Polynucleotides containing a 5'UTR-3'UTR combination and uses thereof

Abstract

The present invention relates to the field of artificial polynucleotides, particularly to RNA polynucleotides comprising a specific 5'UTR-3'UTR gene combination. The present specification provides an RNA polynucleotide comprising at least one 5'UTR element selected from genes such as ANXA1, E. coli enolase, RPS14, RPS25, or TCV, and at least one 3'UTR selected from genes such as HBA1, RPS29, Ube2d2a, or UBL5. The present invention also relates to a pharmaceutical composition comprising one or more RNA polynucleotides according to the present invention, and a method for inducing an improved immune response in a subject using said RNA polynucleotide or pharmaceutical composition.

Description

Technology Field

[0001] The present invention relates to the field of artificial polynucleotides, particularly to RNA polynucleotides comprising a specific 5'UTR-3'UTR gene combination. The present specification provides an RNA polynucleotide comprising at least one 5'UTR element selected from genes such as ANXA1, E. coli enolase, RPS14, RPS25, or TCV, and at least one 3'UTR selected from genes such as HBA1, RPS29, Ube2d2a, or UBL5. The present invention also relates to a pharmaceutical composition comprising one or more RNA polynucleotides according to the present invention, and a method for inducing an improved immune response in a subject using said RNA polynucleotide or pharmaceutical composition. Background Technology

[0002] Synthetic mRNA has emerged as a powerful tool for the transfer of genetic information and is being explored for various therapeutic applications. Many of these applications require the extension of the intracellular persistence of mRNA to improve the bioavailability of the encoded protein. mRNA molecules or polynucleotides are inherently unstable, and their intracellular kinetics are governed by the 5' uncoding region (5' UTR) and 3' uncoding region (3' UTR) that enclose the coding sequence. Structural features of UTRs, such as sequence, length, and secondary structure, have a significant impact on translation initiation and peak expression levels (primarily 5' UTR), as well as on mRNA stability and expression duration (primarily 3' UTR).

[0003] Therefore, the combination of UTRs determines the expression kinetics of mRNA polynucleotides. UTRs used in synthetic RNA are generally derived from α / β-globin, human heat shock proteins, or viruses. However, UTR performance can vary between cell types, and specific UTRs tailored to the target cell type are required.

[0004] Accordingly, the object of the present invention is to provide an RNA polynucleotide comprising an optimal 5'UTR / 3'UTR combination of specific UTRs that results in increased stability and / or translation efficiency. Such RNA polynucleotides are particularly useful for mRNA-based therapies, gene therapy, and / or gene vaccination. The object of the present invention is to provide an RNA polynucleotide in which protein expression from the RNA polynucleotide is increased and prolonged, as well as biodistribution is improved and, preferably, increased translation efficiency is exhibited. These objects of the present invention are achieved by the claimed subject matter of the invention.

[0005] In a first aspect, the present invention provides an RNA polynucleotide comprising the following:

[0006] i) at least one 5' untranslated region (5'UTR) element comprising a nucleic acid sequence derived from the 5'UTR of ANXA1, E. coli enolase, RPS14, RPS25, or TCV gene, or a fragment or variant thereof;

[0007] ii) at least one open reading frame (ORF) encoding a polypeptide;

[0008] iii) at least one 3' untranslated region (3'UTR) element comprising a nucleic acid sequence derived from the 3'UTR of the HBA1, RPS29, Ube2d2a, or UBL5 gene, or a fragment or variant thereof.

[0009] RNA polynucleotides having at least one of these 5' UTR and 3' UTR combinations were found to cause improved expression and biodistribution compared to the control group.

[0010] In a specific embodiment, the 5'UTR of the RNA polynucleotide comprises an RNA sequence corresponding to a DNA sequence selected from SEQ ID NO. 1 [ANXA1], SEQ ID NO. 2 [eno], SEQ ID NO. 3 [RPS14], SEQ ID NO. 4 [RPS25], or SEQ ID NO. 5 [TCV], or a sequence having at least 95% sequence identity with respect to this.

[0011] In another embodiment, the 3'UTR of the RNA polynucleotide comprises an RNA sequence corresponding to a DNA sequence selected from SEQ ID NO. 6 [HBA1], SEQ ID NO. 7 [RPS29], SEQ ID NO. 8 [Ube2d2a], or SEQ ID NO. 9 [UBL5], or a sequence having at least 95% sequence identity with respect to this.

[0012] In another embodiment, the 3'UTR of the RNA polynucleotide comprises an RNA sequence corresponding to a DNA sequence selected from SEQ ID NO. 6 [HBA1], SEQ ID NO. 18 [HBA1], SEQ ID NO. 7 [RPS29], SEQ ID NO. 8 [Ube2d2a], or SEQ ID NO. 9 [UBL5], or a sequence having at least 95% sequence identity with respect to this.

[0013] In a specific embodiment, the 5'UTR and 3'UTR of the RNA polynucleotide comprise an RNA sequence fragment or a variant thereof corresponding to a DNA sequence selected from any one of the following 5' UTR-3' UTR gene combinations:

[0014] - A combination of ANXA1's 5'UTR and HBA1's 3'UTR; or

[0015] - A combination of the eno's 5'UTR and the RPS29's 3'UTR; or

[0016] - A combination of eno's 5'UTR and Ube2d2a's 3'UTR; or

[0017] - A combination of the 5'UTR of RPS14 and the 3'UTR of HBA1; or

[0018] - A combination of the 5'UTR of RPS25 and the 3'UTR of Ube2d2a; or

[0019] - Combination of TCV's 5'UTR and UBL5's 3'UTR.

[0020] In an additional embodiment, the 5' UTR and 3' UTR of the RNA polynucleotide comprise an RNA sequence corresponding to a DNA sequence selected from any one of the following combinations, or a sequence having at least 95% sequence identity therewith:

[0021] - A combination of SEQ ID NO. 1 [ANXA1] for the 5'UTR and SEQ ID NO. 6 [HBA1] for the 3'UTR; or

[0022] - A combination of SEQ ID NO. 2[eno] for the 5'UTR and SEQ ID NO. 7[RPS29] for the 3'UTR; or

[0023] - A combination of SEQ ID NO. 2[eno] for the 5'UTR and SEQ ID NO. 8[Ube2d2a] for the 3'UTR; or

[0024] - A combination of SEQ ID NO. 3 [RPS14] for the 5'UTR and SEQ ID NO. 6 [HBA1] for the 3'UTR; or

[0025] - A combination of SEQ ID NO. 3 [RPS25] for the 5'UTR and SEQ ID NO. 8 [Ube2d2a] for the 3'UTR; or

[0026] - A combination of SEQ ID NO. 1 [ANXA1] for the 5'UTR and SEQ ID NO. 18 [HBA1] for the 3'UTR; or

[0027] - A combination of SEQ ID NO. 3[RPS14] for the 5' UTR and SEQ ID NO. 18[HBA1] for the 3' UTR; or

[0028] - Combination of sequence number 5[TCV] for the 5' UTR and sequence number 9[UBL] for the 3' UTR.

[0029] According to a further embodiment of the present invention, the 5' UTR of the RNA polynucleotide further comprises a Kozak sequence and / or an internal ribosome entry site (IRES). In another further embodiment, the RNA polynucleotide is mRNA or non-coding RNA, preferably mRNA, antisense RNA, siRNA, sgRNA, guide RNA, or CRISPR.

[0030] In another embodiment, the present invention relates to a pharmaceutical composition comprising one or more RNA polynucleotides according to the present invention and at least one pharmaceutically acceptable formulation.

[0031] In a further embodiment, the present invention relates to RNA polynucleotides or pharmaceutical compositions according to the present invention for use in human medicine and / or veterinary medicine. More specifically, RNA polynucleotides or pharmaceutical compositions are provided for use as vaccines or in gene therapy.

[0032] In another embodiment, the present invention relates to using an RNA polynucleotide or a pharmaceutical composition according to the present invention to improve the expression and / or translation of a gene product, particularly to improve translation initiation. Even more specifically, the use of said RNA polynucleotide or said pharmaceutical composition is provided to improve the expression stability and / or duration of a gene product, particularly to improve biodistribution.

