TW202535429A - Polynucleotides comprising 5'utr-3'utr combinations and use thereof - Google Patents

Abstract

The present invention relates to the field of artificial polynucleotides and in particular to RNA polynucleotides comprising specific 5'UTR - 3'UTR gene combinations. Provided herein is an RNA polynucleotide comprising at least one 5'UTR element selected from a gene such as ANXA1, E. Coli enolase, RPS14, RPS25, or TCV and at least one 3'UTR selected from a gene such as HBA1, RPS29, Ube2d2a, or UBL5. The invention further relates to a pharmaceutical composition comprising one or more RNA polynucleotides according to the invention and a method of inducing an enhanced immune response in a subject using said RNA polynucleotides or pharmaceutical composition.

Description

Polynucleotides containing 5'UTR-3'UTR combinations and their uses

This invention relates to the field of artificial polynucleotides, and more particularly to RNA polynucleotides comprising specific 5'UTR-3'UTR gene combinations. This document 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. This invention further relates to a pharmaceutical composition comprising one or more RNA polynucleotides according to this invention, and a method for inducing an enhanced immune response in an individual using such RNA polynucleotides or pharmaceutical compositions.

Synthetic mRNA has become a powerful tool for transmitting genetic information, and its use in various therapeutic applications is being explored. Many of these applications require prolonging the intracellular residence time of mRNA to improve the biological usability of the encoded proteins. mRNA molecules or polynucleotides are inherently unstable, and their intracellular dynamics depend on the 5' untranslated region (5' UTR) and 3' untranslated region (3' UTR) containing the coding sequence. The structural features of the UTR (such as sequence, length, and secondary structure) have a strong influence on transcription initiation and peak expression levels (mainly in the 5' UTR), and also affect mRNA stability and duration of expression (mainly in the 3' UTR).

Therefore, the combination of UTRs determines the dynamics of mRNA polynucleotide expression. UTRs used in RNA synthesis are typically derived from α/β-globulins, human heat shock proteins, or viruses. However, UTR efficacy can vary across cell types, and it is necessary to tailor specific UTRs for the target cell type.

Therefore, one object of the present invention is to provide an RNA polynucleotide that contains an optimal 5'UTR/3'UTR combination of specific UTRs, thereby improving stability and/or translational efficiency. Such RNA polynucleotides are particularly useful in mRNA-based therapies, gene therapies, and/or gene vaccine administration. The object of the present invention is to provide RNA polynucleotides that increase and prolong protein expression and enhance biological distribution, preferably exhibiting improved translational efficiency. The fundamental object of the present invention is addressed through the claimed subject matter.

In the first state, the present invention provides an RNA polynucleotide comprising: i) at least one 5' untranslated 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; iii) at least one 3' untranslated 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.

Compared with the control, RNA polynucleotides with at least one of the 5'UTR and 3'UTR combinations showed enhanced expression and biological distribution.

In a specific instance, the 5'UTR of the RNA polynucleotide contains an RNA sequence corresponding to, or having at least 95% sequence identity with, 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].

In another specific example, the 3'UTR of the RNA polynucleotide contains an RNA sequence corresponding to, or having at least 95% sequence identity with, 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].

In another specific example, the 3'UTR of the RNA polynucleotide contains an RNA sequence corresponding to, or having at least 95% sequence identity with, 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].

In a specific instance, the 5'UTR and 3'UTR of the RNA polynucleotide contain RNA sequence fragments or variants thereof, corresponding to a DNA sequence selected from any of the following 5'UTR-3'UTR gene combinations: - 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.

In another specific example, the 5' UTR and 3' UTR of the RNA polynucleotide contain an RNA sequence or a sequence having at least 95% sequence identity with it, corresponding to a DNA sequence selected from any of the following combinations: - SEQ ID NO: 1 [ANXA1] of the 5' UTR and SEQ ID NO: 6 [HBA1] of the 3' UTR; or - SEQ ID NO: 2 [eno] of the 5' UTR and SEQ ID NO: 7 [RPS29] of the 3' UTR; or - SEQ ID NO: 2 [eno] of the 5' UTR and SEQ ID NO: 8 [Ube2d2a] of the 3' UTR; or - SEQ ID NO: 3 [RPS14] of the 5' UTR and SEQ ID NO: 6 [HBA1] of the 3' UTR; or - SEQ ID NO: 3 [ANXA1] of the 5' UTR and SEQ ID NO: 6 [HBA1] of the 3' UTR. The combination of [RPS25] with SEQ ID NO: 8 [Ube2d2a] of the 3'UTR; or the combination of SEQ ID NO: 1 [ANXA1] of the 5'UTR with SEQ ID NO: 18 [HBA1] of the 3'UTR; or the combination of SEQ ID NO: 3 [RPS14] of the 5'UTR with SEQ ID NO: 18 [HBA1] of the 3'UTR; or the combination of SEQ ID NO: 5 [TCV] of the 5'UTR with SEQ ID NO: 9 [UBL] of the 3'UTR.

According to another specific example of the invention, the 5'UTR of the RNA polynucleotide further includes a KOZAK sequence and/or an internal ribosome entry address (IRES). In yet another specific example, the RNA polynucleotide is mRNA or non-coding RNA, preferably mRNA, antisense RNA, siRNA, sgRNA, guide RNA, or CRISPR.

In another specific example, the invention relates to a pharmaceutical composition comprising one or more RNA polynucleotides according to the invention and at least one pharmaceutically acceptable agent.

In another specific example, the invention relates to RNA polynucleotides or pharmaceutical compositions according to the invention for use in human and/or veterinary medicine. More specifically, RNA polynucleotides or pharmaceutical compositions are provided for use as vaccines or for use in gene therapy.

In another specific example, the invention relates to the use of an RNA polynucleotide or pharmaceutical composition according to the invention for enhancing the expression and/or translation of gene products, particularly enhancing translation initiation. Even more specifically, it provides the use of the RNA polynucleotide or pharmaceutical composition to enhance the stability and/or duration of gene product expression, particularly enhancing biological distribution.

In yet another specific example, the invention relates to a method for inducing an enhanced immune response in an individual, comprising administering a therapeutically effective amount of an RNA polynucleotide or pharmaceutical composition according to the invention.

The invention will now be described further. In the following paragraphs, different aspects of the invention are defined in more detail. Unless clearly indicated to the contrary, each aspect so defined may be combined with any other aspect. Specifically, any feature indicated as preferred or advantageous may be combined with any other feature indicated as preferred or advantageous.

Unless the context clearly requires otherwise, as used in this specification and the accompanying claims, the singular forms “a,” “an,” and “the” include a plural referent. For example, “RNA polynucleotide” means one or more RNA polynucleotides. As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” or “containing,” and are inclusive or open-ended, not excluding additional members, elements, or method steps not listed. Ranges of values expressed by endpoints include all numbers and fractions falling within the corresponding range, as well as the listed endpoints.

When referring to measurable values such as parameters, quantities, durations of time, and similar parameters, the terms "about" or "approximately" as used herein mean a variation of a given value by +/-10% or less, preferably +/-5% or less, better +/-1% or less, and even better +/-0.1% or less, provided that such variation is suitable for implementation in the disclosed invention. It should be understood that the value referred to by the modifiers "about" or "approximately" is itself a specific and preferred disclosure.

Although the term "one or more" (such as one or more members in a group) is self-evident, by further example, the term specifically covers any one or any two or more of such members, such as any ≥3, ≥4, ≥5, ≥6 or ≥7 of such members, and at most all of such members.

