Artificial polynucleotides for expressing proteins

Abstract

The present invention provides a polynucleotide comprising, in a 5' to 3' direction, a 5' untranslated region (5'-UTR) and an open reading frame (ORF), wherein the 5'-UTR comprises at least two tandem repeats of the sequence 5'-GCCNCC-3' operably linked to the ORF, where N is any nucleotide. The present invention also provides compositions comprising lipid nanoparticles and polynucleotides, as well as pharmaceutical compositions and their use in medicines, particularly for use as vaccines or in gene therapy.

Description

[Technical Field]

[0001] This application claims the benefit of European Patent Application No. 22382946.6, filed October 7, 2022.

[0002] The present invention is in the field of polynucleotides, in particular artificial polynucleotides that encode polypeptides. The polynucleotides of the present invention are particularly useful for genetic vaccination. [Background technology]

[0003] Genetic vaccination and gene therapy are two of the most promising and rapidly developing therapeutic areas of modern medicine, both of which are based on the delivery of polynucleotides, such as DNA or RNA molecules, into a patient's cells or tissues to produce polypeptides that have a therapeutic effect.

[0004] The use of RNA in gene therapy and vaccination is generally considered safer than the use of DNA, because RNA does not involve the risk of being stably integrated into the genome of transfected cells.In addition, RNA is more easily degraded in vivo, and as a result, has a relatively short half-life, in contrast to DNA.Therefore, compared with DNA-based therapy, RNA-based therapy has a lower risk of host producing undesirable anti-RNA antibodies, which may reduce therapeutic effect and cause serious side effects.Therefore, RNA is often considered the molecule of choice for gene medicine therapy.

[0005] One of the main limitations of RNA-based therapeutics is their limited protein production efficiency. Because RNA has a relatively short half-life, it is crucial that they provide a very high translation rate in order to produce a sufficient amount of the desired protein before the RNA is degraded. Optimization of UTRs to improve protein production has been attempted through rational design of 5'UTRs. However, the benefits achieved so far are still very limited.

[0006] Thus, despite recent attempts, there remains a need for polynucleotides, particularly RNA, with high translation rates suitable for use in genetic vaccination and gene therapy. Summary of the Invention

[0007] The present inventors have developed a novel nucleic acid sequence that, when inserted into the 3' end of the 5' UTR, significantly enhances the translation efficiency of mRNA transcripts. This novel sequence enables the design of artificial transcripts with extremely high protein production yields, which may be useful for a wide range of applications, including the production of nucleic acid therapeutics or industrial recombinant proteins.

[0008] As shown in the Examples below, we found that inserting a single repeat of the sequence 5'-GCCACC-3' at the 3'-end of the 5'-UTR of a transcript did not affect its ability to produce the protein either in vitro or in vivo (see Figures 1 and 2, R2 vs R1). However, when two tandem repeats of the above sequence were inserted, a significant increase in protein production was observed both in vitro (see Figure 1, R3 vs R1) and in vivo (see Figure 2, R3 vs R1). This was completely unexpected, given the complete absence of effect observed when only a single repeat was present.

[0009] Remarkably, the synergistic effect provided by the presence of the two tandem repeats allowed an approximately 400% increase in in vivo protein expression after 24 hours compared to transcripts that did not contain these two tandem repeats (Figure 2, R3 vs. R1 or R2), and also transcripts that contained regulatory elements of commercially available RNAs (Figure 2, R3 vs. R4 or R6) or previously disclosed RNAs (Figure 2, R3 vs. R5).

[0010] Therefore, from the data provided below, it is clear that the polynucleotides provided by the present invention are important new tools for overcoming the limitations of current artificial RNAs, in particular their low protein production efficiency. Furthermore, the higher translation efficiency of the polynucleotides of the present invention may allow for a reduction in the therapeutic dose of RNA required for genetic vaccination, which may help reduce associated side effects.

[0011] In a first aspect, the present invention provides an artificial polynucleotide comprising, in a 5'→3' direction, a 5' untranslated region (5'-UTR), and an open reading frame (ORF), 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 nucleotide.

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

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

[0014] In a fourth aspect, the present invention provides a cell comprising 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.

[0015] In a fifth aspect, the present invention provides a composition comprising a lipid nanoparticle 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.

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

[0017] When the polynucleotide of the present invention comprises an ORF encoding a protein associated with a disease, all of the above-listed embodiments can be used as a pharmaceutical. Thus, in a seventh embodiment, the present invention provides a polynucleotide according to the first embodiment, a DNA construct according to the second embodiment, an expression vector according to the third embodiment, a cell according to the fourth embodiment, a composition according to the fifth embodiment, or a pharmaceutical composition according to the sixth embodiment, for use in a pharmaceutical.

[0018] In an eighth 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 a method of inducing an immune response in a subject.

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

[0020] In a ninth 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 a method for therapeutic immunization of a subject.

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

[0022] In a tenth 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 as a vaccine or for use in gene therapy.

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

[0024] In a twelfth aspect, the present invention provides 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 a 5' to 3' direction, the method comprising inserting at least two tandem repeats of the sequence 5'-GCCNCC-3' at the 3' end of the 5'-UTR, where N is any nucleotide.

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

[0026] In a fourteenth aspect, the present invention provides a process for producing a pharmaceutical composition according to the sixth aspect, which comprises mixing a polynucleotide, DNA construct, expression vector, or composition of the invention with at least one excipient and / or carrier. [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1 relates to Example 1 and is a bar graph diagram showing luciferase production levels 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 unit. [Figure 2] Figure 2 relates to Example 2 and is a bar graph showing luciferase production levels in the muscle of mice administered LNPs containing the indicated mRNAs after (A) 4 hours, (B) 7 hours, and (C) 24 hours. The structures of mRNAs R1, R2, R3, R4, R5, and R6 are detailed in Table 2 below. [Figure 3] Figure 3 relates to Example 3 and is a bar graph showing luciferase production levels 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 unit. [Figure 4]Figure 4 relates to Example 4 and is a bar graph showing luciferase production levels in (A) HeLa cells and (B) HEK293T cells after transfection with the indicated mRNAs. The structures of mRNAs R4, R5, R9, and R10 are detailed in Table 4 below. "RLU" stands for relative light unit. DETAILED DESCRIPTION OF THE INVENTION

[0028] All terms used herein in this application are to be understood in their ordinary sense as known in the art, unless otherwise specified. Other, more specific definitions for certain terms used in this application are set forth below and are intended to be uniformly applied throughout the specification and claims, unless a definition expressly set forth elsewhere provides a broader definition.

[0029] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more." Unless otherwise specified, definite articles such as "the" as used herein also include plural nouns.