[0033] In addition, in another embodiment, the present invention relates to a method for inducing an improved immune response in a subject, comprising the step of administering a therapeutically effective amount of an RNA polynucleotide or a pharmaceutical composition according to the present invention. Brief explanation of the drawing

[0034] Now, referring specifically to the drawings, it is emphasized that the details presented herein are merely for the purpose of illustrating various embodiments of the present invention and for illustrative consideration. These drawings are presented to explain the principles and conceptual aspects of the present invention in the most useful and understandable way. In this regard, there is no intention to show structural details beyond what is necessary to fundamentally understand the present invention. The description provided with the drawings is intended to enable a person skilled in the art to clearly understand how certain forms of the present invention can be practically implemented. Fig. 1: Flowchart of mRNA screening in the first round. The mRNA library was constructed using a combination of 21 5' UTRs, 21 3' UTRs, and secreted Nanoluc. After transfecting iMoDCs with 100 ng of each mRNA in duplicates, transfection efficiency, cell viability, and Nanoluc expression were evaluated as shown in the center panel of the figure. The duplicates were then pooled, and the area under the curve (AUC) was calculated for each mRNA to obtain the 40 mRNAs exhibiting the best performance through subsequent selection of top performers from each plate. C1, C2, C3, and C4 represent control mRNA transfections. Fig. 2: UTR selection of the second round. Selection of the top 40 mRNAs showing the best performance in A) SkMC, B) iMoDcs donor 1, and C) iMoDcs donor 2. Bars marked with black dots represent the selected mRNA species, and the dotted line represents the baseline to which the data were normalized against control 3. Fig. 3: Identification of 6 top performers using mRNA encoding luciferase in 8 different cell lines. Six UTR combinations showing top performance tested in BJ, iMoDC, SkMC, HEK293T, HepG2, K562, CT26, and TC-1 cell lines. Fig. 4: All six major candidate 5'UTR-3'UTR gene combinations showed higher or equivalent levels in vivo compared to the tested control 5'UTR-3'UTR gene combination ( in vivo ) caused the expression of fireflies luciferase. Results of quantifying whole-body bioluminescence signals over time in BALB / c mice injected with TBS, or hit candidate 5'UTR-3'UTR gene combination firefly luciferase (fLuc) mRNA-LNP or control 5'UTR-3'UTR gene combination fLuc mRNA-LNP. Fig. 5: Ex Vivo expression: In all three mRNA constructs tested (muIL-7, muIL-21, and mu41BBL), both UTR V8 and V10 resulted in significantly higher protein expression compared to the control UTR V5 combination. Fig. 6: In vivo efficacy: The highest efficacy was obtained when using V10 UTR (Fig. 6d) and V8 UTR (Fig. 6c). The control V5 UTR (Fig. 6b) showed significantly lower performance compared to these two UTR combinations of the present invention. TBS was used as a negative example (Fig. 6a). Specific details for implementing the invention

[0035] Detailed description of the invention

[0036] The present invention will now be described in more detail. In the following paragraphs, various aspects of the invention are defined more specifically. Each aspect defined in this way may be combined with any other aspects or aspects unless otherwise explicitly stated. In particular, any feature described as preferred or advantageous may be combined with any other feature or features described as preferred or advantageous.

[0037] The singular forms (“a,” “an,” and “the”) used in this specification and the appended claims include plural objects unless the context clearly indicates otherwise. By way of example, “RNA polynucleotide (a RNA polynucleotide)” means one RNA polynucleotide or one or more RNA polynucleotides. The terms “comprising,” “comprises,” and “comprised of” as used in this specification are synonymous with “including,” “includes,” or “containing,” and “contains,” and have an inclusive or open-ended meaning, so as not to exclude additional members, elements, or method steps not mentioned. References to numeric ranges through both ends include the mentioned ends, including all numbers and fractions contained within each range.

[0038] In this specification, the terms “about” or “approximately” used to refer to measurable values, such as parameters, quantities, and temporal durations, mean that they encompass variations of + / -10% or less of a specific value, preferably + / -5% or less, more preferably + / -1% or less, and even more preferably + / -0.1% or less, and such variations must be appropriate for carrying out the disclosed invention. Furthermore, the value itself referred to by the modifiers “about” or “approximately” should also be understood as specifically and preferably disclosed.

[0039] As with one or more members of a group of members, the term “one or more” is clear in itself, but to give additional examples, this term encompasses references to any one of the members, or any two or more of the members, for example, any three or more, four or more, five or more, six or more, seven or more, etc., and up to all of the members.

[0040] Throughout this specification, references to "one embodiment" or "an embodiment" mean that specific features, structures, or characteristics described in relation to such embodiments are included in at least one embodiment of the present invention. Accordingly, the appearance of phrases such as "in one embodiment" or "in an embodiment" in various places throughout this specification does not necessarily refer to the same embodiment, but may. Furthermore, such specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as will be obvious to a person skilled in the art from this disclosure. Moreover, while some embodiments described in this specification may include some of the features included in other embodiments but exclude others, combining features of different embodiments is also within the scope of the present invention and should be understood as constituting separate embodiments, as will be understood by a person skilled in the art. For example, in the following claims, any of the claimed embodiments may be used in any combination.

[0041] All literature cited in this specification is incorporated herein by reference in its entirety.

[0042] Unless otherwise specified, all terms used to disclose the present invention, including technical and scientific terms, have the meaning generally understood by those skilled in the art to which the present invention pertains. Definitions of terms may be included through additional guidance to better understand the teachings of the present invention.

[0043] To find RNA polynucleotides exhibiting increased stability and / or translation efficiency, the inventors investigated whether specific 5' UTRs and 3' UTRs could influence the expression kinetics of the corresponding RNA polynucleotides. The inventors unexpectedly confirmed that RNA polynucleotides containing specific custom-designed 5' UTR and 3' UTR elements exhibit advantageous effects in terms of stability and translation efficiency. Furthermore, it was additionally confirmed that RNA polynucleotides containing a 5' UTR sequence derived from the 5' UTR of a gene selected from ANXA1, E. coli enolase, RPS14, RPS25, or TCV exhibit particularly superior performance when combined with a 3' UTR sequence derived from the 3' UTR of a gene selected from HBA1, RPS29, Ube2d2a, or UBL5.

[0044] The present invention is particularly suitable for use in pharmaceutical compositions in an isolated state or in the form of lipid nanoparticles, and thus is also suitable for inducing an immune response in subjects. In this context, the RNA polynucleotide according to the present invention is also suitable for use as a vaccine or in gene therapy. The main advantage of using the RNA polynucleotide according to the present invention is that it not only improves the expression and / or translation of gene products, but also, more particularly, improves biodistribution, including the stability and / or duration of gene product expression.

[0045] In the context of the present invention, the term “RNA” refers to a nucleic acid molecule comprising ribonucleotide residues and preferably composed wholly or substantially of ribonucleotide residues. When the term “nucleic acid molecule” is referred to, unless otherwise specified, it means an RNA polynucleotide according to the present invention. In particular, the RNA polynucleotide comprises or is a polymer composed of nucleotide monomers covalently bonded to each other by phosphodiester bonds of a sugar / phosphate backbone.

[0046] "Ribonucleotide" relates to a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. In particular, this term refers to double-stranded RNA, but may also refer to single-stranded RNA, isolated RNA such as partially purified RNA or substantially pure RNA, synthetic RNA, RNA produced by recombinant methods, as well as modified RNA distinguished from naturally occurring RNA through the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material to the end(s) or inside of the RNA, for example, to one or more nucleotides of the RNA. Nucleotides within the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides, chemically synthesized nucleotides, or deoxynucleotides. These modified RNAs may be referred to as analogs or analogs of naturally occurring RNA. In the context of the present invention, where specific sequences are disclosed, they are understood to represent the corresponding RNA sequences in which T is replaced by U.

[0047] According to the present invention, the term "RNA polynucleotide" includes, preferably, "mRNA" meaning "messenger RNA" and is also related to a "transcript" that can be produced using DNA as a template and codes for a peptide or protein. mRNA generally includes a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR).

[0048] In specific embodiments, the RNA polynucleotide of the present invention is mRNA or non-coding RNA, preferably mRNA, antisense RNA, siRNA, sgRNA, guide RNA, or CRISPR. Furthermore, the term "RNA polynucleotide" is not limited to meaning only "one single molecule" and is generally understood to include an assembly of identical molecules. Accordingly, this may relate to a plurality of identical molecules contained in a composition or, for example, lipid nanoparticles.

[0049] To be clear, mRNA encompasses any coding RNA molecule that can be translated into protein by a eukaryotic host. Preferably, mRNA is obtained in vitro using a DNA template ( in vitro ) is generated through transcription. In one embodiment of the present invention, RNA is produced in vitro ( in vitro ) It is obtained through transcription. In a test tube ( in vitroTranscription methods are known to those skilled in the art and may include a purified linear DNA template containing a promoter, a buffer system containing ribonucleotide triphosphate, dithiothreitol (DTT), and magnesium ions, spermidine, and a suitable RNA polymerase such as T7 RNA polymerase. The exact conditions used for the transcription reaction depend on the amount of RNA required for a specific application. Various commercially available in vitro ( in vitro There is a transfer kit.

[0050] Accordingly, the present invention also provides a nucleic acid molecule that can serve as a template for an RNA polynucleotide according to the present invention, which is stabilized and optimized with respect to translation efficiency. In other words, the present invention also relates to an artificial nucleic acid molecule that may be a (modified) RNA or DNA molecule, such as a DNA vector, that can be used for mRNA production. This may be provided as a double-stranded molecule having a sense strand and an antisense strand, for example, as a DNA molecule having a sense strand and an antisense strand. The resulting mRNA can, in turn, be translated to produce a desired peptide or protein encoded by an open reading frame. If the artificial nucleic acid molecule is DNA, this may, for example, be used for continuous and repetitive in vitro ( in vitro ) or in vivo( in vivo It can be used as a double-stranded storage form for production.