Throughout this specification, the reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment. Furthermore, as will be apparent to those skilled in the art from this disclosure, in one or more embodiments, a particular feature, structure, or characteristic can be combined in any suitable manner. Furthermore, although some specific examples described herein include some features included in other specific examples but not all of them, combinations of features of different specific examples are intended to form different specific examples within the scope of this invention, as will be understood by those skilled in the art. For example, any of the specific examples claimed within the scope of the following claims may be used in any combination.

All references cited in this manual are hereby incorporated in their entirety.

Unless otherwise specified, all terms used to disclose the invention (including technical and scientific terms) shall have the meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Definitions of terms may be included for the purpose of further guidance in order to better understand the teachings of the invention.

In searching for RNA polynucleotides exhibiting increased stability and/or translational efficiency, the inventors investigated whether specific 5' UTRs and 3' UTRs might affect the efficacies of such RNA polynucleotides. The inventors unexpectedly discovered that specifically designed RNA polynucleotides containing both 5' UTR and 3' UTR elements had a beneficial effect on stability and translational efficiency. It was further determined that RNA polynucleotides containing 5' UTR sequences derived from the 5' UTRs of genes selected from HBA1, RPS29, Ube2d2a, or UBL5, when combined with 3' UTR sequences derived from the 5' UTRs of genes selected from ANXA1, E. coli enolase, RPS14, RPS25, or TCV, exhibited particularly good performance.

This invention is particularly suitable for use in pharmaceutical compositions in isolated form or as lipid nanoparticles, and therefore also suitable for inducing immune responses in individuals. In this case, the RNA polynucleotide according to this invention is also suitable for use as a vaccine or in gene therapy. The main advantage of using the RNA polynucleotide according to this invention is its enhancement of gene product expression and/or translation, but more particularly, it enhances the stability and/or duration of gene product expression and biodistribution.

In the context of this invention, the term "RNA" refers to a nucleic acid molecule that contains ribonucleotide residues and is preferably composed entirely or substantially of ribonucleotide residues. When referring to the term "nucleic acid molecule," unless otherwise stated, it means the RNA polynucleotide according to this invention. Specifically, an RNA polynucleotide is a polymer containing or composed of nucleotide monomers covalently linked together by phosphodiester bonds of a sugar/phosphate backbone.

"Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-furanose ribosyl group. Specifically, this term refers to double-stranded RNA, but can also refer to single-stranded RNA, isolated RNA (such as partially purified RNA), substantially pure RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA by adding, deleting, substituting, and/or altering one or more nucleotides. Such alterations may include the addition of non-nucleotide substances, such as addition to the ends or interior of RNA, for example, at one or more nucleotides. The nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides, chemically synthesized nucleotides, or deoxynucleotides. Such modified RNA may be called analogues or analogues of naturally occurring RNA. In the context of this invention, when a particular sequence is revealed, such sequences also represent the corresponding RNA sequences, and it should be understood that T is replaced by U.

According to the present invention, the term "RNA polynucleotide" also includes, and preferably refers to, "mRNA," meaning "messenger RNA" and relating to a "transcript" that can be produced using DNA as a template and encodes peptides or proteins. mRNA typically contains a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR).

In a specific instance, the RNA polynucleotide of this 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 "a single molecule," but is typically understood as a collection of homologous molecules. Therefore, it can refer to components or, for example, a plurality of homologous molecules contained in lipid nanoparticles.

For clarity, mRNA encompasses any RNA molecule that can be translated into protein by a eukaryotic host. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one specific example of the invention, RNA is obtained by in vitro transcription. In vitro transcription methods are known to those skilled in the art and may include a purified linear DNA template containing a promoter, ribonucleotide triphosphates; a buffer system including dithiothreitol (DTT) and magnesium ions; spermidine; and a suitable RNA polymerase, such as T7 RNA polymerase. The exact conditions used in the transcription reaction depend on the amount of RNA required for the specific application. Several commercially available in vitro transcription kits are available.

Therefore, 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 has been stabilized and optimized in terms of translation efficiency. In other words, the present invention also relates to an artificial nucleic acid molecule, which can be a (modified) RNA or DNA molecule, such as a DNA vector that can be used to produce mRNA. It can be provided in the form of a double-stranded molecule having a sense strand and an antisense strand, for example, in the form of a DNA molecule having a sense strand and an antisense strand. The available mRNA can then be translated to produce the desired peptide or protein encoded by the open reading frame. If the artificial nucleic acid molecule is DNA, it can be used, for example, as a double-stranded storage form for the continuous and repeated in vitro or in vivo production of mRNA.

In the context of this invention, the term "5'-untranslated region (5' UTR)" should be understood as a specific portion of mRNA located at the 5' end of the protein coding region (i.e., the open reading frame; ORF) of the mRNA. Typically, a 5' UTR begins with a transcription start site and is a single nucleotide terminus preceding the start codon in the open reading frame. The 5' UTR may contain elements used to control gene expression, also known as regulatory elements. Such regulatory elements may be, for example, but not limited to, promoters, enhancers, silencers, insulators, ribosome-binding sites, and 5'-terminal oligopyrimidine segments. The 5' UTR may be modified post-transcriptionally, for example by adding a 5' cap. In the context of this invention, the 5' UTR corresponds to the sequence of mature mRNA located between the 5' cap and the start codon. In the context of this invention, the term "a 5' UTR of a gene," such as "a 5' UTR of the ANXA1 gene," corresponds to the sequence of the 5' UTR of the mature mRNA derived from this gene (i.e., the mRNA obtained through gene transcription and maturation of immature mRNA). The term "a 5' UTR of a gene" encompasses both the DNA and RNA sequences of the 5' UTR.

In the context of this invention, the term "3' untranslated region (3' UTR)" should be understood as a portion of mRNA located between the protein coding region (i.e., ORF) and the poly(A) sequence of the mRNA. The 3' UTR of mRNA is not translated into an amino acid sequence. The 3' UTR sequence is generally encoded by the gene coding sequence that is transcribed into the corresponding mRNA during gene expression. The gene sequence is first transcribed into immature mRNA, which contains introns, if present. The immature mRNA is then further processed into mature mRNA during maturation. In the context of this invention, the 3' UTR corresponds to the sequence of mature mRNA, located at the 3' of the termination codon in the protein coding region. In the context of this invention, the term "a 3' UTR of a gene," such as "a 3' UTR of the HBA1 gene," corresponds to the sequence of the 3' UTR of the mature mRNA derived from this gene (i.e., the mRNA obtained through gene transcription and maturation of immature mRNA). The term "a 3' UTR of a gene" encompasses both the DNA and RNA sequences of the 3' UTR.

As used herein, the term "open reading frame" (ORF) typically refers to a sequence of three nucleotide triplets that can be translated into a peptide or protein. Preferably, the ORF contains a start codon at its 5' end, which is typically a combination of three consecutive nucleotides (ATG or AUG) encoding the amino acid methionine, followed by a region typically a multiple of three nucleotides in length. The ORF is preferably terminated by a termination codon (e.g., TAA, TAG, TGA). Typically, this is the only termination codon of the ORF. The ORF can be isolated, or it can be incorporated into a longer nucleic acid sequence, such as an RNA polynucleotide. The ORF can also be referred to as a "protein coding region." Furthermore, the ORF can be at least partially codon-optimized. Codon optimization is based on the finding that translation efficiency can be determined by the different frequencies of transtransfer RNA (tRNA) in the cell, and is known to those with general knowledge in the field of mRNA production.

