cell with a polynucleotide as defined in any one of embodiments 1 to 25, a DNA construct as defined in embodiment 26, an expression vector as defined in embodiment 27, a composition as defined in embodiments 29 to 30, or a pharmaceutical composition as defined in any one of embodiments 31 to 32.

[0251] 36. An in vitro method for increasing the translation rate of a polynucleotide, wherein the polynucleotide comprises a 5' untranslated region (5'-UTR) and an open reading frame (ORF) in the 5' to 3' direction, the method comprising the step of inserting at least two tandem repeats of the sequence 5'-GCCNCC-3' at the 3'-end of the 5'-UTR, in particular inserting two tandem repeats of the sequence 5'-GCCRCC-3' at the 3'-end of the 5'-UTR.

[0252] References

[0253] Altschul et al.,“Basic local alignment search tool”,1990,J.Mol.Biol,v.215,pages 403-410

[0254] EMBOSS:The European Molecular Biology Open Software Suite,Rice etal.,2000,Trends Genet.16:276-277

[0255] Hassett,K.J.et al.,"Optimization of Lipid Nanoparticles forIntramuscular Administration ofmRNA Vaccines",2019,Mol.Ther.Nucleic Acid,vol.15,pp.1-11

Claims

1. An artificial polynucleotide comprising in the 5' to 3' direction: -5' untranslated region (5'-UTR), and - Open reading frame (ORF), The 5'-UTR comprises at its 3'-end at least two tandem repeats of the sequence 5'-GCCNCC-3' operably linked to the ORF, wherein N is any nucleotide. 2 . The polynucleotide according to claim 1 , wherein the at least two tandem repeat sequences are the sequence 5′-GCCRCC-3′, wherein R is a purine nucleotide.

3. The polynucleotide according to any one of claims 1-2, wherein the 5'-UTR comprises at its 3'-end two tandem repeats of the sequence 5'-GCCRCC-3' operably linked to the ORF; in particular wherein the two tandem repeats form the sequence SEQ ID NO:

3.

4. The polynucleotide according to any one of claims 1 to 3, wherein the 5'-UTR comprises, in the 5' to 3' direction, a sequence from a 5'-UTR of a gene transcript linked to at least two tandem repeats of the sequence 5'-GCCNCC-3'.

5. The polynucleotide according to any one of claims 1 to 4, wherein the sequence from the 5'-UTR of the gene transcript comprises a Kozak sequence, in particular a non-consensus Kozak sequence, at its 3'-end.

6. The polynucleotide according to any one of claims 4-5, wherein the sequence from the 5'-UTR of the gene transcript is from a gene selected from the group consisting of apolipoprotein A2 (APOA2), hemoglobin subunit β (HBB), pre-T cell antigen receptor alpha (PTCRA) and small nuclear ribonucleoprotein D1 polypeptide (SNRPD1).

7. The polynucleotide of any one of claims 1-6, wherein the 5'-UTR comprises a sequence selected from SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 41, or a variant that is at least 85% identical to SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO:

41.

8. The polynucleotide according to any one of claims 1 to 7, further comprising one or more of the following: -5'-cap structure; -3' untranslated region (3'-UTR); and -3' tail sequence.

9. The polynucleotide according to any one of claims 1 to 8, comprising in the 5' to 3' direction: (i) a 5'-cap structure, in particular a cap-1 structure; (ii) a 5' untranslated region (5'-UTR), in particular the 5' untranslated region of SEQ ID NO: 9; (iii) an open reading frame (ORF), in particular the polypeptide it encodes; (iv) a 3' untranslated region (3'-UTR), in particular the 3' untranslated region of SEQ ID NO: 19; and (v) a 3' tail sequence, in particular a 3' tail sequence of SEQ ID NO:

39.

10. The polynucleotide according to any one of claims 1 to 8, comprising in the 5' to 3' direction: (i) a 5'-cap structure, in particular a cap-1 structure; (ii) a 5' untranslated region (5'-UTR), in particular the 5' untranslated region of SEQ ID NO: 9; (iii) an open reading frame (ORF), in particular the polypeptide it encodes; (iv) a 3' untranslated region (3'-UTR), in particular the 3' untranslated region of SEQ ID NO: 18; and (v) a 3' tail sequence, in particular a 3' tail sequence of SEQ ID NO:

39.

11. The polynucleotide according to any one of claims 1 to 8, comprising in the 5' to 3' direction: (i) a 5'-cap structure, in particular a cap-1 structure; (ii) a 5' untranslated region (5'-UTR), in particular the 5' untranslated region of SEQ ID NO: 41; (iii) open reading frame (ORF), particularly encoding a polypeptide; (iv) a 3' untranslated region (3'-UTR), in particular the 3' untranslated region of SEQ ID NO: 18; and (v) a 3' tail sequence, in particular a 3' tail sequence of SEQ ID NO:

39.

12. The polynucleotide according to any one of claims 1 to 11, which is an RNA polynucleotide, in particular a messenger RNA (mRNA).

13. The polynucleotide according to any one of claims 1 to 12, wherein the polynucleotide comprises at least one chemical modification, particularly wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, N6-methyladenosine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxypseudouridine, 4-thiol-1-methylpseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2'-O-methyluridine and combinations thereof.

14. A DNA construct comprising a promoter operably linked to a sequence encoding the polynucleotide according to any one of claims 1-13.

15. A composition comprising: - lipid nanoparticles; and - A polynucleotide as defined in any one of claims 1 to 13 or a DNA construct as defined in claim 14.

16. A pharmaceutical composition comprising a therapeutically effective amount of the polynucleotide according to any one of claims 1 to 13, the DNA construct according to claim 14 or the composition according to claim 15, and at least one pharmaceutically acceptable excipient and / or carrier.

17. An in vitro method for producing a polypeptide in a cell, the method comprising contacting the cell with a polynucleotide as defined in any one of claims 1 to 13, a DNA construct as defined in claim 14, a composition as defined in claim 15 or a pharmaceutical composition as defined in claim 16.

18. An in vitro method for increasing the translation rate of a polynucleotide comprising a 5' untranslated region (5'-UTR) and an open reading frame (ORF) in the 5' to 3' direction, the method comprising the step of inserting at least two tandem repeats of the sequence 5'-GCCNCC-3' at the 3'-end of the 5'-UTR, wherein N is any nucleotide.

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