[0051] In the context of the present invention, the term "5'-untranslated region (5' UTR)" is understood as a specific segment of mRNA located at the 5' of the protein-coding region (i.e., the open reading frame; ORF) of the mRNA. Generally, the 5' UTR starts at the transcription initiation site and ends at the nucleotide immediately preceding the start codon of the open reading frame. The 5' UTR may include elements that regulate gene expression, also referred to as regulatory elements. Such regulatory elements may be, for example, promoters, enhancers, silencers, insulators, ribosome binding sites, and 5'-terminal oligopyrimidine tracts, but are not limited thereto. The 5' UTR may undergo post-transcriptional modification, for example, by the addition of a 5'-cap. In the context of the present invention, the 5' UTR corresponds to the sequence of mature mRNA located between the 5'-cap and the start codon. In the context of the present invention, the term "5' UTR of a gene," such as "5' UTR of the ANXA1 gene," refers to the sequence corresponding to the 5' UTR of mature mRNA derived from the gene, that is, mRNA obtained through the transcription of the gene and the maturation of immature mRNA. The term "5' UTR of a gene" encompasses the DNA sequence and RNA sequence of the 5' UTR.

[0052] In the context of the present invention, the term "3' untranslated region (3' UTR)" should be understood as a part of the mRNA located between the protein-coding region (i.e., ORF) of the mRNA and the poly(A) sequence. The 3' UTR of the mRNA is not translated into an amino acid sequence. The 3' UTR sequence is generally coded by the gene that is transcribed into the corresponding mRNA during the process of gene expression. The genome sequence is first transcribed into pre-mature mRNA, which contains any introns. Subsequently, the pre-mature mRNA undergoes a maturation process and is further processed into mature mRNA. In the context of the present invention, the 3' UTR corresponds to the sequence of the mature mRNA located at the 3' of the stop codon of the protein-coding region. In the context of the present invention, the term "3' UTR of a gene," such as "3' UTR of the HBA1 gene," refers to the sequence corresponding to the 3' UTR of the mature mRNA derived from the corresponding gene, that is, the mRNA obtained through the transcription of the gene and the maturation of the pre-mature mRNA. The term "3' UTR of a gene" encompasses the DNA sequence and RNA sequence of the 3' UTR.

[0053] As used herein, the term “open reading frame (ORF)” may be a sequence of nucleotide triplets that can typically be translated into a peptide or protein. An open reading frame preferably includes a start codon, i.e., a combination of three consecutive nucleotides (ATG or AUG) that typically codes for the amino acid methionine, at its 5’ end and in a subsequent region that is typically a multiple of three nucleotides in length. The ORF is preferably terminated by a stop codon (e.g., TAA, TAG, TGA). Typically, this is the only stop codon within the open reading frame. An open reading frame may be separate or incorporated within a longer nucleic acid sequence, e.g., an RNA polynucleotide. An open reading frame may also be referred to as a “protein coding region.” Additionally, an open reading frame may be at least partially codon-optimized. Codon optimization is based on the fact that translation efficiency can be determined by differences in the frequency of presence of transfer RNA (tRNA) within a cell, a technique known to ordinary technicians in the field of mRNA manufacturing.

[0054] Accordingly, the present invention provides an RNA polynucleotide comprising the following:

[0055] i) at least one 5' untranslated region (5'UTR) element comprising a 5' UTR of a gene selected from ANXA1, E. coli enolase, RPS14, RPS25, or TCV, or a nucleic acid sequence derived from a fragment or variant thereof;

[0056] ii) at least one open reading frame (ORF) encoding a polypeptide;

[0057] iii) at least one 3' untranslated region (3'UTR) element comprising a nucleic acid sequence derived from a 3' UTR of a gene selected from HBA1, RPS29, Ube2d2a, or UBL5, or a fragment or variant thereof.

[0058] RNA polynucleotides having at least one of these 5' UTR and 3' UTR combinations were found to exhibit improved expression and biodistribution compared to the control group.

[0059] Preferably, the at least one 5' UTR element and the at least one 3' UTR element are functionally linked to the ORF. This means that the 5' UTR element and the 3' UTR element are preferably associated with the ORF to exert functions such as a stabilizing function for the expression of the ORF, a function to increase the production of the protein encoded by the ORF, or a stabilizing function for the RNA polynucleotide, preferably in an additive manner, and more preferably in a synergistic manner. Preferably, the 5' UTR element, the ORF, and the 3' UTR element are associated in the 5' to 3' direction. Thus, preferably, the RNA polynucleotide comprises a structure of 5'-5' UTR element-(optional) linker-ORF-(optional) linker-3' UTR element-3', wherein the linker may be present or absent. For example, the linker may be a sequence composed of one or more nucleotides, e.g., 1-50 or 1-20 nucleotides.

[0060] In a specific embodiment, the present invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element comprising a nucleic acid sequence derived from a 5' UTR of the ANXA1 gene and at least one 3' UTR element comprising a nucleic acid sequence derived from a 3' UTR of a gene selected from HBA1, RPS29, Ube2d2a, or UBL5.

[0061] In another specific embodiment, the present invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element comprising a nucleic acid sequence derived from the 5' UTR of an Escherichia coli (E. coli) enolase gene and at least one 3' UTR element comprising a nucleic acid sequence derived from the 3' UTR of a gene selected from HBA1, RPS29, Ube2d2a, or UBL5.

[0062] In yet another embodiment, the present invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element comprising a nucleic acid sequence derived from the 5' UTR of the RPS14 gene and at least one 3' UTR element comprising a nucleic acid sequence derived from the 3' UTR of a gene selected from HBA1, RPS29, Ube2d2a, or UBL5.

[0063] In a specific embodiment, the present invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element comprising a nucleic acid sequence derived from the 5' UTR of the RPS25 gene and at least one 3' UTR element comprising a nucleic acid sequence derived from the 3' UTR of a gene selected from HBA1, RPS29, Ube2d2a, or UBL5.

[0064] In an additional embodiment, the present invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element comprising a nucleic acid sequence derived from the 5' UTR of a TCV gene and at least one 3' UTR element comprising a nucleic acid sequence derived from the 3' UTR of a gene selected from HBA1, RPS29, Ube2d2a, or UBL5.

[0065] As previously explained, embodiments of the present invention also include fragments and variants of the gene. In the context of the present invention, the term “fragment thereof” encompasses the entire 5’ UTR or 3’ UTR sequence of the gene, i.e., the sequence corresponding to the full length 5’ UTR or 3’ UTR sequence, and the sequence corresponding to the fragment of the 5’ UTR or 3’ UTR sequence of the gene. Preferably, the 5’ UTR or 3’ UTR fragment of the gene comprises a continuous nucleotide stretch corresponding to a continuous nucleotide stretch within the full length 5’ UTR or 3’ UTR of the gene, which accounts for at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full length 5’ UTR or 3’ UTR of the gene. In the sense of the present invention, such fragments are preferably functional fragments as described in this specification.

[0066] In the context of the present invention, the term “variant thereof” refers to a variant of a 5’ UTR or 3’ UTR of a naturally occurring gene, preferably a variant of a 5’ UTR or 3’ UTR of a vertebrate gene, preferably a variant of a 5’ UTR or 3’ UTR of a mammalian gene, more preferably a variant of a 5’ UTR or 3’ UTR of a human or mouse gene. Such variants may be modified 5’ UTRs or 3’ UTRs of the gene. For example, a variant 5’ UTR or 3’ UTR may exhibit one or more nucleotide deletions, insertions, additions, and / or substitutions compared to the naturally occurring 5’ UTR or 3’ UTR from which the variant is derived. Preferably, a variant of the 5' UTR or 3' UTR of a gene has at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% identity with the naturally occurring 5' UTR or 3' UTR from which the variant is derived. Preferably, the variant is a functional variant as described herein. As used herein, the term "variant" also encompasses the term "homologs," which should be understood as sequences corresponding to the same gene but derived from different species.

[0067] Additionally, in each embodiment, the RNA polynucleotide may comprise one or more 5' UTR elements or 3' UTR elements as described above. For example, an RNA polynucleotide molecule according to the present invention may comprise one, two, three, four, or more 5' UTR elements and / or one, two, three, four, or more 3' UTR elements, wherein the individual 5' UTR or 3' UTR elements may be identical to each other or may be different. For example, the RNA polynucleotide according to the present invention may comprise a combination of two substantially identical 5' UTR elements, for example, two 5' UTR elements comprising or composed of nucleic acid sequences derived from the Escherichia coli (E. coli) enolase gene, or a fragment or variant thereof, and two 3' UTR elements comprising or composed of nucleic acid sequences derived from the HBA1 gene, or a fragment or variant thereof.

[0068] As previously defined, the 5' UTR of the ANXA1, E. coli enolase, RPS14, RPS25, or TCV gene corresponds to the 5' UTR sequence of the mature mRNA derived from the respective ANXA1, E. coli enolase, RPS14, RPS25, or TCV gene, which preferably extends from the nucleotide located at 3' of the 5' cap to the nucleotide located at 5' of the start codon. The length of the 5' UTR of said genes may vary within the range of 10 to a maximum of 500 nucleotides, generally less than about 300 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 150 nucleotides. Within the meaning of the present invention, exemplary 5' UTRs of said genes are nucleic acid sequences according to SEQ ID NOs. 1 to 5, or fragments or variants thereof. Preferably, within the meaning of the present invention, the 5' UTR element codes for a nucleotide sequence that functions as a 5' UTR or performs the function of a 5' UTR. The 5' UTR element is preferably suitable for increasing protein production from artificial RNA polynucleotides.