Therefore, the present invention provides an RNA polynucleotide comprising: i) at least one 5' untranslated 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; iii) at least one 3' untranslated 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.

It was found that RNA polynucleotides with at least one of these 5'UTR and 3'UTR combinations enhanced expression and biological distribution compared to the control.

Preferably, at least one 5' UTR element and at least one 3' UTR element are functionally linked to the ORF. This "preferably" means that the 5' UTR element and the 3' UTR element are associated with the ORF to enable it to function, preferably in an additive manner, and more preferably in a synergistic manner, such as stabilizing the ORF's performance, enhancing the protein production of proteins encoded by the ORF, or stabilizing the RNA polynucleotide. Preferably, the 5' UTR element, the ORF, and the 3' UTR element are coupled in a 5' to 3' orientation. Therefore, preferably, the RNA polynucleotide comprises the structure 5'-5' UTR element-(if present) linker-ORF-(if present) linker-3' UTR element-3', wherein the linker may or may not be present. For example, a linker can be one or more nucleotides, such as a chain of 1-50 or 1-20 nucleotides.

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

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

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

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

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

As previously described, specific examples also include fragments and variants of such genes. In the context of this invention, the term "fragment thereof" encompasses both the sequence corresponding to the entire 5' UTR or 3' UTR sequence (i.e., the full-length 5' UTR or 3' UTR sequence of such genes) and the sequence of a fragment corresponding to the 5' UTR or 3' UTR sequence of such genes. Preferably, the fragment of the 5' UTR or 3' UTR gene comprises a continuous nucleotide chain corresponding to a continuous nucleotide chain in the full-length 5' UTR or 3' UTR of the gene, representing 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 this invention, the fragment is preferably the functional fragment described herein.

In the context of this invention, the term "variant thereof" means a variant of the 5' UTR or 3' UTR of a naturally occurring gene, preferably a variant of the 5' UTR or 3' UTR of a vertebrate gene, preferably a variant of the 5' UTR or 3' UTR of a mammalian gene, and more preferably a variant of the 5' UTR or 3' UTR of a human or mouse gene. Such variants may be modified 5' UTRs or 3' UTRs of genes. 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 of a derived variant. Preferably, the variant of the 5' UTR or 3' UTR of the gene is 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% identical to the naturally occurring 5' UTR or 3' UTR from which the variant is derived. Preferably, the variant is the functional variant described herein. As used herein, the term "variant" also encompasses the term "homologs," which should be understood as sequences of the same gene but derived from other species.

Furthermore, in specific examples, the RNA polynucleotide may contain more than one 5' UTR element or 3' UTR element as described above. For example, the RNA polynucleotide molecule according to the invention may contain 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 the same or may be different. For example, the RNA polynucleotide according to the present invention may contain two substantially identical 5' UTR elements, such as two 5' UTR elements comprising a nucleic acid sequence or fragment or variant thereof derived from the E. coli enolase gene or composed of a nucleic acid sequence or fragment or variant thereof derived from the E. coli enolase gene, and a combination of, for example, two 3' UTR elements comprising a nucleic acid sequence or fragment or variant thereof derived from the HBA1 gene or composed of a nucleic acid sequence or fragment or variant thereof derived from the HBA1 gene.

As defined above, the 5' UTR of the ANXA1, E. coli enolase, RPS14, RPS25, or TCV genes corresponds to the 5' UTR sequence of the mature mRNA derived from the ANXA1, E. coli enolase, RPS14, RPS25, or TCV genes, respectively, preferably extending from the nucleotide at the 3' of the 5' cap to the nucleotide at the 5' of the start codon. The length of the 5' UTR of these genes can vary between 10 and 500 nucleotides, and is typically less than about 300 nucleotides, preferably less than about 200 nucleotides, and more preferably less than about 150 nucleotides. In the sense of this invention, exemplary 5' UTRs of these genes are nucleic acid sequences according to SEQ ID NO. 1-5 or fragments or variants thereof. Preferably, the 5' UTR element in the present invention functions as a 5' UTR or encodes a nucleotide sequence that implements the function of a 5' UTR. The 5' UTR element is preferably suitable for increasing the production of proteins containing artificial RNA polynucleotides.

Similarly, the 3' UTR of the HBA1, RPS29, Ube2d2a, or UBL5 genes corresponds to the 3' UTR sequence of the mature mRNA derived from the HBA1, RPS29, Ube2d2a, or UBL5 genes, respectively. The length of the 3' UTR of these genes can vary between 10 and 500 nucleotides, and is typically less than about 300 nucleotides, preferably less than about 200 nucleotides, and more preferably less than about 150 nucleotides. In the sense of the invention, exemplary 3' UTRs of these genes are nucleic acid sequences according to SEQ ID NO. 6-9 or fragments or variants thereof. Preferably, the 3' UTR element in the sense of the invention functions as a 3' UTR or a nucleotide sequence encoding a 3' UTR function. The 3' UTR element is preferably used to enhance the stability of mRNA molecules.

In a particular specific example, the invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element and at least one 3' UTR element, wherein the 5' UTR element comprises an RNA sequence corresponding to or having at least 95%, 96%, 97%, 98%, or 99% sequence identity with a DNA sequence selected from SEQ ID NO: 1 [ANXA1], and the 3' UTR element comprises 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 having at least 95%, 96%, 97%, 98%, or 99% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 9 [UBL5].

In a particular specific example, the invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element and at least one 3' UTR element, wherein the 5' UTR element comprises an RNA sequence corresponding to or having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the DNA sequence selected from SEQ ID NO: 2 [Escherichia coli enolase gene], and the 3' UTR element comprises 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 having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the same.

In a particular specific example, the invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element and at least one 3' UTR element, wherein the 5' UTR element comprises an RNA sequence corresponding to or having at least 95%, 96%, 97%, 98%, or 99% sequence identity with a DNA sequence selected from SEQ ID NO: 3 [RPS14], and the 3' UTR element comprises 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 having at least 95%, 96%, 97%, 98%, or 99% sequence identity with a DNA sequence selected from SEQ ID NO: 9 [UBL5].

In a particular specific example, the invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element and at least one 3' UTR element, wherein the 5' UTR element comprises an RNA sequence corresponding to or having at least 95%, 96%, 97%, 98%, or 99% sequence identity with a DNA sequence selected from SEQ ID NO: 4 [RPS25], and the 3' UTR element comprises 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 having at least 95%, 96%, 97%, 98%, or 99% sequence identity with a DNA sequence selected from SEQ ID NO: 9 [UBL5].

In a particular specific example, the invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element and at least one 3' UTR element, wherein the 5' UTR element comprises an RNA sequence corresponding to or having at least 95%, 96%, 97%, 98%, or 99% sequence identity with a DNA sequence selected from SEQ ID NO: 5 [TCV], and the 3' UTR element comprises 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 having at least 95%, 96%, 97%, 98%, or 99% sequence identity with a DNA sequence selected from SEQ ID NO: 9 [UBL5].

In a particular specific example, the invention relates to an RNA polynucleotide comprising a combination of at least one 5' UTR element and at least one 3' UTR element, wherein the 5' UTR element comprises an RNA sequence corresponding to or having at least 95%, 96%, 97%, 98%, or 99% sequence identity with 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], or SEQ ID NO: 5 [TCV], and the 3' UTR element comprises a sequence corresponding to or having at least 95%, 96%, 97%, 98%, or 99% sequence identity with 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] is a DNA sequence or an RNA sequence or a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to it.