[0030] The term "polynucleotide" is used interchangeably with "nucleic acid" and refers to a polymer of nucleotides, either ribonucleotides or deoxyribonucleotides. Polynucleotides formed from ribonucleotides may be referred to as "RNA polynucleotides," "ribonucleic acid," or simply "RNA." Polynucleotides formed from deoxyribonucleotides may be referred to as "DNA polynucleotides," "deoxyribonucleic acid," or simply "DNA." Polynucleotides may be single-stranded or double-stranded, and optionally incorporate synthetic, non-natural, or modified nucleotides that can be incorporated into DNA or RNA. An "artificial polynucleotide" refers to a polynucleotide having a sequence that does not occur in nature or that has been altered by human intervention. A "purine nucleotide" is a nucleotide that contains purine, particularly adenine or guanine, as the nitrogenous base. When the sequence of a polynucleotide is shown, it represents an information-providing molecule, and only one strand is shown. Nevertheless, those skilled in the art will understand that they can be in the form of single-stranded or double-stranded nucleic acid polymers; for example, mRNA is single-stranded; a DNA construct is double-stranded.

[0031] As used herein, the term "messenger RNA" or "mRNA" or "transcript" refers to any RNA polynucleotide that encodes a polypeptide of interest and can be translated to produce the encoded polypeptide of interest in vitro, in vivo, or ex vivo. Typically, mRNA is single-stranded and contains a 5'-cap structure, a 5' UTR, an ORF, a 3' UTR, and a 3' tailing sequence.

[0032] As used herein, the "untranslated region" or "UTR" of a polynucleotide refers to a region located upstream (i.e., 5') or downstream (i.e., 3') of an open reading frame (ORF) and not translated by ribosomes. Thus, the "5' untranslated region" or "5'-UTR" refers to a region located upstream (i.e., 5') of the start codon of an ORF, which is transcribed but not translated into an amino acid sequence. The 5'-UTR usually corresponds to a sequence extending from the nucleotide located immediately 3' to the 5'-cap to the nucleotide located immediately 5' to the start codon of the ORF. The "3' untranslated region" or "3'UTR" refers to a region located downstream of the stop codon of an ORF, which is transcribed but not translated into an amino acid sequence. The 3'-UTR usually corresponds to a sequence extending from the nucleotide immediately 3' to the stop codon of an ORF to the nucleotide immediately 5' to the poly(A) sequence.

[0033] As used herein, "open reading frame" or "ORF" refers to a sequence of several nucleotide triplets that encodes a polypeptide, i.e., can be translated into a polypeptide sequence. An open reading frame usually includes a start codon, i.e., a three-nucleotide combination (e.g., ATG or AUG) that usually encodes the amino acid methionine at its 5' end, and a subsequent region that usually has a length that is a multiple of three nucleotides. An ORF is usually terminated by a stop codon (e.g., TAA, TAG, TGA). An open reading frame may also be referred to as a "protein coding region."

[0034] A "5'-cap structure" refers to an entity, typically a modified nucleotide entity, that generally closes the 5'-end of a mature mRNA. The 5'-cap can usually be formed by a modified nucleotide, particularly a derivative of a guanine nucleotide. Preferably, the 5'-cap is linked to the 5'-end via a 5'-5'-triphosphate bond. The 5'-cap can be methylated, e.g., m7GpppN, where N is the 5'-terminal nucleotide of the nucleic acid bearing the 5'-cap, typically the 5'-end of the RNA. 5'-cap structures include, but are not limited to, "cap-0," which refers to a methylated guanosine (on the nitrogen at position 7, N7) added to an mRNA in a 5'-5' linkage; "cap-1," which refers to a cap-0 with a methyl group added to the 2'-carbon of the ribose on the 5'-end nucleotide (N1) of the chain; and "cap-2," which refers to a cap-1 with another 2'-methyl group added to the next nucleotide (N2).

[0035] "3' tailing sequence" refers to a sequence located at the 3' end of an mRNA that enhances mRNA stability, is usually rich in adenine nucleotides, and is at least 80 nucleotides in length.

[0036] As used herein, "operatively linked" refers to the linkage of sequences such that expression of the coding sequence is achieved under conditions compatible with the control sequences. Operatively linked sequences include both expression control sequences contiguous with the coding sequence of interest and expression control sequences that act in trans or at a distance to control the coding sequence of interest. For example, at least two tandem repeats of the sequence 5'-GCCNCC-3' located at the 3'-end of the 5'-UTR are expression control sequences operably linked to an ORF located downstream because they enhance translation of the ORF. In certain embodiments, "operably linked sequences" refer to directly linked sequences.

[0037] As used herein, the term "tandem repeat" refers to a sequence that occurs consecutively. For example, two tandem repeats of the sequence 5'-GCCNCC-3' refer to a sequence that includes two consecutive units of 5'-GCCNCC-3' (i.e., 5'-GCCNCCGCCNCC-3').

[0038] As used herein, a "Kozak sequence" refers to a sequence located within an mRNA that helps the ribosomal translation machinery recognize where translation of a transcript should begin. Kozak sequences have the 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 a "strong Kozak sequence" refers to a sequence that closely matches the consensus sequence described above. A "non-consensus Kozak sequence" or a "weak Kozak sequence" refers to a sequence that does not closely match the consensus sequence described above, particularly at positions +4 and -3 relative to the adenosine (+1) at the 5' end of the disclosed codon.

[0039] 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 5'-UTR is not found in combination with that ORF in the natural sequence.

[0040] In the present invention, the term "identical" or "identity" refers to the percentage of positions that are identical in two sequences when the sequences are optimally aligned. In optimal alignment, if a position in a first sequence is occupied by the same nucleotide as the corresponding position in a second sequence, the sequences are said to be identical at that position. The percentage of identity determines the number of identical nucleotides over a defined length in a given alignment. Therefore, the level of identity between two sequences ("percent sequence identity") is measured as the ratio of the number of identical positions shared by the sequences to the number of positions compared (i.e., percent sequence identity = (number of identical positions / total number of positions compared) × 100). Gaps, i.e., positions in the alignment where a nucleotide is present in one sequence but not the other, are considered positions with non-identical nucleotides and are considered as positions to be compared.

[0041] Many mathematical algorithms for quickly obtaining optimal alignment and calculating the identity between two or more sequences are known and are incorporated into many available software programs.For the purpose of the present invention, the sequence identity between two nucleic acid sequences is preferably determined using a global alignment-based algorithm, such as the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277); or the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), which is implemented by using default settings in the BLAST Global Alignment tool (Altschul et al., "Basic local alignment search tool", 1990, J. Mol. Biol., v. 215, pages 403-410).Local alignment can also be used when the sequences to be compared are substantially the same length.

[0042] As used herein, a "DNA construct" refers to an artificial polynucleotide comprising a sequence of interest operably linked to an expression promoter that controls the expression of the sequence. An "expression vector" refers to a vector used to introduce a particular nucleic acid, usually a DNA construct, into a target cell for expression of the nucleic acid by the cell. Examples of suitable expression promoters and expression vectors include those conventionally used in molecular biology and known to those skilled in the art.

[0043] The term "polypeptide" refers to any peptide or protein containing two or more amino acids joined together by peptide bonds or modified peptide bonds (i.e., peptide equivalents). "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides, or oligomers, and to longer chains, commonly referred to as proteins.