[0069] Similarly, the 3' UTR of the HBA1, RPS29, Ube2d2a, or UBL5 gene corresponds to the 3' UTR sequence of the mature mRNA derived from the respective HBA1, RPS29, Ube2d2a, or UBL5 gene. The 3' UTR length of the said genes may vary from 10 nucleotides to a maximum of 500 nucleotides, and is generally less than about 300 nucleotides, preferably less than about 200 nucleotides, and more preferably less than about 150 nucleotides. Within the meaning of the present invention, the exemplary 3' UTR of said genes is a nucleic acid sequence according to SEQ ID NOs. 6 to 9, or a fragment or variant thereof. Preferably, within the meaning of the present invention, the 3' UTR element codes for a nucleotide sequence that functions as a 3' UTR or performs the function of a 3' UTR. The said 3' UTR element is preferably suitable for improving the stability of the mRNA molecule.

[0070] In a specific embodiment, the present invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element comprising an RNA sequence corresponding to a DNA sequence selected from SEQ ID NO. 1 [ANXA1], or a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith, and at least one 3' UTR element comprising a nucleic acid sequence selected from SEQ ID NO. 6 [HBA1], SEQ ID NO. 18 [HBA1], SEQ ID NO. 7 [RPS29], SEQ ID NO. 8 [Ube2d2a], or SEQ ID NO. 9 [UBL5], or a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

[0071] In a specific embodiment, the present invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element comprising an RNA sequence corresponding to a DNA sequence selected from SEQ ID NO. 2 [E. coli enolase gene], or a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith, and at least one 3' UTR element comprising a nucleic acid sequence selected from SEQ ID NO. 6 [HBA1], SEQ ID NO. 18 [HBA1], SEQ ID NO. 7 [RPS29], SEQ ID NO. 8 [Ube2d2a], or SEQ ID NO. 9 [UBL5], or a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

[0072] In a specific embodiment, the present invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element comprising an RNA sequence corresponding to a DNA sequence selected from SEQ ID NO. 3 [RPS14], or a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith, and at least one 3' UTR element comprising a nucleic acid sequence selected from SEQ ID NO. 6 [HBA1], SEQ ID NO. 18 [HBA1], SEQ ID NO. 7 [RPS29], SEQ ID NO. 8 [Ube2d2a], or SEQ ID NO. 9 [UBL5], or a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

[0073] In a specific embodiment, the present invention relates to an RNA polynucleotide comprising at least one 5' UTR element comprising an RNA sequence corresponding to a DNA sequence selected from SEQ ID NO. 4 [RPS25], or a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith, and at least one 3' UTR element comprising a nucleic acid sequence selected from SEQ ID NO. 6 [HBA1], SEQ ID NO. 18 [HBA1], SEQ ID NO. 7 [RPS29], SEQ ID NO. 8 [Ube2d2a], or SEQ ID NO. 9 [UBL5], or a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

[0074] In a specific embodiment, the present invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element comprising an RNA sequence corresponding to a DNA sequence selected from SEQ ID NO. 5 [TCV], or a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith, and at least one 3' UTR element comprising a nucleic acid sequence selected from SEQ ID NO. 6 [HBA1], SEQ ID NO. 18 [HBA1], SEQ ID NO. 7 [RPS29], SEQ ID NO. 8 [Ube2d2a], or SEQ ID NO. 9 [UBL5], or a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

[0075] In a specific embodiment, the present invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element comprising an RNA sequence corresponding to a DNA sequence selected from SEQ ID NO. 1 [ANXA1], SEQ ID NO. 2 [E. Coli enolase gene], SEQ ID NO. 3 [RPS14], SEQ ID NO. 4 [RPS25], and SEQ ID NO. 5 [TCV], or a sequence having at least 95%, 96%, 97%, 98%, and 99% sequence identity therewith, and at least one 3' UTR element comprising an RNA sequence corresponding to a DNA sequence selected from SEQ ID NO. 6 [HBA1], SEQ ID NO. 18 [HBA1], SEQ ID NO. 7 [RPS29], SEQ ID NO. 8 [Ube2d2a], or SEQ ID NO. 9 [UBL5], or a sequence having at least 95%, 96%, 97%, 98%, and 99% sequence identity therewith.

[0076] Accordingly, a nucleic acid sequence derived from the 5' UTR or 3' UTR of a gene is a eukaryotic gene, preferably a plant or animal gene, more preferably a chordate gene, even more preferably a vertebrate gene, most preferably a mammalian gene, e.g., a human or mouse gene ( For example, Table 1It is derived from ). For example, the 5' UTR or 3' UTR element comprises or is composed of a nucleic acid sequence selected from the group consisting of each SEQ ID NO. 1 to 5 or SEQ ID NO. 6 to 9, 18 (e.g., an RNA sequence corresponding to the corresponding DNA sequence), a fragment or variant thereof, or a nucleic acid sequence derived from a homolog of SEQ ID NO. 1 to 5 or SEQ ID NO. 6 to 9, 18. In the context of the present invention, the term “homologs” means a sequence of another species having homology to the sequence according to SEQ ID NO. 1 to 5 or SEQ ID NO. 6 to 9, 18, for example, a species other than Homo sapiens (human) or Mus musculus (mouse). For example, SEQ ID NO. 1 is associated with a sequence containing the 5' UTR of human Annexin A1 (ANXA). In the context of the present invention, a homolog of SEQ ID NO. 1 is any sequence derived from an ANXA gene of another species other than humans, e.g., any vertebrate or plant gene, preferably any mammalian ANXA gene other than the human ANXA gene, e.g., mouse, rat, rabbit, monkey, etc.

[0077] In a specific embodiment, the 5' UTR element and the 3' UTR element are heterologous, for example, preferably the 5' UTR element and the 3' UTR element are derived from different genes of the same or different species. In a preferred embodiment, the 5' UTR element and the 3' UTR element are selected to exert at least an additive, preferably synergistic, function for protein production from the ORF of the RNA polynucleotide molecule. Preferably, protein production is increased at least additively, preferably synergistically, by the 5' UTR element and the 3' UTR element. Thus, after the initiation of ORF expression, for example, after transfection of a test cell or cell line, the protein amount of the protein encoded by the ORF at a specific time point, such as a reporter protein like luciferase, is at least equal to, or preferably greater than, the amount expected when the protein production-increasing effect of the 5' UTR element and the 3' UTR element is purely additive. These additive effects, preferably synergistic effects, can be measured, for example, through a luminescence assay.

[0078] Preferably, the at least one 5' UTR element and the at least one 3' UTR element act synergistically to stabilize and / or increase protein production from the RNA polynucleotide according to the present invention, for example, the mRNA according to the present invention, as described above. In the context of the present invention, the term "stabilizing and / or increase protein production" refers to reference or control 5' UTR and 3' UTR elements ( For example, Table 2 This means that compared to protein production from a reference mRNA containing ), protein production from the corresponding mRNA is stabilized and / or increased.

[0079] In this specification, it has been revealed that an RNA polynucleotide comprising a 5'UTR and 3'UTR RNA sequence fragment or a variant thereof corresponding to a DNA sequence selected from any one of the following combinations is particularly suitable for increasing expression in various cell types compared to a reference RNA polynucleotide:

[0080] - A combination of ANXA1's 5'UTR and HBA1's 3'UTR; or

[0081] - A combination of the eno's 5'UTR and the RPS29's 3'UTR; or

[0082] - A combination of eno's 5'UTR and Ube2d2a's 3'UTR; or

[0083] - A combination of the 5'UTR of RPS14 and the 3'UTR of HBA1; or

[0084] - A combination of the 5'UTR of RPS25 and the 3'UTR of Ube2d2a; or

[0085] - A combination of the 5'UTR of TCV and the 3'UTR of UBL5. Because these RNA polynucleotide molecules can increase or prolong the expression of proteins encoded by the open reading frame of the RNA polynucleotide, they are particularly useful for mRNA-based therapies, gene therapy, and / or gene vaccination.

[0086] Particularly desirable combinations are the combination of the 5'UTR of ANXA1 and the 3'UTR of HBA1, and the combination of the 5'UTR of -RPS14 and the 3'UTR of HBA1.

[0087] More specifically, these advantageous effects were particularly pronounced when using an RNA polynucleotide containing a 5' UTR and a 3' UTR corresponding to a DNA sequence selected from any one of the following combinations, an RNA sequence, or a sequence having at least 95% sequence identity with it:

[0088] - A combination of SEQ ID NO. 1 [ANXA1] for the 5'UTR and SEQ ID NO. 6 [HBA1] for the 3'UTR; or

[0089] - A combination of SEQ ID NO. 2[eno] for the 5'UTR and SEQ ID NO. 7[RPS29] for the 3'UTR; or

[0090] - A combination of SEQ ID NO. 2[eno] for the 5'UTR and SEQ ID NO. 8[Ube2d2a] for the 3'UTR; or

[0091] - A combination of SEQ ID NO. 3 [RPS14] for the 5'UTR and SEQ ID NO. 6 [HBA1] for the 3'UTR; or

[0092] - A combination of SEQ ID NO. 3 [RPS25] for the 5'UTR and SEQ ID NO. 8 [Ube2d2a] for the 3'UTR; or

[0093] - A combination of SEQ ID NO. 1 [ANXA1] for the 5'UTR and SEQ ID NO. 18 [HBA1] for the 3'UTR; or

[0094] - A combination of SEQ ID NO. 3 [RPS14] for the 5'UTR and SEQ ID NO. 18 [HBA1] for the 3'UTR; or

[0095] - A combination of sequence number 5 [TCV] for the 5'UTR and sequence number 9 [UBL] for the 3'UTR.