Therefore, the 5' UTR or 3' UTR nucleotide sequence derived from a gene is derived from a eukaryotic gene, preferably a plant or animal gene, preferably a chordate gene, even more preferably a vertebrate gene, and best of all mammal genes, such as human or mouse genes ( e.g., Table 1 ). For example, a 5' UTR or 3' UTR element comprises or is composed of the following nucleotide sequences derived from a group of nucleotide sequences (e.g., RNA sequences corresponding to DNA sequences) selected from the group consisting of fragments or variants of SEQ ID NO. 1-5 or SEQ ID NO. 6-9, 18, or homologs of SEQ ID NO. 1-5 or SEQ ID NO. 6-9, 18, respectively. In the context of this invention, the term "homologs" refers to sequences from other species, such as those from Homo sapiens (humans) or house mice (mice), that are homologous to sequences according to SEQ ID NO. 1-5 or SEQ ID NO. 6-9, 18. For example, SEQ ID NO. 1 relates to a sequence containing the 5' UTR of human annexin A1 (ANXA). In the context of this invention, a homolog of SEQ ID NO. 1 is any such sequence derived from an ANXA gene from another species other than humans, such as any vertebrate or plant gene, preferably any mammalian ANXA gene other than the human ANXA gene, such as mouse, rat, rabbit, monkey, etc.

In a specific instance, the 5' UTR element and the 3' UTR element are heterologous; for example, preferably, the 5' UTR and the 3' UTR are derived from different genes of the same or different species. In a preferred instance, the 5' UTR element and the 3' UTR element are selected to exert at least an additive function, and preferably a synergistic function, in the production of proteins from the ORF of RNA polynucleotide molecules. Preferably, protein production is increased by the 5' UTR element and the 3' UTR element in a at least additive, preferably synergistic manner. Therefore, at some point in time after the onset of ORF expression (e.g., after transfection of test cells or cell lines), the protein quality of proteins encoded by the ORF (e.g., reporter proteins, such as luciferase) is preferably at least the same, and preferably higher than the expected protein quality if the increase in protein quality between the 5' UTR element and the 3' UTR element is purely additive. The additive effect, or preferably the synergistic effect, can be determined, for example, by luminescence analysis.

Preferably, at least one 5' UTR element and at least one 3' UTR element work together to stabilize and/or increase the protein production of the RNA polynucleotide according to the invention, such as the mRNA according to the invention, as described above. In the context of the invention, the term "stabilizing and/or increasing protein production" means that the protein production of the mRNA is stable and/or increased compared to the protein production of a reference mRNA, such as containing reference or control 5' UTR and 3' UTR elements ( e.g., Table 2 ).

RNA polynucleotides containing 5'UTR and 3'UTR RNA sequence fragments or variants thereof have been discovered, which correspond to DNA sequences selected from any of the following combinations: - 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. These are particularly suitable for enhancing expression in different cell types compared to reference RNA polynucleotides. These RNA polynucleotide molecules are particularly useful in mRNA-based therapies, gene therapies, and/or gene vaccines because they can increase and/or prolong the expression of proteins encoded by the open reading frames of the RNA polynucleotide.

The particularly preferred 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.

More specifically, these advantageous effects are particularly evident when using RNA sequences containing 5' UTR and 3' UTR, or RNA polynucleotide sequences having at least 95% sequence identity with them, the 5' UTR and 3' UTR corresponding to DNA sequences selected from any of the following combinations: - SEQ ID NO: 1 [ANXA1] of the 5' UTR and SEQ ID NO: 6 [HBA1] of the 3' UTR; or - SEQ ID NO: 2 [eno] of the 5' UTR and SEQ ID NO: 7 [RPS29] of the 3' UTR; or - SEQ ID NO: 2 [eno] of the 5' UTR and SEQ ID NO: 8 [Ube2d2a] of the 3' UTR; or - SEQ ID NO: 3 [RPS14] of the 5' UTR and SEQ ID NO: 6 [HBA1] of the 3' UTR; or - The combination of SEQ ID NO: 3 [RPS25] of 5'UTR and SEQ ID NO: 8 [Ube2d2a] of 3'UTR; or the combination of SEQ ID NO: 1 [ANXA1] of 5'UTR and SEQ ID NO: 18 [HBA1] of 3'UTR; or the combination of SEQ ID NO: 3 [RPS14] of 5'UTR and SEQ ID NO: 18 [HBA1] of 3'UTR; or the combination of SEQ ID NO: 5 [TCV] of 5'UTR and SEQ ID NO: 9 [UBL] of 3'UTR.

Particularly preferred combinations are the combination of SEQ ID NO: 1 [ANXA1] of the 5'UTR and SEQ ID NO: 6 [HBA1] of the 3'UTR; and the combination of SEQ ID NO: 3 [RPS14] of the 5'UTR and SEQ ID NO: 6 [HBA1] of the 3'UTR.

The preferred alternative combinations are the combination of SEQ ID NO: 1 [ANXA1] of the 5'UTR and SEQ ID NO: 18 [HBA1] of the 3'UTR; and the combination of SEQ ID NO: 3 [RPS14] of the 5'UTR and SEQ ID NO: 18 [HBA1] of the 3'UTR.

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: RPS14 5'UTR - HBA1 3'UTR, specifically, SEQ ID NO: 3 [RPS14] - SEQ ID NO: 18 [HBA1]; ANXA1 5'UTR - HBA1 3'UTR, specifically, SEQ ID NO: 1 [ANXA1] - SEQ ID NO: 18 [HBA1]

Particularly suitable RNA polynucleotides are those containing at least one 5' UTR element, which comprises a nucleic acid sequence derived from the 5' UTR of the Escherichia coli enolase gene or the ANXA gene.

Furthermore, the combination of at least one 5' UTR element and at least one 3' UTR element works synergistically to increase the biodistribution of RNA polynucleotides according to the invention, such as the biodistribution of mRNA according to the invention, as described above. In the context of the invention, the term "biodistribution" means the translocation and accumulation of mRNA or lipid nanoparticles containing mRNA in the body, organs, or tissues, compared to the biodistribution of a reference or control mRNA, such as mRNA containing reference 5' UTR and 3' UTR elements (see, for example, RNA polynucleotides containing control 5'UTR/3' UTRs as described in Table 2 ).

More specifically, when compared with reference RNA polynucleotide molecules, the RNA polynucleotides according to the invention exhibit more uniform protein production and biodistribution over a predetermined time period, preferably 2 hours, such as 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, preferably 12 hours, more preferably 24 hours, even more preferably 48 hours, and optimally 72 hours. Therefore, for example in mammalian systems, the level of protein produced from the RNA polynucleotides according to the invention will not decrease to the level observed with reference RNA polynucleotides (such as reference mRNA) as described above. For example, the amount of protein (encoded by ORF) observed 5 hours after the onset of expression was comparable to the amount observed 48 hours after the onset of expression (e.g., 48 hours after transfection). In particular, biodistribution was found to increase from 5 hours to 24 hours, decrease slightly after 48 and 72 hours, but remained generally higher than that of reference RNA polynucleotides.