[0044] The expression "therapeutically effective amount" as used herein refers to the amount of compound that is sufficient to prevent or alleviate to some extent one or more symptoms of the disease being treated when administered.The specific dose of the compound administered according to the present invention will naturally be determined by the specific circumstances surrounding the case, including the compound to be administered, the route of administration, the specific condition to be treated, and similar considerations.The term "pharmaceutical" also encompasses the concept of "veterinary composition".Therefore, these relate to compositions that are therapeutically effective when administered to any animal, including humans, by any desired or applicable route.

[0045] In the present invention, the term "antigen" is a compound that can be recognized by the immunoglobulin receptor of a B cell or by a T cell receptor when complexed with an MHC. Preferably, an "antigen" is a polypeptide.

[0046] The term "nanoparticle" as used herein refers to a particle having at least two dimensions at the nanoscale, particularly all three dimensions at the nanoscale, where the nanoscale is in the range of about 1 nm to about 300 nm. In particular, when the nanoparticle is rod-shaped with a substantially circular cross section, such as a nanowire or nanotube, the term "nanoparticle" refers to a particle having at least two dimensions at the nanoscale, where these two dimensions are the cross section of the nanoparticle. The term "lipid nanoparticle" as used herein refers to a nanoparticle whose outer envelope is made entirely or partially of lipid. Suitable lipid nanoparticles that can be used in the compositions of the present invention are described, for example, 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.

[0047] As described above, in a first aspect, the present invention provides an artificial polynucleotide comprising, in a 5'→3' direction, a 5' untranslated region (5'-UTR) and an open reading frame (ORF), 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 nucleotide.

[0048] In certain embodiments of the first aspect, optionally in combination with any of the above or below embodiments, the at least two tandem repeats are tandem repeats of the sequence 5'-GCCRCC-3', where R is a purine nucleotide.

[0049] In one embodiment of the first aspect, the artificial polynucleotide comprises, in a 5'→3' direction, a 5' untranslated region (5'-UTR) and an open reading frame (ORF), wherein the 5'-UTR comprises at its 3' end at least two tandem repeats of the sequence 5'-GCCRCC-3' operably linked to the ORF, and wherein R is a purine nucleotide.

[0050] In one embodiment of the first aspect, the polynucleotide comprises, contiguously and in a 5' to 3' direction, a 5' untranslated region (5'-UTR) and an open reading frame (ORF), 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 N is any nucleotide. In a more particular embodiment, the two tandem repeats are two tandem repeats of the sequence 5'-GCCRCC-3', and R is a purine nucleotide.

[0051] In one embodiment of the first aspect, the polynucleotide comprises, in a 5'→3' direction, a 5' untranslated region (5'-UTR) and an open reading frame (ORF), wherein the 5'-UTR comprises at its 3' end at least two tandem repeats of the sequence 5'-GCCRCC-3' operably linked to the ORF, and wherein R is an adenine nucleotide or a guanine nucleotide.

[0052] In one embodiment of the first aspect, at least two tandem repeats of the sequence 5'-GCCNCC-3' or the sequence 5'-GCCRCC-3' are directly linked to the ORF (i.e., the at least two tandem repeats are located immediately upstream of the first codon of the ORF).

[0053] In one embodiment of the first aspect, the 5'-UTR comprises two tandem repeats of the sequence 5'-GCCNCC-3' operably linked to an ORF at its 3' end. In one embodiment of the first aspect, the 5'-UTR comprises three tandem repeats of the sequence 5'-GCCNCC-3' operably linked to an ORF at its 3' end. In another embodiment, the 5'-UTR comprises two or three tandem repeats of the sequence 5'-GCCNCC-3' operably linked to an ORF at its 3' end.

[0054] In one embodiment of the first aspect, at least two tandem repeats of the sequence 5'-GCCNCC-3' increase the translation efficiency of the ORF.

[0055] 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 particular embodiment, the 5'-UTR comprises SEQ ID NO: 3 at its 3' end.

[0056] In one embodiment of the first aspect, the 5'-UTR 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 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145 and variants thereof that are 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 at their 3' end, and in particular, these variants substantially maintain or improve the translation enhancing effect of 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.

[0057] Polynucleotide sequence variants are well understood to those of skill in the art and can include sequence modifications, including nucleotide deletions, insertions, or alterations.

[0058] In certain embodiments of the first aspect, at least two tandem repeats of the sequence 5'-GCCNCC-3' form 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 particular SEQ ID NO:3.

[0059] In one embodiment of the first aspect, the 5'-UTR comprises or consists of a sequence derived from the 5'-UTR of a transcript of a gene linked in the 5'→3' direction to at least two tandem repeats of the sequence 5'-GCCNCC-3'. In one embodiment of the first aspect, the 5'-UTR comprises or consists of a sequence derived from the 5'-UTR of a transcript of a gene linked in the 5'→3' direction to at least two tandem repeats of the sequence 5'-GCCNCC-3'. In another embodiment, the 5'-UTR comprises or consists of a sequence derived from the 5'-UTR of a transcript of a gene linked in the 5'→3' direction to at least two tandem repeats of the sequence 5'-GCCNCC-3'. In another embodiment, the 5'-UTR comprises or consists of a 5'-UTR of a transcript of a gene directly linked in the 5'→3' direction to at least two tandem repeats of the sequence 5'-GCCNCC-3'. In more particular embodiments, the at least two tandem repeats are tandem repeats of the sequence 5'-GCCRCC-3'. In more particular embodiments, the gene is a naturally occurring gene. In even more particular embodiments, the gene is a mammalian gene or a human gene.

[0060] In another specific embodiment of the first aspect, the 5'-UTR comprises or consists of a sequence from the 5'-UTR of a gene transcript linked in the 5'→3' direction to at least two tandem repeats of the sequence 5'-GCCNCC-3', wherein the sequence from the 5'-UTR of the gene transcript comprises a Kozak sequence, particularly a non-consensus Kozak sequence. Thus, the Kozak sequence is directly linked 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 the 5'-UTR of a gene transcript linked in the 5'→3' direction to two tandem repeats of the sequence 5'-GCCNCC-3', wherein the sequence from the 5'-UTR of the gene transcript comprises a Kozak sequence, particularly a non-consensus Kozak sequence. Thus, the polynucleotide may comprise, consecutively and in the 5' to 3' direction, nucleotides of the Kozak sequence upstream of the start codon, (at least) two tandem repeats of the sequence 5'-GCCNCC-3', and then the start codon. In an even more particular embodiment, the two tandem repeats are tandem repeats of the sequence 5'-GCCRCC-3'.

[0061] In certain embodiments of the first aspect, the 5'-UTR comprises or consists of a sequence from the 5'-UTR of a transcript of a gene linked, in the 5'→3' direction, to at least two tandem repeats of the sequence 5'-GCCNCC-3', wherein the gene is selected from the group consisting of apolipoprotein A2 (APOA2), hemoglobin subunit beta (HBB), pre-T cell antigen receptor alpha (PTCRA), and small nuclear ribonucleoprotein D1 polypeptide (SNRPD1). In more particular embodiments, the gene is a human gene selected from the group consisting of 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) (GeneID: 171558, updated on August 5, 2022), small nuclear ribonucleoprotein D1 polypeptide (SNRPD1) (GeneID: 6632, updated on September 7, 2023). In more particular embodiments, the at least two tandem repeats are tandem repeats of the sequence 5'-GCCRCC-3'.