[0096] Particularly desirable combinations are the combination of sequence number 1 [ANXA1] for the 5'UTR and sequence number 6 [HBA1] for the 3'UTR; and the combination of sequence number 3 [RPS14] for the 5'UTR and sequence number 6 [HBA1] for the 3'UTR.

[0097] Alternative desirable combinations are the combination of sequence number 1 [ANXA1] for the 5'UTR and sequence number 18 [HBA1] for the 3'UTR; and the combination of sequence number 3 [RPS14] for the 5'UTR and sequence number 18 [HBA1] for the 3'UTR.

[0098] In a very specific embodiment, the present invention provides one or more mRNA molecules encoding IL-7, IL-21 and / or 4-1BBL, comprising a combination of UTRs selected from the following:

[0099] RPS14 5'UTR - HBA1 3'UTR - Specifically, Sequence No. 3[RPS14] - Sequence No. 18[HBA1]

[0100] ANXA1 5'UTR - HBA1 3'UTR - Specifically, Sequence No. 1 [ANXA1] - Sequence No. 18 [HBA1]

[0101] A particularly suitable RNA polynucleotide is an RNA polynucleotide comprising at least one 5' UTR element containing a nucleic acid sequence derived from the 5' UTR of the E. coli enolase gene or the ANXA gene.

[0102] In addition, as previously explained, the combination of at least one 5' UTR element and at least one 3' UTR element acts synergistically in increasing the biodistribution of the RNA polynucleotide according to the present invention, for example, the mRNA according to the present invention. In the context of the present invention, the term "biodistribution" refers to a reference or control mRNA, for example, an mRNA comprising reference 5' UTR and 3' UTR elements (e.g., Table 2 This means that compared to the biodistribution of the control RNA polynucleotide containing 5' UTR / 3' UTR described in [reference], the delivery of mRNA or lipid nanoparticles containing said mRNA into the body, organs, or tissues and the accumulation thereof are increased.

[0103] More specifically, the RNA polynucleotide according to the present invention exhibits more uniform protein production and biodistribution over a predetermined time period, preferably, for example, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, preferably 12 hours or more, more preferably 24 hours or more, even more preferably 48 hours or more, most preferably 72 hours or more, when compared to a reference RNA polynucleotide molecule. Accordingly, the level of protein production from the RNA polynucleotide according to the present invention, for example in a mammalian system, does not drop as sharply as the level observed for a reference RNA polynucleotide, such as the reference mRNA described above. For example, the amount of protein (coded by the ORF) observed at 5 hours after the onset of expression may be equivalent to the amount of protein observed after 48 hours after the onset of expression, for example, after 48 hours following transfection. In particular, bioavailability was found to improve between 5 and 24 hours and decrease slightly after 48 and 72 hours, but remained at an overall higher level compared to the reference RNA polynucleotide.

[0104] The increase in stability of the RNA polynucleotide, the increase in protein production stability, the extension of the protein production duration, the increase in protein production yield, and / or the increase in biodistribution are preferably achieved by reference 5' UTR and 3' UTR elements, for example, a 5' UTR element comprising an RNA sequence corresponding to a DNA sequence selected from SEQ ID NOs 10 to 13, or a 3' UTR element comprising an RNA sequence corresponding to a DNA sequence selected from SEQ ID NOs 14 to 17 ( Table 2 It is determined by comparison with each reference RNA polynucleotide containing (see reference).

[0105] In the context of the present invention, 'increased protein expression' may refer to an increase in protein expression at a specific point in time after the onset of expression or an increase in total protein production within a certain period after the onset of expression compared to a control molecule. Accordingly, the protein level observed at a specific point in time after the onset of expression, for example, after transfection, for example, after 2, 3, 4, 5, 7, 8, 12, 24, 48, or 72 hours after transfection of the RNA polynucleotide according to the present invention, or the total protein produced within a specific period, for example, 24, 48, or 72 hours, is preferably higher than the protein level observed at the same point in time after the onset of expression, for example, after transfection, or the total protein produced within the same period for a reference RNA polynucleotide, such as a reference mRNA containing a reference 5' and / or reference 3' UTR. As described above, influencing the increase in protein production from RNA polynucleotides is a particularly desirable function of the 5' UTR element. Preferably, compared to an RNA polynucleotide containing a reference 5' UTR and 3' UTR, the increase in protein production influenced by the 5' UTR element at a given time after the initiation of expression is at least 1.5 times, more preferably at least 2 times, more preferably at least 3 times, even more preferably at least 4 times, and most preferably at least 5 times.

[0106] Furthermore, it is desirable that the 3' UTR element and the 5' UTR element exhibit at least an additive effect, preferably a synergistic effect, on the total protein production from the corresponding artificial nucleic acid molecule within a specific period after the initiation of expression, e.g., 24 hours, 48 ​​hours, or 72 hours.

[0107] Accordingly, the present invention also relates to the use of RNA polynucleotides or pharmaceutical compositions according to the present invention for improving the expression and / or translation of a gene product, particularly for improving translation initiation. More specifically, said RNA polynucleotides or said pharmaceutical compositions are provided for use in improving the stability and / or duration of gene product expression, particularly for improving biodistribution.

[0108] According to another embodiment of the present invention, the 5' UTR of the RNA polynucleotide further comprises a KOZAK sequence and / or an internal ribosome entry site (IRES). In the context of the present invention, the term "KOZAK sequence" refers to a nucleic acid motif that functions as a protein translation initiation site and mediates ribosome assembly to ensure that an accurate protein sequence is translated. In the context of the present invention, the term "internal ribosome entry site (IRES)" should be understood as a sequence or motif that separates multiple open reading frames, for example, when the RNA polynucleotide codes for two or more peptides or proteins. The IRES sequence may be particularly useful when the corresponding mRNA is bi-cistronic or multi-cistronic RNA.

[0109] Additionally, the RNA polynucleotide may further include additional elements known to a person skilled in the art of mRNA manufacturing, for example, a 5'-cap and a poly(A) tail. An optional 5'-cap is preferably attached to the 5' of a 5' UTR element. The 5'-cap may be added to the 5' end of the RNA after transcription. Preferably, an optional poly(A) sequence is located at the 3' of an ORF or at least one 3' UTR element and is preferably linked to the 3' end of the ORF or 3' UTR element. This linkage may be direct or indirect and may be linked, for example, through a nucleotide stretch of 2, 4, 6, 8, 10, 20, etc., e.g., 1 to 50, preferably 1 to 20 nucleotides, for example, through a linker comprising or composed of one or more restriction enzyme recognition sites. In one embodiment, the selective polyadenylation signal is located within the 3' UTR element. The length of the poly(A) sequence may vary. For example, the poly(A) sequence may have a length of about 20 adenine nucleotides to a maximum of about 300 adenine nucleotides, preferably about 40 to about 200 adenine nucleotides, more preferably about 50 to about 100 adenine nucleotides, e.g., about 60, 70, 80, 90, or 100 adenine nucleotides.

[0110] Furthermore, the RNA polynucleotide according to the present invention may comprise one or more typical mRNA modifications, referred herein as 'modified mRNA nucleosides,' which are understood to comprise one or more modified nucleosides exhibiting useful properties such as a substantial lack of induction of the innate immune response of cells into which the mRNA is introduced. These modified nucleic acids not only improve protein production efficiency, intracellular retention of nucleic acids, and viability of contacted cells, but also exhibit reduced immunogenicity.

[0111] In some embodiments, the modified nucleobase in the nucleic acid (e.g., RNA nucleic acid such as mRNA nucleic acid) comprises 1-methyl-pseudouridine (m1 y), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (y). In some embodiments, the modified nucleobase in the nucleic acid (e.g., RNA nucleic acid such as mRNA nucleic acid) comprises 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine, and / or 5-methoxycytidine. In some embodiments, the polyribonucleotide comprises a combination of at least two (e.g., two, three, four, or more) of the above-described modified nucleobases, including but not limited to chemical modifications.

[0112] In another embodiment, the present invention relates to a pharmaceutical composition comprising one or more RNA polynucleotides according to the present invention and at least one pharmaceutically acceptable formulation.

[0113] In the context of the present invention, the term “pharmaceutical composition” means a composition having pharmaceutical properties. That is, it refers to a composition that provides pharmacological and / or physiological effects. A pharmaceutical composition may include one or more pharmaceutically acceptable agents, such as excipients, carriers, and diluents. These may be in the form of a solution, suspension, liquid, or aqueous formulation, or any combination thereof. In some embodiments, pharmaceutically acceptable agents include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents for obtaining a composition available in a dosage form. Additional suitable pharmaceutical carriers and diluents, and the pharmaceutical details required for their use, are described in the literature [Remington's Pharmaceutical Sciences]. Unless otherwise specified in this specification, the term “excipient” should be understood to mean any substance formulated together with the active compound for the purpose of long-term stabilization, such as preventing denaturation or aggregation over the expected shelf life; for the purpose of bulking a liquid or solid formulation containing a small amount of potent active compound (hence often referred to as “bulking agents,” “fillers,” or “diluents”); or for the purpose of imparting an improvement to the active compound in the final dosage form, such as promoting absorption, reducing viscosity, or improving solubility.