The increase in RNA polynucleotide stability, the increase in protein production stability, the extension of protein production, the increase in protein production and/or the increase in biological distribution are preferably determined by comparison with a corresponding reference RNA polynucleotide containing reference 5' UTR and 3' UTR elements (e.g., containing a 5' UTR element corresponding to an RNA sequence selected from DNA sequences selected from SEQ ID NO: 10-13 or a 3' UTR element corresponding to an RNA sequence selected from DNA sequences selected from SEQ ID NO: 14-17 ( see Table 2 )).

In the context of this invention, "increased protein expression" can refer to an increase in protein expression at a time point after the onset of expression compared to a reference molecule, or an increase in total protein production over a period of time after the onset of expression. Therefore, the protein levels observed at a time point after the onset of expression of the RNA polynucleotide according to this invention (e.g., post-transfection), such as after transfection of the mRNA according to this invention, for example, at 2, 3, 4, 5, 7, 8, 12, 24, 48, or 72 hours post-transfection, or the total protein produced over a time span of, for example, 24, 48, or 72 hours, are preferably higher than the protein levels observed at the same time point after the onset of expression of the reference RNA polynucleotide (e.g., post-transfection), or the total protein produced over the same time span. As explained above, a particularly desirable function of the 5' UTR element is to influence the increase in protein production of RNA polynucleotides. Preferably, at a given time point after the onset of expression, compared to RNA polynucleotides containing both a reference 5' UTR and a 3' UTR, the protein production increase achieved by the 5' UTR element 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.

Furthermore, preferably, the 3' UTR element and the 5' UTR element have at least an additive and better synergistic effect on the total protein production of artificial nucleic acid molecules over a certain time span, such as 24 hours, 48 hours or 72 hours after the start of performance.

Therefore, this invention also relates to the use of the RNA polynucleotide or pharmaceutical composition according to this invention for enhancing the expression and/or translation of gene products, particularly enhancing translation initiation. Even more specifically, it provides the use of the RNA polynucleotide or pharmaceutical composition to enhance the stability and/or duration of gene product expression, particularly enhancing biological distribution.

According to another specific example of the present invention, the 5'UTR of the RNA polynucleotide further includes 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 acts as a protein transcription start site and mediates ribosome assembly to ensure the correct transcription of a protein sequence. In the context of the present invention, the term "internal ribosome entry site (IRES)" should be understood as a sequence or motif consisting of several open reading frames, for example, if the RNA polynucleotide encodes two or more peptides or proteins. The IRES sequence may be particularly useful if the mRNA is bicistronic or polycistronic.

Furthermore, the RNA polynucleotide may further include additional elements known to those in the art in mRNA production, such as a 5' cap and a poly(A) tail. The 5' cap, if present, is preferably linked to the 5' side of a 5' UTR element. The 5' cap may be added to the 5' end of the RNA after transcription. Preferably, the poly(A) sequence, if present, is located at the 3' end of the ORF or at least one 3' UTR element, and is preferably linked to the 3' end of the ORF or 3' UTR element. The linking may be direct or indirect, for example, via a linker of 2, 4, 6, 8, 10, 20 nucleotides, such as via a linker of 1-50, preferably 1-20 nucleotides, for example, containing one or more restriction sites or consisting of one or more restriction sites. In one specific example, the poly(A) signal, if present, is located within the 3' UTR element. The length of the poly(A) sequence can vary. For example, the length of the poly(A) sequence can be from about 20 adenine nucleotides to about 300 adenine nucleotides, preferably about 40 to about 200 adenine nucleotides, more preferably about 50 to about 100 adenine nucleotides, such as about 60, 70, 80, 90, or 100 adenine nucleotides.

Furthermore, the RNA polynucleotides according to this invention may contain one or more typical mRNA modifications, referred to herein as "modified mRNA nucleosides," which should be understood as containing one or more modified nucleosides that possess useful properties, such as a lack of substantial induction of the innate immune response to the introduced mRNA in cells. These modified nucleic acids enhance the efficiency of protein production, the intracellular retention of nucleic acids and the viability of contacting cells, and have reduced immunogenicity.

In some specific examples, the modified nucleotides in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) include 1-methyl-pseudouridine (m1y), 1-ethyl-pseudouridine (e1\|/), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine (y). In some specific examples, the modified nucleotides in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) include 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine, and/or 5-methoxycytidine. In some specific examples, the polynucleotides include combinations of at least two (e.g., 2, 3, 4, or more) of any of the aforementioned modified nucleotides, including but not limited to chemical modifications.

In another specific example, the invention relates to a pharmaceutical composition comprising one or more RNA polynucleotides according to the invention and at least one pharmaceutically acceptable agent.

In the context of this invention, the term "pharmaceutical composition" refers to a composition having pharmaceutical properties. In other words, it refers to a composition that provides pharmacological and/or physiological effects. A pharmaceutical composition may comprise one or more pharmaceutically acceptable reagents, such as excipients, carriers, and diluents. It may be a solution, suspension, liquid, or aqueous formulation, or any combination thereof. In some specific examples, pharmaceutically acceptable reagents include, but are not limited to, biocompatible mediators, adjuvants, additives, and diluents to obtain a composition usable as a dosage form. Additional suitable pharmaceutical carriers and diluents, as well as the pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences. As used herein and unless otherwise specified, the term "excipient" shall be understood to mean any substance formulated together with an active compound for the purpose of long-term stability, such as preventing denaturation or aggregation within the expected shelf life, increasing the volume of a liquid or solid formulation containing a small amount of the active compound (therefore commonly referred to as a "building agent," "filler," or "thinner"), or enhancing the active compound in the final formulation, such as promoting absorption, reducing viscosity, or increasing solubility.

In another specific example, the present invention provides RNA peptides formulated in liposomes or nanoparticles, such as lipid nanoparticles or polymeric nanoparticles; particularly lipid nanoparticles. RNA peptides as defined herein can be formulated in lipid nanoparticles (LNPs) that encapsulate the structure to protect it from degradation and promote cellular uptake. In the context of the present invention, the term "lipid nanoparticle" or LNP refers to nanoscale particles composed of one or more lipids (e.g., combinations of different lipids). Possible lipids used in LNPs may be, for example, but not limited to, at least one phospholipid, at least one modified lipid (such as PEGylated lipids), at least one ionizable lipid, or at least one sterol. The lipid nanoparticles and their components disclosed herein are generally known in the art.

In particular, as defined herein, the term "pharmaceutical formulation" is used especially in the context of an LNP comprising a combination of the RNA polynucleotide of the present invention and at least one pharmaceutically acceptable carrier or adjuvant. On the other hand, the term "pharmaceutical formulation" may also refer to the naked RNA polynucleotide of the present invention suspended in a buffer solution and at least one pharmaceutically acceptable carrier or adjuvant (i.e., not encapsulated within an LNP). This combination may, for example, be formulated into an LNP and is particularly intended to prolong nucleic acid stability and/or improve delivery.

LNPs may contain one or more single-type RNA polynucleotides (i.e., RNA polynucleotides with the same 5' UTR, 3' UTR combination), or may contain two or more RNA polynucleotides with similar or different 5' UTR-3' UTR combinations. In some specific examples, two or more different RNA polynucleotides are formulated in the same lipid nanoparticle.