[0062] In certain embodiments of the first aspect, the 5'-UTR 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% identical to SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, and SEQ ID NO:41, or 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% identical to SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, and SEQ ID NO:41. , or variants thereof that are 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, and in particular, the variants substantially maintain or improve the translation enhancing effect of SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, or SEQ ID NO:41.

[0063] In certain embodiments of the first aspect, the polynucleotide further comprises one or more of: a 5'-cap structure; a 3' untranslated region (3'-UTR); and a 3' tailing sequence.

[0064] In certain embodiments of the first aspect, the polynucleotide comprises or consists of, in the 5'→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' tailing sequence.

[0065] In particular embodiments of the first aspect, the polynucleotide is an RNA polynucleotide, in particular a messenger RNA (mRNA).

[0066] In certain embodiments of the first aspect, the 5'UTR is heterologous to the ORF and / or the 3'UTR.

[0067] In particular embodiments of the first aspect, the 3'-UTR comprises or consists of a sequence from the 3'-UTR of a transcript of a gene, in particular a mammalian gene or a human gene.

[0068] In certain embodiments of the first aspect, the 3'-UTR comprises or consists of at least two tandem repeats of a sequence derived from the 3'-UTR of a transcript of a gene, particularly two tandem repeats of a sequence derived from the 3'-UTR of a transcript of a gene, wherein the gene is selected from the group consisting of HBB, PTCRA, APOA2, and SNRPD1.

[0069] In certain embodiments of the first aspect, the 3'-UTR comprises or consists of a 3'-UTR of a transcript of a gene, particularly a mammalian or human gene. More specifically, the 3'-UTR comprises or consists of two tandem repeats of the 3'-UTR of a transcript of a gene, particularly the gene selected from the group consisting of HBB, PTCRA, APOA2, and SNRPD1.

[0070] In one embodiment of the first aspect, the 3'-UTR 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 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119 ... or a variant thereof that is at least 98%, at least 98.5%, 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.

[0071] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 9, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 18.

[0072] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 9, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 19.

[0073] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 9, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 20.

[0074] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 9, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 21.

[0075] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 9, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 22.

[0076] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 9, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 23.

[0077] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 15, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 18.

[0078] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 15, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 19.

[0079] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 15, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 20.

[0080] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 15, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 21.

[0081] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 15, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 22.

[0082] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 15, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 23.

[0083] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 12, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 18.

[0084] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 12, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 19.

[0085] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 12, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 20.

[0086] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 12, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 21.

[0087] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 12, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 22.

[0088] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 12, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 23.

[0089] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 41, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 18.

[0090] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 41, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 19.

[0091] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 41, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 20.

[0092] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 41, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 21.

[0093] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 41, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 22.

[0094] In one embodiment of the first aspect, the polynucleotide comprises, in 5'→3' direction, a 5'-UTR, an ORF, and a 3'-UTR, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 41, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 23.

[0095] In one embodiment of the first aspect, the ORF encodes a polypeptide, and in particular, the polypeptide is an antigen. More particularly, the antigen is selected from the group consisting of a viral protein, a bacterial protein, and a tumor-associated antigen. In a particular embodiment of the first aspect, the polypeptide is an antibody or a fragment thereof. In a more particular embodiment, the antibody or fragment thereof is a therapeutic antibody or a fragment thereof.

[0096] In one embodiment of the first aspect, the 5'-cap structure is selected from the group consisting of cap-0, cap-1, cap-2, ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In a more particular embodiment, the 5'-cap structure is cap-1.

[0097] In one embodiment of the first aspect, the 3' tailing sequence is a poly(A) region, and in particular the poly(A) region is at least 80 nucleotides, at least 90 nucleotides, or at least 100 nucleotides in length. In more particular embodiments, the 3' tailing sequence is identical to the sequence of SEQ ID NO: 39, or 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%, or a variant thereof that is 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 thereto.

[0098] In one embodiment of the first aspect, the polynucleotide comprises at least one of the following: a 5' untranslated region (5-UTR) of the sequence of SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, or SEQ ID NO:41; a 3' untranslated region (3'-UTR) of the sequence of 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' tailing sequence of the sequence of SEQ ID NO:39.

[0099] In one embodiment of the first aspect, the polynucleotide comprises at least one of the following: a 5' untranslated region (5'-UTR) of the sequence of SEQ ID NO: 9; a 3' untranslated region (3'-UTR) of the sequence of SEQ ID NO: 18 or 19; and a 3' tailing sequence of the sequence of SEQ ID NO: 39.

[0100] In one embodiment of the first aspect, the polynucleotide comprises a 5' untranslated region (5'-UTR) of the sequence of SEQ ID NO: 9; a 3' untranslated region (3'-UTR) of the sequence of SEQ ID NO: 18 or 19; and a 3' tailing sequence of the sequence of SEQ ID NO: 39.

[0101] In one embodiment of the first aspect, the polynucleotide comprises at least one of the following: a 5' untranslated region (5'-UTR) of the sequence of SEQ ID NO: 41; a 3' untranslated region (3'-UTR) of the sequence of SEQ ID NO: 18 or 19; and a 3' tailing sequence of the sequence of SEQ ID NO: 39.

[0102] In one embodiment of the first aspect, the polynucleotide comprises a 5' untranslated region (5'-UTR) of the sequence of SEQ ID NO: 41; a 3' untranslated region (3'-UTR) of the sequence of SEQ ID NO: 18 or 19; and a 3' tailing sequence of the sequence of SEQ ID NO: 39.

[0103] In one embodiment of the first aspect, the polynucleotide comprises or consists, in the 5'→3' direction, of: (i) a 5'-cap1 structure; (ii) a 5' untranslated region (5'-UTR), particularly of the sequence of SEQ ID NO: 9 or 41; (iii) an open reading frame (ORF), particularly encoding a polypeptide; (iv) a 3' untranslated region (3'-UTR), particularly of the sequence of SEQ ID NO: 18 or 19; and (v) a 3' tailing sequence, particularly of the sequence of SEQ ID NO: 39.

[0104] In one embodiment of the first aspect, the polynucleotide comprises at least one chemical modification. In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine (also referred to as 1-methylpseudouridine (mIΨ), N6-methyladenosine (also referred to as m6A), 2-thiouridine (also referred to as s2U), 4'-thiouridine, 5-methylcytosine (also referred to as 5mC), 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4'-thiouridine, 5'-methylcytosine (also referred to as 5mC), 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudour ... The chemical modification is selected from methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine (also referred to as m5U), 5-methoxyuridine, 2'-O-methyluridine, and combinations thereof. In even more particular embodiments, the chemical modification is N1-methylpseudouridine, 5-methoxyuridine, or a combination thereof; particularly, the chemical modification is N1-methylpseudouridine.