[0114] In another embodiment, the present invention provides an RNA polypeptide according to the present invention, formulated as a liposome or nanoparticle, e.g., lipid nanoparticle or polymer nanoparticle; in particular, as a lipid nanoparticle. The RNA polypeptide defined herein may be formulated as a lipid nanoparticle (LNP) that encapsulates the construct to protect the construct from degradation and to promote cellular uptake. In the context of the present invention, the term “lipid nanoparticle” or LNP refers to a nanosized particle composed of one or more lipids, e.g., a combination of different lipids. The lipids that may be used in the LNP may be, for example, at least one phospholipid, at least one modified lipid, e.g., PEG lipid, at least one ionizable lipid, and at least one sterol. The lipid nanoparticles of the present disclosure and their compositions are generally known in the art.

[0115] In particular, as defined herein, the term “pharmaceutical composition” is used in the context of referring to a form in which an LNP comprising the RNA polynucleotide of the present invention is combined with at least one pharmaceutically acceptable carrier or excipient. Meanwhile, the term “pharmaceutical formulation” may refer to the RNA polynucleotide of the present invention suspended in a buffer solution and at least one pharmaceutically acceptable carrier or excipient (i.e., not encapsulated in an LNP). Such combination may be formulated, for example, into an LNP and is used particularly for the purpose of extending the stability of the nucleic acid and / or improving delivery.

[0116] The LNP may comprise one or more single-type RNA polynucleotides (i.e., RNA polynucleotides having the same 5' UTR and 3' UTR combination), or may comprise two or more RNA polynucleotides having similar or different types of 5' UTR-3' UTR combinations. In some embodiments, two or more different RNA polynucleotides are formulated within the same lipid nanoparticle.

[0117] In a further aspect, the present invention provides RNA polynucleotides or pharmaceutical compositions according to the present invention for use in human or animal medical applications. Such pharmaceutical compositions are particularly suitable for vaccines or gene therapy. Vaccines may be for prophylactic purposes (e.g., to prevent or mitigate the effects of future infections caused by any natural or 'wild' pathogens) or for therapeutic purposes (e.g., to actively treat or alleviate the symptoms of an ongoing disease). The administration of a vaccine is referred to as vaccination. Finally, the present invention provides a method for preventing and treating diseases in humans and animals by administering a pharmaceutical composition or a vaccine to a subject in need.

[0118] In the context of the present invention, the term “gene therapy” should be understood as treatment of a patient’s body or isolated elements of the patient’s body, e.g., isolated tissues / cells, by means of a nucleic acid encoding a peptide or protein, preferably an RNA polynucleotide according to the present invention. In the context of the present invention, the term “genetic vaccination” may generally be understood as vaccination through the administration of a nucleic acid molecule encoding an antigen, an immunogen, or a fragment thereof. When transfected into specific cells of the body or isolated cells, the antigen or immunogen is expressed by these cells and subsequently presented to the immune system, which can induce an adaptive immune response, i.e., an antigen-specific immune response.

[0119] Therefore, gene therapy and gene vaccination generally involve a) administering the RNA polynucleotide or pharmaceutical composition defined herein—through any route of administration—directly to a subject or in vitro ( in vitro Administered to isolated cells / tissues of the subject in ) and in vivo ( in vivo ) / Extracellular( ex vivo ) or in a test tube ( in vitro The method may include at least one of the following steps: a) causing transfection of the target's cells; b) transcription and / or translation of the introduced nucleic acid molecule; and optionally c) isolation and re-administration of the transfected cells to the target if the nucleic acid is not directly administered to the target.

[0120] In another aspect, the present invention relates to a method for inducing an improved immune response in a subject, comprising administering an RNA polynucleotide or a pharmaceutical composition according to the present invention in a therapeutically effective amount. The term “immune response” as used throughout this specification is not intended to be limited to the types of immune responses exemplified herein, such as specific immune responses to disease-associated antigens or cells expressing disease-associated antigens. Accordingly, the term encompasses an immune response to all infectious agents for which vaccination may be beneficial to the subject.

[0121] In the context of the present invention, the term 'therapeutically effective amount' should be understood as an amount sufficient to induce a pharmaceutical effect, such as an immune response, a change in the pathological level of an expressed peptide or protein, or, for example, the replacement of a deficient gene product in the case of a pathological situation.

[0122] The present invention also provides a method for treating or preventing a disease or disorder as described above, comprising administering an RNA polynucleotide according to the present invention or a pharmaceutical composition according to the present invention to a subject in need thereof.

[0123] In the context of this application, the terms “treatment,” “treating,” “treat,” and similar terms refer to obtaining a desired pharmacological and / or physiological effect. Such effect may be preventive in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it provides partial or complete stabilization or healing of the disease and / or harmful effects attributable to the disease. “Treatment” encompasses any treatment for a disease in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease or symptoms in subjects who may be predisposed to the disease or symptoms but have not yet been diagnosed with them; (b) suppressing the symptoms of the disease, i.e., halting the progression of the disease; or (c) alleviating the symptoms of the disease, i.e., causing the regression of the disease or symptoms.

[0124] The RNA polynucleotide or pharmaceutical composition according to the present invention may be administered orally, parenterally, via inhalation spray, topically, rectally, nasally, orally, vaginally, or via an implanted reservoir. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intramedullary, intrahepatic, intralesional, intracranial, percutaneous, intradermal, intrapulmonary, intraperitoneal, intracardiac, intra-arterial, and sublingual injection or infusion techniques.

[0125] Examples

[0126] Example 1: In vitro screening

[0127] Materials and Methods

[0128] iMoDC differentiation and transfection

[0129] Immature human monocyte-derived dendritic cells (Mo-iDC) were differentiated from monocytes enriched by positive selection using CD14+ beads (StemCell Technologies, EasySep Human CD14 Positive Selection Kit II, Cat. #17858) in buffy coats obtained from two different donors. Subsequently, CD14+ cells were cultured for 7 days in X-VIVO15 medium (VWR, Cat. #LONZBE02-060F) supplemented with 250 U / ml IL-4 (Miltenyi Biotec, Cat. #130-093-922), 800 U / ml Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (Miltenyi Biotec, Cat. #130-093-867), and 1% human AB serum (Merck Life Science, Cat. #H4522-100ML), with fresh cytokines added twice during the differentiation period. On day 7, cells were harvested using phosphate-buffered saline (PBS) / EDTA (Fisher Scientific, Catalog #15433589) and frozen in Cryostor CS10 medium (StemCell Technologies, Catalog #7930). For UTR screening, cells from two donors were thawed and pooled, and 400 UTR mRNAs were transfected in double replicates using Lipofectamine MessengerMAX Transfection Reagent (Thermo Fisher Scientific, catalog #LMRNA008) according to the manufacturer's instructions (100 ng / well, 100 μL, ratio 1:3) in 96-well plates containing 100 x 10 e 3 iMoDCs / well. To ensure the same transfection efficiency in all wells, target Nanoluc mRNA with different UTRs and 10% mCitrine mRNA were co-encapsulated.Cell viability was measured using the CellTiter-Fluor Cell Viability Assay (Promega, catalog number G6081). Viability and transfection efficiency (Teff) were quantified after 24 hours, at which point Nanoluc secretion levels were measured at 6, 24, 48, 72, and 96 hours. Subsequently, the area under the Nanoluc secretion curve (AUC) was normalized against transfection efficiency and cell density, and then the top 40 (top 10%) mRNAs with superior performance were down-selected for a round of screening targeting another iMoDC donor and human skeletal muscle cells. In the second round of screening, two different iMoDC donors were evaluated for the expression of the top 40 UTRs using redouts similar to those in the first round of screening. In the validation experiment, cells were transfected with Fluc, and redouts were performed 24 hours after transfection.

[0130] SkMC culture, differentiation, and transfection

[0131] Human skeletal muscle cells (SkMCs) were cultured according to the manufacturer's instructions (Promo Cell, Catalog No. C-12530). In summary, cells were subcultured in skeletal muscle cell growth medium (Bio-connect, Catalog # C-23060). For differentiation, cells were cultured in the skeletal muscle cell growth medium until they reached a confluence of approximately 60–80%. Subsequently, the growth medium was replaced with skeletal muscle cell differentiation medium (Bio-connect, Catalog # C-23061). After incubation in the skeletal muscle cell differentiation medium for 5 days, these cells were used to transfect 40 UTRs exhibiting peak performance, formulated with Lipofectamine MessengerMAX Transfection Reagent (Thermo Fisher Scientific, Catalog # LMRNA008), according to the manufacturer's instructions (50 ng / well, 1:3 ratio in 100 μL). As with the selection in the first round, transfection efficiency and cell viability were tracked, and Nanoluc expression was collected at 6, 24, 48, 72, and 96 hours. In the validation experiment, cells were transfected with Fluc, and a full readout was performed after 24 hours.