In another embodiment, the invention provides RNA polynucleotides or pharmaceutical compositions according to the invention for use in human and/or veterinary medicine. Pharmaceutical compositions are particularly suitable as vaccines or for use in gene therapy. Vaccines can be preventative (e.g., preventing or mitigating the effects of future infection by any natural or "wild" pathogen) or therapeutic (e.g., actively treating or alleviating symptoms of an ongoing disease). The administration of a vaccine is called vaccination. Finally, the invention provides a method for the prevention and treatment of human and veterinary diseases by administering a pharmaceutical composition or vaccine to an individual in need.

In the context of this invention, the term "gene therapy" should be understood as the treatment of a patient's body or isolated elements of the patient's body, such as isolated tissues/cells, by means of nucleic acids encoding peptides or proteins, preferably RNA polynucleotides according to this invention. In the context of this invention, the term "genetic vaccination" can be typically understood as vaccination by administering nucleic acid molecules or fragments thereof that encode antigens or immunogens. Following transfection of certain cells in the body or transfection of isolated cells, antigens or immunogens can be expressed by those cells and subsequently presented to the immune system, inducing adaptation, i.e., an antigen-specific immune response.

Therefore, gene therapy and gene vaccine administration typically include at least one of the following steps: a) direct administration of an RNA polynucleotide or pharmaceutical composition as defined herein to an individual via any administration route, or in vitro administration of an RNA polynucleotide or pharmaceutical composition as defined herein to an individual's isolated cells/tissues, resulting in in vivo / in vitro or in vitro transfection of the individual's cells; b) transcription and/or translation of the introduced nucleic acid molecules; and, if applicable, c) re-administration of isolated, transfected cells to an individual if nucleic acids have not yet been directly administered to the individual.

In another embodiment, the invention relates to a method for inducing an enhanced immune response in an individual, comprising administering a therapeutically effective amount of an RNA polynucleotide or pharmaceutical composition according to the invention. The term "immune response" as used throughout this specification is not intended to be limited to the types of immune responses that may be exemplified herein, such as specific immune responses against disease-associated antigens or to cells expressing disease-associated antigens. Therefore, the term encompasses all infectious agents to which vaccination will be beneficial to an individual.

In the context of this invention, the term "therapeutically effective amount" should be understood as an amount sufficient to induce medicinal effects (such as immune responses), alter the pathological level of expressed peptides or proteins, or replace deficient gene products (e.g., in pathological conditions).

The present invention also provides a method for treating or preventing diseases or conditions as described above, comprising administering to an individual in need an RNA polynucleotide or a pharmaceutical composition according to the present invention.

In the context of this application, the terms “treatment,” “treating,” “treat,” and similar terms refer to the attainment of desired pharmacological and/or physiological effects. Such effects may be preventative or therapeutic in order to completely or partially prevent a disease or its symptoms, and/or therapeutic in order to partially or completely stabilize or cure a disease and/or in order to address adverse effects attributable to that disease. “Treatment” encompasses any treatment of a disease in mammals (especially humans), including: (a) preventing a disease or its symptoms from occurring in individuals who may be susceptible to the disease or its symptoms but have not yet been diagnosed with it; (b) suppressing disease symptoms, i.e., halting their development; or (c) alleviating disease symptoms, i.e., resolving the disease or its symptoms.

The RNA polynucleotides or pharmaceutical compositions according to this invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via implantable devices. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrasheath, intrahepatic, intralesional, intracranial, percutaneous, intradermal, intrapulmonary, intraperitoneal, intracardiac, intraarterial, and sublingual injection or infusion techniques. Example 1 : In vitro screening of materials and methods for iMoDC differentiation and transfection

Using positively selected CD14+ beads from two different donors (StemCell Technologies, EasySep Human CD14 Positive Selection Kit II, Catalog No. 17858), immature human monocytogenes-derived dendritic cells (Mo-iDCs) were differentiated from monocytogenes enriched from erythrocyte sedimentation rate (ESR) brown layer. CD14+ cells were then cultured for 7 days in X-VIVO15 medium (VWR, catalog number LONZBE02-060F) supplemented with 250 U/ml IL-4 (Miltenyi Biotec, catalog number 130-093-922), 800 U/ml granulocyte-macrophage community-stimulating factor (GM-CSF) (Miltenyi Biotec, catalog number 130-093-867), and 1% human AB serum (Merck Life Science, catalog number H4522-100ML). Fresh interferon was added twice during the differentiation period. Cells were harvested on day 7 in phosphate-buffered saline (PBS)/EDTA (Fisher Scientific, catalog number #15433589) and frozen in Cryptotor CS10 medium (StemCell Technologies, catalog number 7930). For UTR selection, cells from two donors were thawed, combined, and transfected in duplicate with 400 UTR mRNA in 96-well plates containing 100*10e3 iMoDC/well using Lipofectamine MessengerMAX transfection reagent (Thermo Fisher Scientific, catalog number LMRNA008) according to the manufacturer's instructions (100 ng/well, 100 μl, ratio 1:3). To ensure equivalent transfection efficacy across all wells, 10% of mCitrine mRNA was co-encapsulated with NanoLuc mRNA, which had different UTRs. Cell viability was monitored using CellTiter-Fluor assay (Promega, catalog number G6081). Viability and transfection efficacy (Teff) were quantified after 24 hours, with Nanoluc secretion levels tracked at 6, 24, 48, 72, and 96 hours. Subsequently, the area under the Nanoluc secretion curve was normalized relative to transfection efficacy and cell density, and the top 40 performing mRNAs (top 10%) were selected for the next round of screening in another iMoDC donor and human skeletal muscle cells. In the second round of screening, the top 40 UTRs of two other iMoDC donors were evaluated, with read counts similar to those in the first round. In the confirmatory experiment, cells were transfected with Flux, and reads were taken 24 hours post-transfection. SkMC culture, differentiation, and transfection were also performed.

Culture human skeletal muscle cells (SkMCs) according to the manufacturer's instructions (Promo Cell, catalog number C-12530). In short, passage the cells in skeletal muscle cell growth medium (Bio-connect, catalog number C-23060). For differentiation, culture the cells in skeletal muscle cell growth medium until they reach approximately 60-80% confluence. Then replace the growth medium with skeletal muscle cell differentiation medium (Bio-connect, catalog number C-23061). After culturing in skeletal muscle cell differentiation medium for 5 days, cells were transfected using 40 optimally performing UTRs prepared according to the manufacturer's instructions (50 ng/well, 1:3 ratio in 100 μL). Transfection efficacy and cell viability were monitored as usual in the first round of screening, with Nanoluc expression collected at 6, 24, 48, 72, and 96 hours. In the confirmatory experiment, cells were transfected with Fluc, and complete readouts were performed 24 hours later. Cancer cell line culture and transfection.

Four different human cell lines were cultured according to the manufacturer's instructions: HeP2G (hepatocellular carcinoma cell line (ATCC, catalog number HB-8065), K562 (leukemia cell line (ATCC, catalog number CCL-243), BJ (fibroblasts (ATCC, catalog number CRL-2522), HEK293 (human embryonic kidney immortalized cell line (ATCC, catalog number CRL-1573)) and two mouse cell lines: CT26 (colon and rectal cancer (ATCC, catalog number CRL-2638)) and TC-1 (immortalized lung epithelial cell line (a gift from Thorbald Van Hall Laboratory, Leiden University)). Lipofectamine MessengerMAX transfection reagent (Thermo Fisher Scientific) was used. Scientific (catalog number LMRNA008) transfected cells with mRNA encoding firefly luciferase (Fluc) at two different doses (100 ng vs. 200 ng/well). In the confirmatory experiments, only the 24-hour time point was evaluated. Data Analysis

In all experiments, Nanoluc values were normalized relative to transfection efficacy, and cell aggregation was also considered in some experiments. The curve areas at all time points were then evaluated using Graph Pad Prism (version 10.0.2(232)). Statistical significance was assessed using one-way ANOVA, assuming all assumptions were met, followed by a post-hoc Tukey test to correct for multiple comparisons. Standard error and degrees of freedom +1 were used for statistical evaluation in the case of AUC. Results: First round of screening (Figure 1 ).