[0105] In certain embodiments of the first aspect, the polynucleotide is partially modified with N1-methylpseudouridine. In another specific embodiment, the polynucleotide is 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% partially modified with N1-methylpseudouridine. In another specific embodiment, the polynucleotide is fully modified with N1-methylpseudouridine, 5-methoxyuridine, or a combination thereof. In a more specific embodiment, the polynucleotide is fully modified with N1-methylpseudouridine.

[0106] In certain embodiments of the first aspect, the polynucleotide is an isolated, artificial polynucleotide.

[0107] As disclosed above, in a second aspect, the present invention provides a DNA construct comprising a promoter operably linked to a sequence encoding a polynucleotide of the first aspect, i.e., this aspect provides a DNA construct whose transcription results in a polynucleotide defined in the first aspect.

[0108] All embodiments of the polynucleotide of the first aspect are intended to also apply to the DNA construct of the second aspect.

[0109] In one embodiment of the second aspect, the polynucleotide is an RNA polynucleotide, in particular an mRNA.

[0110] Those skilled in the art know how to produce the polynucleotides, DNA constructs, or expression vectors of the invention without the exercise of any original skill by defined methods well known in the art, for example by chemical synthesis or by molecular biology techniques.

[0111] As indicated above, in a fifth aspect, the present invention provides a composition comprising lipid nanoparticles and a polynucleotide defined in the first aspect, a DNA construct defined in the second aspect, or an expression vector defined in the third aspect. That is, the composition comprises lipid nanoparticles encapsulating a polynucleotide defined in the first aspect, a DNA construct defined in the second aspect, or an expression vector defined in the third aspect.

[0112] The method for synthesis of the composition formed by lipid nanoparticles that encapsulates polynucleotide, DNA construct or expression vector is well known to those skilled in the art and has been formally established in molecular biology protocols.Specific conditions are shown in examples.Those skilled in the art will know which lipid nanoparticles should be used to encapsulate polynucleotide, DNA construct or expression vector according to the intended use of composition.

[0113] In one embodiment of the fifth aspect, the lipid nanoparticles comprise at least one selected from the group consisting of a PEG-modified lipid, a non-cationic lipid, a sterol, and an ionizable cationic lipid. In one embodiment of the fifth aspect, the lipid nanoparticles comprise or consist of a PEG-modified lipid, a non-cationic lipid, a sterol, and an ionizable cationic lipid. In a more specific embodiment, the lipid nanoparticles comprise at least one of SM-102, distearolyphosphatidycholine (DSPC), cholesterol, and DMG-PEG2000. In a more specific embodiment, the lipid nanoparticles comprise or consist of SM-102, DSPC, cholesterol, and DMG-PEG2000.

[0114] In certain embodiments of the fifth aspect, the lipid nanoparticles are ionizable or charged.

[0115] In certain embodiments of the fifth aspect, the composition comprises a polynucleotide, a DNA construct, or an expression vector of the invention encapsulated in a lipid nanoparticle.

[0116] In certain embodiments of the fifth aspect, the composition is administered in the form of a pharmaceutical composition.

[0117] As indicated above, in a sixth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a polynucleotide, DNA construct, expression vector, or composition of the invention and at least one pharmaceutically acceptable excipient and / or carrier.

[0118] The phrase "pharmaceutically acceptable excipient and / or carrier" refers to a pharmaceutically acceptable material, composition, or vehicle. Each component must be pharmaceutically acceptable in the sense of being compatible with the other components of the pharmaceutical composition. It must also be suitable for use in contact with the tissues or organs of humans and non-human animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0119] Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc. Except insofar as any conventional excipient medium is incompatible with the substance or its derivatives, for example, by producing any undesirable biological effect or by interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated within the scope of the present invention.

[0120] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition of the present invention will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered.

[0121] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Excipients such as coloring agents, coating agents, sweetening agents, and flavoring agents may be present in the composition, at the discretion of the formulator.

[0122] The polynucleotides or compositions described herein may be used in vaccine therapy, enhancing the efficacy of conventional vaccines, and / or as novel vaccine forms for use against infectious pathogens such as viruses, bacteria, fungi, protozoa, prions, and helminths (worms), or for use in treating diseases such as cancer.

[0123] In one embodiment of the sixth aspect, the pharmaceutical composition is a vaccine. In a more particular embodiment, the pharmaceutical composition is a vaccine and further comprises an adjuvant. Based on their general knowledge, those skilled in the art know which excipients, carriers and adjuvants should be included in the vaccine according to the intended use.

[0124] In a further embodiment, the polynucleotide, DNA construct, expression vector, cell, composition, or pharmaceutical composition of the invention is for use in the prevention of COVID-19.

[0125] In one embodiment 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 by intraarticular administration.

[0126] As indicated above, in an eleventh aspect, the present invention also provides an in vitro method for producing a polypeptide in a cell, the method comprising contacting the cell with a polynucleotide, a DNA construct, an expression vector, a composition, or a pharmaceutical composition of the present invention under particularly suitable conditions. Those skilled in the art will be able to determine the optimal operational conditions for producing the polypeptide by using routine experimentation.

[0127] The present invention also provides a method for increasing the translation rate of a polynucleotide in a twelfth aspect. Those skilled in the art will know how to insert tandem repeat sequences into the sequence of a polynucleotide using conventional molecular biology techniques.

[0128] In certain embodiments of the twelfth aspect, the method comprises inserting at least two tandem repeats of the sequence 5'-GCCRCC-3' at the 3'-end of the 5'-UTR, where 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.

[0129] In particular embodiments of the twelfth aspect, the method comprises inserting two tandem repeats of the sequence 5'-GCCRCC-3' at the 3'-end of the 5'-UTR, in particular two tandem repeats of the sequence 5'-GCCACC-3' at the 3'-end of the 5'-UTR.

[0130] All embodiments of the first aspect relating to polynucleotide sequences are also intended to apply to this twelfth aspect.

[0131] Throughout this specification and claims, the word "comprise" and variations of that word are not intended to exclude other technical features, additives, ingredients, or steps. Furthermore, the word "comprise" encompasses the instances of "consisting of." Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of this specification or may be learned by practice of the invention. The following examples and figures are provided for illustrative purposes and are not intended to limit the invention. Furthermore, the present invention encompasses all possible combinations of the specific and preferred embodiments described herein.

[0132] The sequences of the present invention are disclosed in Table 1 below. [Table 1]

[0133] The following examples are provided for illustrative purposes and are not intended to limit the invention. Furthermore, the present invention encompasses all possible combinations of specific and preferred embodiments described herein.

[0134] Example Example 1: Protein expression in cells transfected with RNA of the invention In vitro transcription of RNA The plasmids used in this experiment are listed in Table 2 below. [Table 2]

[0135] All plasmids encoded transcripts containing the firefly luciferase coding sequence under the control of different UTRs, as shown in Table 2. Plasmid P2 was generated by inserting the sequence 5'-GCCACC-3' at the 3'-end of the 5'-UTR of P1, and plasmid P3 was generated by inserting two tandem repeats of the sequence 5'-GCCACC-3' at the 3'-end of the 5'-UTR of P1. Plasmids P4-P6 encoded transcripts with regulatory elements (i.e., UTRs) of commercially available RNAs and were used for comparison purposes along with P1 and P2.