[0132] Cancer cell line culture and transfection

[0133] Four human cell lines of different origins (HeP2G - liver cancer cell line (ATCC, catalog # HB-8065), K562 - leukemia cell line (ATCC, catalog # CCL-243), BJ - fibroblast (ATCC, catalog # CRL-2522), HEK293 - human embryonic kidney immortalized cell line (ATCC, catalog # CRL-1573)) and two mouse cell lines (CT26 - colorectal carcinoma (ATCC, catalog # CRL-2638), TC-1 - immortalized lung epithelial cell line (kindly provided by Thorbald Van Hall Laboratory, University of Leiden)) were cultured according to the manufacturer's instructions. Cells were transfected with mRNA encoding firefly luciferase (Fluc) at two different doses (100 ng and 200 ng / well) using Lipofectamine MessengerMAX Transfection Reagent (Thermo Fisher Scientific, catalog # LMRNA008). In the validation experiment, only the 24-hour time point was evaluated.

[0134] Data analysis

[0135] In all experiments, Nanoluc values ​​were normalized with respect to transfection efficiency, and in some experiments, cell confluency was also considered. Subsequently, the area under the curve (AUC) over all time points was evaluated using GraphPad Prism (version 10.0.2(232)). Statistical significance was assessed via one-way ANOVA, provided all assumptions were met, followed by a post-HOC Tukey test to adjust for multiple comparisons. For AUC, the standard error and degrees of freedom +1 were used for statistical evaluation.

[0136] result:

[0137] Screening of the first round (Fig. 1).

[0138] To select 5-prime and 3-prime UTR combinations that may exhibit higher mRNA expression and longer intracellular persistence compared to control UTRs (see Table 2, referred to as control UTRs hereinafter), the inventors constructed mRNA libraries by combining 21 (X) 5-prime UTRs and 21 (X) 3-prime UTRs with the coding sequences of secretory NanoLuc. NanoLuc expression from 441 mRNAs was monitored in human immature monocyte-derived dendritic cells because this cell type is commonly targeted in the context of lipid nanoparticle (LNP)-based therapies, and mRNA expression is often cell type-specific.

[0139] The inventors corrected Nanoluc expression levels based on transfection efficiency obtained by co-transfecting cells with a 10% mRNA load consisting of mCitrine mRNA. The inventors also monitored cell viability to exclude the most toxic mRNA species, which may be observed, for example, in cases of potent innate activation. After normalizing the transfection efficiency (Teff), cumulative Nanoluc expression levels were calculated for 6, 24, 48, 72, and 96 hours based on the area under the curve. Subsequently, mRNAs with an AUC value in the 60th percentile were selected from each 96-well plate as top performers, thereby obtaining a total of 40 mRNAs for screening and verification in the next round.

[0140] Selection of the second round in primary human cells (Fig. 2a, b, c).

[0141] Down-selected UTRs were tested in primary human cells, namely skeletal muscle cells (skMCs). These primary cells are associated with an approach to intramuscular immunization utilizing delivery via lipid nanoparticles. The top 40 mRNAs were transfected into mature skMCs in double replicates using 50 ng of mRNA formulated with Messenger Max at a 1:3 ratio. Nanoluc secretion was evaluated at 6, 24, 48, 72, and 96 hours, and transfection efficiency, along with cell viability, was assessed 24 hours after transfection. Kinetic data were corrected using Teff values, and cell density was also taken into account. The baseline values ​​of the three control mRNAs were subtracted from the AUC of the integrated Nanoluc measurements in GraphPadPrism using division, and the average of all replicate values ​​was used in the calculation. Next, four top performers were selected: 1) ANXA1-secNluc-HBA1, 2) eno-secNluc-FTH1-201 and eno-secNluc-Ube2d2a, 3) eno-secNluc-TPRKB and eno-secNluc-UBL5, 4) tabA-secNluc-Ube2d2a (Fig. 2a).

[0142] Accordingly, to compensate for the high variability inherent in different donors and to reconfirm initial observations, 40 compression-selected mRNAs were transfected into immature MoDCs from two other donors. This experiment was performed identically to the first round of screening, with additional normalization for cell density. For Donor 1, the four top performers were as follows: 1) RPS25-secNluc-Ube2d2a, 2) RPS14-secNluc-HBA1, 3) Eno-secNluc-Ube2d2a, and 4) REG1A-secNluc-Ube2d2a (Fig. 2B). For donor 2, they appeared in the following order: 1) TCV-secNluc-HBB-001, 2) HBA1-secNluc-HBA1, 3) TCV-secNluc-UBL5, 4) TCV-secNluc-Med13 and ANXA1-secNluc-HBG2 (Fig. 2c).

[0143] One combination, eno-secNluc-Ube2d2a, was duplicated in SkMC and iMoDC. The HBA1 3-prime UTR has been proven to exhibit superior mRNA expression in all donors and cell types when combined with various 5-prime sequences, suggesting that the corresponding UTR can boost mRNA characteristics in various tissues. Therefore, combinations of the ANXA1 and RPS14 5-prime UTRs with the HBA1 3-prime UTR were selected from the top lists of SkMC and iMoDC, respectively. Similarly, Ube2d2a was also frequently observed in the list and was selected for combination with RPS25. For one specific iMoDC donor, the TCV 5-prime UTR appeared very frequently, so this 5' UTR was maintained in combination with another major 3' UTR sequence, UBL5 (observed twice). The 6th compression-selected UTRs are summarized in Table 1, and the control group UTRs are summarized in Table 2.

[0144]

[0145]

[0146] Validation of the top 6 performers using different cell lines and mRNA types (Fig. 3)

[0147] To evaluate novel UTR combinations in the context of various coding mRNA sequences, the inventors synthesized mRNA encoding firefly luciferase (Fluc) ORFs with six selected UTR combinations. Additionally, to determine whether the cellular environment influences mRNA expression induced by UTRs, the inventors evaluated Fluc expression in six different human cell lines and mouse cell lines. The novel mRNA was transfected at different concentrations of 100 ng / well and 200 ng / well, respectively, into six human cell lines of different origin: HeP2G (hepatocellular carcinoma cell line), K562 (leukemia cell line), BJ (fibroblast), HEK293 (human embryonic kidney-derived immortalized cell line), Mo-iDC (immature monocyte-derived dendritic cell line), and SkMC (skeletal muscle cell line), and two mouse cell lines: CT26 colorectal carcinoma cell line and TC-1 immortalized lung epithelial cell line. FLuc luminescence, mCitrine fluorescence, and viability were quantified after 24 hours. For viability, fluorescence was converted into a percentage of viability (%) by assuming the viability of untransfected wells to be 100%. To account for transfection efficiency, Nanoluc expression levels (RLU) at 200 ng / well transfection were divided by mCitrine mRNA expression levels (RFU), indicated by the p / s ratio normalized in Figure 3. The novel UTR combinations consistently showed high Fluc mRNA expression levels across a wide range of cell lines, and the top three UTR combinations were: 1) ANXA-HBA1, 2) eno-RSP29, and 3) eno-Ube2d2. In particular, TCV-UBL5 was demonstrated to exhibit high expression in HepG2 cell lines, suggesting that UTRs act in a tissue-specific manner. The UTR combinations that showed the best performance in the second round of screening targeting SkMC and iMoDC are listed in Table 3.

[0148]

[0149] Example 2: In vivo ( In vivo ) Screening

[0150] Materials and Methods

[0151] Bioluminescence imaging

[0152] 50 μL of TBS or 40 μg / mL of firefly luciferase (fLuc) mRNA-LNP was administered intramuscularly, and bioluminescent imaging was performed using an IVIS Lumina S5 imaging system (PerkinElmer) at 5, 24, 48, and 72 hours. For non-invasive imaging, 100 μL of 30 mg / mL D-luciferin (Promega, catalog number # E1605) dissolved in sterile water was injected intraperitoneally into 6 to 8-week-old female BALB / c mice (Charles River). 5 minutes after D-luciferin administration, the mice were anesthetized in an anesthesia induction chamber using 5% isoflurane and positioned on an imaging platform while maintaining anesthesia using 3% isoflurane. Whole-body images were taken 10 to 15 minutes after D-luciferin administration using automatic exposure settings. Quantification of bioluminescent signals was performed using Living Image software (PerkinElmer).

[0153] result

[0154] 6 hit candidate 5'UTR - 3'UTR gene combinations in vivo ( in vivoTo investigate the effects on RNA polynucleotide stability and / or translation efficiency, 50 μL of TBS, one of six hit candidate fLuc mRNA-LNPs with 5'UTR-3'UTR combinations, or one of three control fLuc mRNA-LNPs with 5'UTR-3'UTR gene combinations were injected intramuscularly into 6 to 8-week-old female BALB / c mice. D-luciferin was injected at 5, 24, 48, and 72 hours after administration, and fLuc expression was measured by quantifying whole-body bioluminescence signals using the IVIS platform. Overall, across all time points, all six hit candidate 5'UTR - 3'UTR gene combinations tended to exhibit superior or equivalent performance to the tested in vivo / benchmark 5'UTR - 3'UTR gene combinations, and the top three UTR combinations were 1) eno-RSP29, 2) eno-Ube2d2, and 3) TCV-UBL5. For the initial time point, ANXA1-HBA1 in vivo ( in vivo Although it was the bottom performer in the expression study, the RPS25-Ube2d2a combination was associated with the lowest bioluminescence signal at the later stage (Fig. 4).