To select combinations of 5' and 3' UTRs that achieve higher messenger RNA expression and longer intracellular persistence compared to the control UTR (see Table 2, further referred to as the control UTR), we generated an mRNA library in which 21 × 5' UTRs and 21 × 3' UTRs were combined with the coding sequence of secreted NanoLuc. The expression of NanoLuc in 441 mRNAs was monitored in human immature mononuclear globulus-derived dendritic cells, as this cell type is frequently targeted in the context of lipid nanoparticle-driven therapy, and mRNA expression is often cell type-specific.

We corrected Nanoluc performance based on transfection efficiency obtained by co-transfecting cells with mRNAs composed of 10% mCitrine mRNA. We also monitored cell viability to exclude the most toxic mRNAs, such as those observed under strong innate activation. After normalizing transfection efficiency (Teff), Nanoluc performance was integrated at 6, 24, 48, 72, and 96 hours based on curve under-integration. Subsequently, in each 96-well plate, mRNAs with AUC within the 60th percentile were selected as the best performers, resulting in a total of 40 mRNAs selected for the next round of screening and confirmation. The second round of selection in primary human cells (Figures 2A , B , and C ).

Downselective UTR was tested in primary human skeletal muscle cells (skMCs). These primary cells are associated with intramuscular immunization using lipid nanoparticles. Mature skMCs were transfected with the first 40 mRNAs at a 1:3 ratio of 50 ng to Messenger Max, in duplicate. Nanoluc secretion was assessed at 6, 24, 48, 72, and 96 hours, and transfection efficacy and viability were assessed 24 hours after transfection. Teff values were used to correct for kinetic data, and cell density was also considered. Baseline values for control 3 mRNAs were subtracted from the AUC measured by Nanoluc integration in GraphPadPrism, with all replicates averaged and the mean used for calculation. Subsequently, four best performers were selected: 1) ANXA1-secNluc-HBA1, 2) eno-secNluc-FTH1-201 and eno-secNluc-Ube2d2a, 3) eno-secNluc-TPRKB and eno-secNluc-UBL5, and 4) tabA-secNluc-Ube2d2a (Figure 2A).

Therefore, 40 downselected mRNAs were transfected into two other immature MoDC donors to compensate for the inherent high variability of different donors and to replicate the original observations. Experiments were performed exactly as in the first round of screening, with additional normalization of cell aggregation. For donor 1, the four best performers were: 1) RPS25-secNluc-Ube2d2a, 2) RPS14-secNluc-HBA1, 3) Eno-secNluc-Ube2d2a, and 4) REG1A-secNluc-Ube2d2a (Figure 2B). For donor 2, the following are in order: 1) TCV-secNluc-HBB-001, 2) HBA1-secNluc-HBA1, 3) TCV-secNluc-UBL5, 4) TCV-secNluc-Med13 and ANXA1-secNluc-HBG2 (Figure 2C).

One combination overlaps in SkMC and iMoDC, namely eno-secNluc-Ube2d2a. The HBA1 3' UTR, combined with different 5' sequences, exhibited superior mRNA expression across all donor and cell types, indicating that UTRs can enhance mRNA properties in different tissues. Therefore, combinations of ANXA1 and RPS14 5' UTRs with the HBA1 3' UTR were selected from the top lists of SkMC and iMoDC, respectively. Similarly, Ube2d2a was frequently observed in the lists and selected in combination with RPS25. For one of the iMoDC donors, the TCV 5' UTR is very common, so we retained the combination of this 5' UTR with another popular 3' UTR sequence, UBL5 (observed twice). The sixth down-rotated UTR is summarized in Table 1, and the corresponding UTRs are summarized in Table 2. Table 1. Selection of 5' and 3' UTR 5'UTR 5'UTR sequence - DNA sequence ( T = U* in the corresponding RNA sequence ) SEQ ID NO: ANXA1 (Human) AGTGTGAAATCTTCAGAGAAGAATTTCTCTTTAGTTCTTTGCAAGAAGGTAGAGATAAAGACACTTTTTCAAAA 1 Eno (Escherichia coli) TTAACTAGTGACTTGAGGAAAACCTAA 2 RPS14 (Human) GTTAACGCACTGGGTCGTGCTCATTGGTCTGATTTGAAGATAGGAACATTTAACTTCGTACACCCAAGACTTACACTTGAAAA 3 RPS25 (Human) CTTTTTGTCCGACATCTTGACGAGGCTGCGGTGTCTGCTGCTATTCTCCGAGCTTCGCA 4 TCV (Turnip Wrinkle Virus) GGTAATCTGCAAATCCCTGGCACCCGCCTAAAATTGCCCTCATCAACCTTCTCTCTATTCACG 5 3'UTR 3'UTR sequence - DNA sequence ( T = U* in the corresponding RNA sequence ) HBA1 (Human) GCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA 6 RPS29-001 (Human) ATGCTCTTCCTTCAGAGGATTATCCGGGGCATCTACTCAATGAAAAACCATGATAATTCTTTGTATATAAAATAAACATTTGAAAAAACC 7 Ube2d2a (mouse) TACTAAAAAAGATATAAAAGTAATTTTATTAAACAAGGTAGAGGCATAACCTTAAAGGCATTTTCATGAA 8 UBL5 (Human) TTGAGAATTCCTCATCTTCCTGCCCCGCTTTCCTCTCCCATCCTCATCCCCCACACTGGGATAGATGCTTGTTTGTAAAAACTCACCTTAATAAAGACTTAGATGTTG 9 HBA1 (Human) - WT GCTGGAGCCTCGGTGGCCATGCTTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA 18 * U indicates uridine or its modified form, such as pseudouridine, especially N1-methylpseudouridine. Table 2. Comparison of 5' and 3' UTRs 5'UTR 5'UTR sequence - DNA sequence ( T = U* in the corresponding RNA sequence ) SEQ ID No: Comparison 1 ETR (10×gtx 5'UTR element) CCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATT 10 Comparison 2 MDRN (artificial) AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCC 11 Compare with 3 BNT (HBA1) GAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCC 12 Comparison 4 CV (HSD17B4) GCTCATCTTCCTACCAGAAATCGGCAAGTCACTGACCCTCGTCCCGCCCCCGCCATTCCCCGCCTCCTCCTGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGCAGGCCTTATTC 13 3'UTR 3'UTR sequence - DNA sequence ( T = U* in the corresponding RNA sequence ) Comparison 1 ETR (KSHV-ENE) CTCGAGTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGTCGAC 14 Control 2 MDRN (HBA1) GCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA 15 Control 3 BNT (AES + 12S rRNA) CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCAGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACG CAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC 16 Comparison 4 CV (PSMB3+HSL) CCCTGTTCCCAGAGCCCACTTTTTTTTCTTTTTTTGAAATAAAATAGCCTGTCTTCACAAAGGCTCTTTTCAGAGCCACCA 17 * U represents uridine or its modified form, such as pseudouridine, especially N1-methylpseudouridine. Six best performers were identified using other cell lines and mRNA types ( Figure 3 ).