[0136] One microgram of RNAse-free plasmid was digested with BspQI restriction enzyme for P1, P2, P3, and P5, and with NotL for P4 and P6. The restriction enzyme cut the plasmid immediately after the transcribed segment. Plasmid linearization reactions were purified using the Wizard® SV Gel and PCR Clean-Up (Promega A7270) purification kit protocol according to the manufacturer's instructions. The recovery of plasmid linearization was at least 50% in all cases.

[0137] The purified linear DNA was then used for RNA production by in vitro transcription using T7 RNA polymerase according to the manufacturer's instructions. Briefly, transcription was carried out for 3 hours at 37°C using the following reagents: Template DNA (50 μg / mL) T7 polymerase (5,000 U / mL; HONGENE® ON-004) RNase inhibitor (1.000 U / mL, HONGENE® ON-039) Inorganic pyrophosphatase (2 U / mL, HONGENE® ON-025) ATP (5 μg / mL, HONGENE® R1331) GTP (5 μg / mL, HONGENE® R2331) CTP (5 μg / mL, HONGENE® R3331) N1-methylpseudouridine (5 μg / mL, HONGENE® R5-027) CleanCap® AG (4 μg / mL, TRILINK® N-7113-10) inorganic pyrophosphatase (2 U / mL, HONGENE® ON-025) ATP (5 μg / mL, HONGENE® R1331) GTP (5 μg / mL, HONGENE® R2331) OTP (5 μg / mL, HONGENE® R3331) N1-methylpseudouridine (5 μg / mL, HONGENE® R5-027) CleanCap® AG (4 μg / mL, TRILINK® N-7113-10)

[0138] The generated transcripts were purified by DNase I incubation (NEB® M0303L) according to the manufacturer's instructions, followed by LiCl precipitation and washing with 75% ethanol. The concentration of the reconstituted RNA in sodium citrate buffer was determined by measuring the optical density at 260 nm. The yield of RNA production was at least 80 μg of RNA per μg of linearized plasmid DNA.

[0139] All RNA samples were analyzed by denaturing agarose gel electrophoresis for quality assurance. RNA was aliquoted and stored at -80°C until use. Each RNA type was synthesized in at least two independent transcription experiments, and all experiments were performed with at least two different batches of mRNA.

[0140] As shown in Table 2 above, in vitro transcription of P1 produced transcript R1; P2 produced transcript R2; P3 produced transcript R3; P4 produced transcript R4; P5 produced transcript R5; and P6 produced transcript R6.

[0141] Cell transfection The day before transfection, HeLa cells (ACC57) or HEK293T cells (CRL-3216) were transfected at 1 × 10 4 Cells were seeded in 96-well plates at a density of 10 cells / well. The culture medium used for both cells was DM EM high glucose (Merck D6429) supplemented with 10% fetal bovine serum (Sigma F7524), 1% Antibiotic Antimycotic Solution (Sigma A5955), and 2 mM Glutamax (Fisher 35050038).

[0142] For transfection, the corresponding culture medium was replaced with 90 pL of fresh medium. Next, a mixture of 10 μL of mRNA R1-R6 (0.1 μg / well) produced as described above and Lipofectamine™ MessengerMAX Reagent (Invitrogen 15397974; 0.2 μL / well) was pre-incubated in OptiMEM medium and added to the cell culture. The cells were incubated with the mRNA-Lipofectamine MessengerMAX mixture at 37°C in a 5% CO atmosphere for 24 hours for further analysis.

[0143] Quantification of firefly luciferase activity Transfected cells were dissolved in 100 μL of PBS-Triton 0.1%, and 98 μL was transferred to an opaque 96-well white plate. Next, 102 μL of buffered d-luciferin (GoldBio LUCK-100) (100 mM Tris-HCl pH 7.8, 5 mM MgCl, 250 μM CoA, 150 μM ATP) was added to the 96-well white plate at a final concentration of 150 μg / mL. Luminescence was measured after 5 minutes of incubation at room temperature using a FLUOstar Omega plate reader according to the manufacturer's instructions.

[0144] As shown in Figure 1A and 1B, different mRNAs produced luciferase at different rates depending on the combination of 5'-UTR and 3'-UTR sequences flanking the coding region. Addition of a single repeat of the sequence 5'-GCCACC-3' immediately before the ORF in R1 (i.e., R2) did not improve protein production. However, addition of two 5'-GCCACC-3' sequences in tandem immediately before the ORF in R1 (i.e., R3) significantly improved protein production in both HeLa and HEK293T cells, reaching the highest production levels of all mRNAs tested.

[0145] These results clearly suggest that the presence of at least two 5'-GCCACC-3' sequences in tandem at the 3' end of the 5'-UTR synergistically enhances the rate of mRNA translation, thereby allowing for higher protein production levels.

[0146] Example 2: Protein expression in muscles of mice administered RNA of the present invention RNA encapsulation in lipid nanoparticles For in vivo administration, mRNA R1–R6 produced as described above were encapsulated into lipid nanoparticles (LNPs) as described in Hassett, KJ et al., “Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines,” 2019, Mol. Ther. Nucleic Acids, vol. 15, pp. 1–11. Briefly, purified capped mRNA was first diluted in sodium citrate buffer at pH 4 to a final concentration of 266 μg / ml. Separately, the lipids SM-102 (BOCSI 2089251-47-6):DSPC (Merck 850365P):cholesterol (Sigma C3045):DMG-PEG2000 (Cayman 33945-1)) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5, with an N:P ratio of 5.5:1.

[0147] 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 the encapsulated mRNA was assessed using Quant-IT® Ribogreen (Invitrogen R11490) according to the manufacturer's instructions.

[0148] The LNP solution was then adjusted to a final mRNA concentration of 100 μg / ml. Size distribution, polydispersity, and zeta potential were measured by dynamic light scattering (DLS). Typical values ​​obtained for these parameters were in the following ranges: size distribution, 90-120 nm; polydispersity, 0.08-1.5; zeta potential, -10-+10 mV. RNA encapsulation was assessed using Quant-IT® Ribogreen according to the manufacturer's instructions, and encapsulation efficiencies of approximately 80%-95% were typically obtained for all mRNAs.

[0149] Finally, the LNP solution was passed through a 0.22 mm filter and the LNPs were stored at -80°C until required.

[0150] Administration of mRNA (LNP) to mice Female BALB / c mice (Charles River Laboratories), 8–10 weeks old and weighing 18–23 g, were acclimated to the new conditions for 3–7 days upon arrival at the experimental facility. The conditions were room temperature (20–24°C), humidity (50–70%), light intensity (60 lux), and a 12-hour light / dark cycle.

[0151] For measurement of firefly luciferase activity in mice, LNPs prepared as described above and containing 5 μg of the indicated mRNA in a final volume of 50 μl were injected intramuscularly.

[0152] Between 4 and 72 hours after RNA-LNP inoculation, mice were anesthetized by inhalation of 4% isoflurane using a vaporizer. Anesthesia was maintained with 1.5% isoflurane. D-luciferin (Quimigen, Ref:12507) was then injected intraperitoneally at 150 mg / kg—typically approximately 200 μL of a 15 mg / mL stock in PBS for a 20 g mouse. Luciferase images were acquired 10 minutes after luciferin inoculation using an IVIS Lumina XRMS Imaging System according to the manufacturer's instructions.