[0155] Example 3: In vitro ( Ex Vivo) expression

[0156] In this embodiment, the inventors, in order to confirm whether expression is improved in MC-38 cells, compared the new muIL-7, muIL-21, and mu41BBL UTRs (V8 and V10) with the legacy UTR (V5) in vitro ( ex vivo ) evaluated expression.

[0157] UTR V8 corresponds to RPS14 5'UTR - HBA1 3'UTR

[0158] UTR V10 corresponds to ANXA1 5'UTR - HBA1 3'UTR

[0159] UTR V5 corresponds to the UTR disclosed in WO2015071295 as a comparative example.

[0160] At the 24-hour mark, tumors were collected, and ex vivo expression was evaluated using Luminex, procartaplex, which can simultaneously detect muIL-21, muIL-7, and mu41BBL.

[0161] Experiment setup:

[0162] · Dose: Inject the formulated LNP at a volume of 20 μl into 15 μg / mouse

[0163] · Reading type: Tumor is collected after 24 hours and protein expression is measured using Luminex

[0164] 0.5*10e6 tumor cells were inoculated into C57BL / 6J mice (unilateral; left flank).

[0165] When the average tumor size reached 50 to 100 mm³ (10 days after inoculation), mice were randomly assigned and LNP was injected via IT.

[0166] As can be seen in Figure 5, in all three mRNA constructs tested (muIL-7, muIL-21, and mu41BBL), both UTR V8 and V10 showed significantly higher protein expression compared to the control UTR V5 combination.

[0167] Example 4: In vivo ( in vivo ) Efficacy Research

[0168] In this embodiment, the inventors, in order to determine whether expression is improved in MC-38 cells, compared the in vivo (V8 and V10) of the new muIL-7, muIL-21, and mu41BBL UTRs with the existing UTR (V5). in vivo ) evaluated efficacy.

[0169] Experiment setup:

[0170] · Dosage: Inject 10 μg / mouse with the formulated LNP at a dose of 20 μl.

[0171] · Interpretation type: Tumors are collected after 24 hours, and mouse survival is verified up to 35 days after inoculation.

[0172] 0.5*10e6 tumor cells were inoculated into C57BL / 6J mice (unilateral; left flank).

[0173] When the average tumor size exceeded 75 mm³ (+ / - 11 days after inoculation), mice were randomized and LNP was injected via IT. The injection was repeated on days 3 and 7 after randomization.

[0174] As demonstrated in Fig. 6, the highest efficiency was obtained when using the V10 UTR (Fig. 6d) and V8 UTR (Fig. 6c). The control V5 UTR (Fig. 6b) clearly showed inferior performance compared to this combination of the two UTRs of the present invention. TBS was used as the voice example (Fig. 6a).

Claims

Claim 1 RNA polynucleotide comprising: i) at least one 5' non-translating region (5'UTR) element comprising a nucleic acid sequence derived from the 5'UTR of a gene selected from ANXA1, E. coli enolase, RPS14, RPS25, or TCV, or a fragment or variant thereof; ii) at least one open reading frame (ORF) encoding a polypeptide; and iii) at least one 3' non-translating region (3'UTR) element comprising a nucleic acid sequence derived from the 3'UTR of a gene selected from HBA1, RPS29, Ube2d2a, or UBL5, or a fragment or variant thereof. Claim 2 The RNA polynucleotide of claim 1, wherein the 5'UTR of the RNA polynucleotide comprises an RNA sequence corresponding to a DNA sequence selected from SEQ ID NO. 1 [ANXA1], SEQ ID NO. 2 [eno], SEQ ID NO. 3 [RPS14], SEQ ID NO. 4 [RPS25], or SEQ ID NO. 5 [TCV], or a sequence having at least 95% sequence identity therewith. Claim 3 RNA polynucleotide according to claim 1 or 2, wherein the 3'UTR of the RNA polynucleotide comprises an RNA sequence corresponding to a DNA sequence selected from SEQ ID NO. 6 [HBA1], SEQ ID NO. 18 [HBA1], SEQ ID NO. 7 [RPS29], SEQ ID NO. 8 [Ube2d2a], or SEQ ID NO. 9 [UBL5], or a sequence having at least 95% sequence identity therewith. Claim 4 An RNA polynucleotide according to any one of claims 1 to 3, wherein the 5'UTR and the 3'UTR of the RNA polynucleotide comprise an RNA sequence corresponding to a DNA sequence selected from any one of the following 5' UTR - 3' UTR gene combinations, a fragment thereof, or a variant thereof: - a combination of the 5'UTR of ANXA1 and the 3'UTR of HBA1; or - a combination of the 5'UTR of eno and the 3'UTR of RPS29; or - a combination of the 5'UTR of eno and the 3'UTR of Ube2d2a; or - a combination of the 5'UTR of RPS14 and the 3'UTR of HBA1; or - a combination of the 5'UTR of RPS25 and the 3'UTR of Ube2d2a; or - a combination of the 5'UTR of TCV and the 3'UTR of UBL5. Claim 5 An RNA polynucleotide according to any one of claims 1 to 3, wherein the 5'UTR and the 3'UTR of the RNA polynucleotide comprise an RNA sequence corresponding to a DNA sequence selected from any one of the following 5' UTR - 3' UTR gene combinations, a fragment thereof, or a variant thereof: - a combination of the 5'UTR of ANXA1 and the 3'UTR of HBA1; or - a combination of the 5'UTR of RPS14 and the 3'UTR of HBA1. Claim 6 An RNA polynucleotide according to any one of claims 1 to 3, wherein the 5'UTR and the 3'UTR of the RNA polynucleotide comprise an RNA sequence corresponding to a DNA sequence selected from any one of the following combinations, or a sequence having at least 95% sequence identity therewith: - a combination of SEQ ID NO. 1 [ANXA1] for the 5'UTR and SEQ ID NO. 6 [HBA1] for the 3'UTR; or - a combination of SEQ ID NO. 2 [eno] for the 5'UTR and SEQ ID NO. 7 [RPS29] for the 3'UTR; or - a combination of SEQ ID NO. 2 [eno] for the 5'UTR and SEQ ID NO. 8 [Ube2d2a] for the 3'UTR; or - a combination of SEQ ID NO. 3 [RPS14] for the 5'UTR and SEQ ID NO. 6 [HBA1] for the 3'UTR; or - a combination of SEQ ID NO. 3 [RPS25] for the 5'UTR and SEQ ID NO. 8 [Ube2d2a] for the 3'UTR; or - a combination of SEQ ID NO. 1 [ANXA1] for the 5'UTR and SEQ ID NO. 18 [HBA1] for the 3'UTR; or - a combination of SEQ ID NO. 3 [RPS14] for the 5'UTR and SEQ ID NO. 18 [HBA1] for the 3'UTR; or - a combination of SEQ ID NO. 5 [TCV] for the 5'UTR and SEQ ID NO. 9 [UBL] for the 3'UTR. Claim 7 An RNA polynucleotide according to any one of claims 1 to 3, wherein the 5' UTR and the 3' UTR of the RNA polynucleotide comprise an RNA sequence corresponding to a DNA sequence selected from any one of the following combinations, or a sequence having at least 95% sequence identity therewith: - a combination of SEQ ID NO. 1 [ANXA1] for the 5' UTR and SEQ ID NO. 6 [HBA1] for the 3' UTR; or - a combination of SEQ ID NO. 3 [RPS14] for the 5' UTR and SEQ ID NO. 6 [HBA1] for the 3' UTR; - a combination of SEQ ID NO. 1 [ANXA1] for the 5' UTR and SEQ ID NO. 18 [HBA1] for the 3' UTR; or - a combination of SEQ ID NO. 3 [RPS14] for the 5' UTR and SEQ ID NO. 18 [HBA1] for the 3' UTR. Claim 8 An RNA polynucleotide according to any one of claims 1 to 7, wherein the 5'UTR of the RNA polynucleotide further comprises a KOZAK sequence and / or an internal ribosome entry site (IRES). Claim 9 An RNA polynucleotide according to any one of claims 1 to 7, wherein the RNA polynucleotide is mRNA or non-coding RNA, preferably mRNA, antisense RNA, siRNA, sgRNA, guide RNA, or CRISPR. Claim 10 A pharmaceutical composition comprising one or more RNA polynucleotides according to any one of claims 1 to 9 and at least one pharmaceutically acceptable formulation. Claim 11 RNA polynucleotide according to any one of claims 1 to 9, or a pharmaceutical composition according to claim 10, used in human medicine and / or veterinary medicine. Claim 12 An RNA polynucleotide according to any one of claims 1 to 9, or a pharmaceutical composition according to claim 10, used as a vaccine or for gene therapy. Claim 13 Use of an RNA polynucleotide according to any one of claims 1 to 9, or a pharmaceutical composition according to claim 10, for improving the expression and / or translation of a gene product, particularly for improving the initiation of translation. Claim 14 Use of an RNA polynucleotide according to any one of claims 1 to 9, or a pharmaceutical composition according to claim 10, for improving the stability and / or duration of expression of a gene product, particularly for improving its distribution in vivo. Claim 15 A method for inducing an improved immune response in a subject, comprising the step of administering to the subject a therapeutically effective amount of an RNA polynucleotide according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10.

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