[0153] As shown in Figure 2, the presence of two tandem 5′-GCCACC-3′ sequences (R3) significantly increased in vivo protein production compared with LNPs with mRNAs containing only one or none of the repeats.

[0154] These results indicate that the presence of two 5'-GCCACC-3' sequences in tandem in polynucleotides for producing proteins significantly enhances their expression potential, which may improve the therapeutic efficiency of known and future protein expression vectors.

[0155] Example 3: Protein expression in cells transfected with RNA of the invention Protein expression assays were performed as described in Example 1 using the plasmids listed in Table 3 below: [Table 3]

[0156] As shown in Figure 3A and B, different mRNAs produced luciferase at different rates depending on the combination of 5'UTR and 3'UTR sequences flanking the coding region. The addition of two 5'-GCCACC-3' sequences in tandem immediately before the ORF in R7 (i.e., R8) significantly improved protein production in both HeLa and HEK293T cells.

[0157] These results indicate that the presence of at least two 5'-GCCACC-3' sequences in tandem at the 3' end of a 5'-UTR consistently enhances the translation rate of mRNAs with different 3'-UTRs.

[0158] Example 4: Protein expression in cells transfected with mRNA of the present invention Protein expression assays were performed as described in Example 1, except that 0.05 μg / well of mRNA was added to the cell cultures instead of 0.1 μg / well of mRNA for cell transfection. The plasmids and mRNAs used are shown in Table 4 below: [Table 4]

[0159] As shown in Figure 4A and B, different mRNAs produced luciferase at different rates depending on the combination of 5'UTR and 3'UTR sequences flanking the coding region. The addition of two 5'-GCCACC-3' sequences in tandem immediately before the ORF in R9 (i.e., R10) significantly improved protein production in both HeLa and HEK293T cells.

[0160] These results indicate that the presence of at least two 5'-GCCACC-3' sequences in tandem at the 3'-ends of different 5'-UTRs consistently increases the rate of mRNA translation, thereby enabling higher protein production levels.

[0161] The present invention includes the following embodiments.

[0162] Additional notes 1. In the 5'→3' direction, a 5' untranslated region (5'-UTR); Open reading frame (ORF) Including, 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; Artificial polynucleotides.

[0163] Appendix 2. The polynucleotide of Appendix 1, wherein the at least two tandem repeats are tandem repeats of the sequence 5'-GCCRCC-3', and R is any purine nucleotide.

[0164] Item 3. The 5'-UTR comprises, at its 3' end, two tandem repeats of the sequence 5'-GCCRCC-3' operably linked to the ORF, or alternatively, 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, operably linked to an ORF; A polynucleotide according to any one of appendix 1 and 2.

[0165] Item 4. A polynucleotide according to any one of items 1 to 3, wherein the 5'-UTR comprises or consists of a sequence from the 5'-UTR of a transcript of a gene linked in the 5' to 3' direction to at least two tandem repeats of the sequence 5'-GCCNCC-3'; in particular, the at least two tandem repeats are tandem repeats of the sequence 5'-GCCRCC-3'.

[0166] Appendix 5. A polynucleotide according to Appendix 4, wherein the sequence derived from the 5'-UTR of the transcript of said gene comprises a Kozak sequence, in particular a non-consensus Kozak sequence.

[0167] Appendix 6: A polynucleotide according to any one of Appendixes 4 to 5, wherein the sequence derived from the 5'-UTR of the transcript of the gene is derived from a gene selected from the group consisting of apolipoprotein A2 (APOA2), hemoglobin subunit β (HBB), pre-T cell antigen receptor α (PTCRA), and small nuclear ribonucleoprotein D1 polypeptide (SNRPD1).

[0168] Appendix 7. A polynucleotide according to any one of Appendixes 1 to 6, wherein at least two tandem repeats of the sequence 5'-GCCNCC-3' form 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.

[0169] Appendix 8: The polynucleotide according to any one of Appendixes 1 to 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.

[0170] Additional Note 9. 5'-cap structure 3' untranslated region (3'-UTR); and 3' tailing sequence 9. The polynucleotide according to any one of appendix 1 to 8, further comprising one or more of the following:

[0171] Additional Note 10. In the 5'→3' direction, (i) 5′-cap structure; (ii) the 5' untranslated region (5-UTR); (iii) open reading frame (ORF); (iv) the 3′ untranslated region (3′-UTR); and (v) 3' tailing sequence 10. The polynucleotide according to any one of appendixes 1 to 9, comprising or consisting of:

[0172] Appendix 11: The polynucleotide of any one of appendices 1 to 10, which is an RNA polynucleotide, particularly a messenger RNA (mRNA).

[0173] Appendix 12. The polynucleotide of any one of embodiments 1 to 11, wherein the 5'UTR is heterologous to the ORF and / or 3'UTR.

[0174] Appendix 13: The polynucleotide according to any one of appendices 9 to 12, wherein the 3'-UTR comprises or consists of a sequence derived from the 3'-UTR of a transcript of a gene, particularly a mammalian gene.

[0175] Appendix 14. A polynucleotide according to any one of appendices 9 to 13, wherein the 3'-UTR comprises or consists of at least two tandem repeats of a sequence derived from the 3'-UTR of a gene transcript, in particular two tandem repeats of a sequence derived from the 3'-UTR of a gene transcript.

[0176] Appendix 15. A polynucleotide according to any one of appendices 9 to 14, wherein the 3'-UTR comprises or consists of a sequence derived from the 3'-UTR of a transcript of a gene selected from the group consisting of apolipoprotein A2 (APOA2), hemoglobin subunit beta (HBB), pre-T cell antigen receptor alpha (PTCRA), and small nuclear ribonucleoprotein D1 polypeptide (SNRPD1).

[0177] Appendix 16: The polynucleotide according to any one of Appendixes 9 to 15, wherein the 3'-UTR comprises or consists of 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.

[0178] Appendix 17: The polynucleotide according to any one of appendices 1 to 16, wherein the ORF encodes a polypeptide, particularly an antigen.

[0179] Appendix 18. The polynucleotide of appendix 17, wherein the antigen is a SARS-CoV-2 antigen, in particular a SARS-CoV-2 spike antigen.

[0180] Appendix 19. A polynucleotide according to any one of appendices 9 to 18, wherein the 5'-cap structure is selected from the group consisting of cap-0, cap-1, cap-2, ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine, and in particular, the 5'-cap structure is cap-1.

[0181] Appendix 20: The polynucleotide according to any one of appendices 9 to 19, wherein the 3' tailing sequence is a polyA region, particularly a polyA region of the sequence of SEQ ID NO:39.

[0182] Item 21. The following: the 5' untranslated region (5-UTR) of the sequence of SEQ ID NO: 9 or 41; The 3' untranslated region (3'-UTR) of the sequence of SEQ ID NO: 18 or 19; and 3' tailing sequence of the sequence of SEQ ID NO: 39 21. The polynucleotide according to any one of appendixes 1 to 20, comprising at least one of the following:

[0183] Addendum 22. In the 5'→3' direction, (i) 5′-cap structure; (ii) the 5' untranslated region (5-UTR) of the sequence of SEQ ID NO: 9 or 41; (iii) an open reading frame (ORF) encoding a polypeptide; (iv) the 3' untranslated region (3'-UTR) of the sequence of SEQ ID NO: 18 or 19; and (v) a 3' tailing sequence of SEQ ID NO: 39 22. The polynucleotide according to any one of appendixes 1 to 21, comprising or consisting of:

[0184] Appendix 23. The polynucleotide of any one of appendixes 1 to 22, wherein the polynucleotide comprises at least one chemical modification, in particular the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methylpseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2'-O-methyluridine, N6-methyladenosine, and combinations thereof.

[0185] Appendix 24. The polynucleotide of Appendix 23, wherein the chemical modification is N1-methylpseudouridine, 5-methoxyuridine, or a combination thereof, and in particular, the chemical modification is N1-methylpseudouridine.

[0186] Appendix 25. The polynucleotide according to any one of appendixes 23 to 24, wherein the polynucleotide is fully modified with N1-methylpseudouridine, 5-methoxyuridine, or a combination thereof, particularly wherein the polynucleotide is fully modified with N1-methylpseudouridine.

[0187] Appendix 26. A DNA construct comprising a promoter operably linked to a sequence encoding a polynucleotide defined in any one of appendices 1 to 25.

[0188] Appendix 27. An expression vector comprising a DNA construct as defined in Appendix 26.

[0189] Item 28. A cell comprising a polynucleotide as defined in any one of items 1 to 25, a DNA construct as defined in item 26, or an expression vector as defined in item 27.

[0190] Item 29. A composition comprising lipid nanoparticles and a polynucleotide defined in any one of items 1 to 25, a DNA construct defined in item 26, or an expression vector defined in item 27.

[0191] Appendix 30. The composition of Appendix 29, wherein the lipid nanoparticles comprise at least one selected from the group consisting of PEG-modified lipids, non-cationic lipids, sterols, and ionizable cationic lipids.

[0192] Item 31. A pharmaceutical composition comprising a therapeutically effective amount of a polynucleotide defined in any one of items 1 to 25, a DNA construct defined in item 26, an expression vector defined in item 27, or a composition defined in any one of items 29 to 30, and at least one pharmaceutically acceptable excipient and / or carrier.

[0193] Appendix 32. The pharmaceutical composition of appendix 31, which is a vaccine, optionally further comprising an adjuvant.

[0194] Appendix 33. A polynucleotide as defined in any of Appendixes 1 to 25, a DNA construct as defined in Appendix 26, an expression vector as defined in Appendix 27, a cell as defined in Appendix 28, a composition as defined in any of Appendixes 29 to 30, or a pharmaceutical composition as defined in any of Appendixes 31 to 32 for use in medicine, for use in a method for inducing an immune response in a subject, for use in a method for therapeutic immunization of a subject, or for use as a vaccine or in gene therapy.

[0195] Appendix 34. A polynucleotide, DNA construct, expression vector, composition, or pharmaceutical composition according to appendix 33 for use in the prevention and / or treatment of COVID-19.

[0196] Appendix 35. An in vitro method for producing a polypeptide in a cell, comprising contacting the cell with a polynucleotide defined in any one of appendices 1 to 25, a DNA construct defined in appendices 26, an expression vector defined in appendices 27, a composition defined in appendices 29 to 30, or a pharmaceutical composition defined in any one of appendices 31 to 32.

[0197] Appendix 36. 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 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.

[0198] Citation List Altschul et al., “Basic local alignment search tool”, 1990, J. Mol. Biol, v. 215, pages 403-410 EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277 Hassett, KJ et al., “Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines”, 2019, Mol. Ther. Nucleic Acid, vol. 15, pp. 1-11

Claims

1. In the 5'→3' direction, a 5' untranslated region (5'-UTR); Open reading frame (ORF) and An artificial polynucleotide comprising: 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; Artificial polynucleotides.

2. 2. The polynucleotide of claim 1, wherein the at least two tandem repeats are tandem repeats of the sequence 5'-GCCRCC-3', where R is a purine nucleotide.

3. 3. The polynucleotide according to claim 1, wherein the 5'-UTR comprises, at its 3' end, two tandem repeats of the sequence 5'-GCCRCC-3' operably linked to an ORF, in particular the two tandem repeats forming the sequence of SEQ ID NO:

3.

4. 4. The polynucleotide of claim 1, wherein the 5'-UTR comprises, in a 5'→3' direction, a sequence derived from the 5'-UTR of a transcript of a gene 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 said sequence derived from the 5'-UTR of a gene transcript comprises a Kozak sequence, in particular a non-consensus Kozak sequence, at its 3' end.

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

7. 7. The polynucleotide of any one of claims 1 to 6, wherein 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 variant thereof 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. 5'-cap structure; a 3' untranslated region (3'-UTR); and 3' tailing sequence The polynucleotide of any one of claims 1 to 7, further comprising one or more of:

9. In the 5'→3' direction, (i) 5′-cap structures, particularly cap-1 structures; (ii) the 5' untranslated region (5'-UTR), in particular of the sequence of SEQ ID NO: 9; (iii) an open reading frame (ORF), particularly encoding a polypeptide; (iv) the 3' untranslated region (3'-UTR), in particular of the sequence of SEQ ID NO: 19; and (v) a 3' tailing sequence, in particular of the sequence of SEQ ID NO: 39 The polynucleotide according to any one of claims 1 to 8, comprising:

10. In the 5'→3' direction, (i) 5′-cap structures, particularly cap-1 structures; (ii) the 5' untranslated region (5'-UTR), in particular of the sequence of SEQ ID NO: 9; (iii) an open reading frame (ORF), particularly encoding a polypeptide; (iv) the 3' untranslated region (3'-UTR), in particular of the sequence of SEQ ID NO: 18; and (v) a 3' tailing sequence, in particular of the sequence of SEQ ID NO: 39 The polynucleotide according to any one of claims 1 to 8, comprising:

11. In the 5'→3' direction, (i) 5′-cap structures, particularly cap-1 structures; (ii) the 5' untranslated region (5'-UTR), in particular of the sequence of SEQ ID NO: 41; (iii) an open reading frame (ORF), particularly encoding a polypeptide; (iv) the 3' untranslated region (3'-UTR), in particular of the sequence of SEQ ID NO: 18; and (v) a 3' tailing sequence, in particular of the sequence of SEQ ID NO: 39 The polynucleotide according to any one of claims 1 to 8, comprising:

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

13. 13. The polynucleotide of any one of claims 1 to 12, wherein the polynucleotide comprises at least one chemical modification, and in particular the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, N6-methyladenosine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-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 a polynucleotide as defined in any one of claims 1 to 13.

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

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

17. 19. An in vitro method for producing a polypeptide in a cell, the method comprising the step of 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. 1. 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 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. method.

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