To evaluate novel combinations of UTRs in the context of different mRNA sequences, we synthesized an mRNA encoding the firefly luciferase (Fluc) ORF with six selected UTR combinations. Furthermore, to examine whether the cellular environment can affect mRNA expression due to UTRs, we evaluated the expression of Flux in six different human and mouse cell lines. Six different human cell lines—HeP2G (hepatocellular carcinoma), K562 (leukemia), BJ (fibroblasts), HEK293 (human embryonic kidney immortalized cell line), Mo-iDC (immature mononuclear globulus-derived dendritic cells), SkMC (skeletal muscle cells)—and two mouse cell lines—CT26 (colon and rectal cancer) and TC-1 (immature lung epithelial cells)—were transfected with novel mRNA at concentrations of 100 ng/well and 200 ng/well, respectively. After 24 hours, FLuc luminescence, mCitrine fluorescence, and viability were quantitatively measured. For viability, based on the assumption that untransfected wells had 100% viability, fluorescence was converted to a percentage of viability. To account for transfection efficiency, the expression (RLU) of Nanoluc transfected at 200 ng/well was divided by the mCitrine mRNA expression level (RFU), shown as a normalized p/s ratio in Figure 3. The novel UTR combinations showed consistently high expression levels in a wide range of cell lines containing Fluc mRNA. The top three UTR combinations were: 1) ANXA-HBA1, 2) eno-RSP29, and 3) eno-Ube2d2. Notably, TCV-UBL5 showed high expression in the HEP2G cell line, indicating that the UTRs function in a tissue-specific manner. The best-performing UTR combinations after a second round of screening in SkMC and iMoDC are listed in Table 3. Table 3. Best-performing UTR combinations ID 5'-3' UTR 1 ANXA-HBA1 2 eno-RPS29 3 eno-Ube2d2 4 RPS14-HBA1 5 RPS25-Ube2d2 6 TCV-UBL5 Example 2 : In vivo screening of materials and methods using bioluminescence imaging

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

To investigate the effects of six candidate 5'UTR-3'UTR gene combinations on in vivo RNA polynucleotide stability and/or translation efficiency, female 6-8 week old BALB/c mice were intramuscularly injected with 50 μL of TBS, one of the six candidate 5'UTR-3'UTR gene combinations containing firefly luciferase (fLuc) mRNA-LNPs, or one of the three control 5'UTR-3'UTR gene combinations containing fLuc mRNA-LNPs. Systemic bioluminescence signals following D-luciferin injection were quantified using the IVIS platform, and fLuc performance was measured at 5, 24, 48, and 72 hours post-injection. Overall, at all time points, all six hit candidate 5'UTR-3'UTR gene combinations tended to perform better or equal to the tested internal/reference 5'UTR-3'UTR gene combinations, with the top three UTR combinations being 1) eno-RSP29, 2) eno-Ube2d2, and 3) TCV-UBL5. For earlier time points, ANXA1-HBA1 performed the worst in this in vivo expression study, while the RPS25-Ube2d2a UTR combination was associated with the lowest bioluminescence signal at later time points (Figure 4). Implementation 3 : In vivo expression

In this embodiment, we evaluated the in vivo performance of novel muIL-7, muIL-21, and mu41BBL UTRs (V8 and V10) compared to the conventional UTR (V5) to examine whether there were any improved performances in MC-38. UTR V8 corresponds to RPS14 5'UTR - HBA1 3'UTR, UTR V10 corresponds to ANXA1 5'UTR - HBA1 3'UTR, and UTR V5 corresponds to the UTR disclosed in WO2015071295, as comparative examples.

Tumors were collected after 24 hours, and their in vivo appearance was evaluated using Luminex and procartaplex, allowing for the simultaneous detection of muIL-21, muIL-7, and mu41BBL. Experimental setup : • Dosage: 15 µg/mouse, injection volume 20 µl, prepared in LNP. • Readout type: Tumors were collected after 24 hours, and protein expression was measured on Luminex.

C57BL/6J mice were inoculated with 0.5*10e6 tumor cells (unilateral; left ventral side).

When the average tumor size reached 50-100 mm³ (day 10 post-inoculation), the mice were randomly divided into groups and injected with LNP to treat the tumor IT.

As is evident in Figure 5, for all three tested mRNA (muIL-7, muIL-21, and mu41BBL) constructs, UTR V8 and V10 induced significantly higher protein expression compared to the comparative UTR V5 combination. Example 4 : In vivo efficacy study

In this embodiment, we evaluated the in vivo efficacy of novel muIL-7, muIL-21, and mu41BBL UTRs (V8 and V10) compared to the conventional UTR (V5) to examine whether there was any improved performance in MC-38. Experimental setup : • Dosage: 10 µg/mouse, injection volume 20 µl, mixed in LNP • Readout type: Tumors were harvested at 24 hours and mouse survival was confirmed until 35 days post-inoculation.

C57BL/6J mice were inoculated with 0.5*10e6 tumor cells (unilateral; left ventral side).

When the average tumor size reached >75 mm³ (+/- day 11 post-inoculation), mice were randomly assigned to groups and injected with LNP to treat tumor IT. Repeat injections were given on days 3 and 7 after randomization.

As shown in Figure 6, the highest efficiency was achieved using the V10 UTR (Figure 6D) and V8 UTR (Figure 6C). Compared to these two UTR combinations of the present invention, the comparative V5 UTR (Figure 6B) performed significantly worse. TBS is used as a counterexample (Figure 6A).

without

Referring now specifically to the diagrams, it must be emphasized that the details shown are illustrative and are used only to illustrate different specific examples of the invention. Their presentation is intended to provide what is believed to be most useful and easily described for understanding the principles and conceptual nature of the invention. In this regard, no attempt is made to demonstrate the structural details of the invention in more detail than is necessary for a basic understanding of the invention. The accompanying illustrations make it apparent to those skilled in the art how the various forms of the invention can be embodied in practice.

[ Figure 1] : Flowchart of the first round of mRNA screening. The mRNA library consisted of a combination of 21 5' UTRs and 21 3' UTRs, plus secreted Nanoluc. iMoDC transfected each mRNA in duplicate with 100 ng each, and then evaluated transfection efficacy, viability, and Nanoluc performance, as shown in the small central graph. Duplicates were then merged, and the curve underside of each mRNA was calculated. The best-performing mRNAs were then selected from each plate, resulting in 40 top-performing mRNAs. C1, C2, C3, and C4 refer to control mRNA transfections.

[ Figure 2] : Second round of UTR selection . The top 40 performing mRNAs were selected from A) SkMC, B) iMoDcs donor 1 and C) iMoDcs donor 2. The selected mRNA species are indicated by bars marked with black dots, and the baseline is indicated by dashed lines, where the data is normalized relative to control 3.

[ Figure 3] : Identification of the six best-performing mRNAs encoding luciferase using eight different cell lines. The top six best-performing UTR combinations tested in BJ, iMoDC, SkMC, HEK293T, HepG2, K562, CT26, and TC-1 cell lines.

[ Figure 4] : All six major candidate 5'UTR-3'UTR gene combinations resulted in higher or equal in vivo luciferase expression compared to the tested control 5'UTR-3'UTR gene combination. Whole-body bioluminescence signals were quantified over time in BALB / c mice injected with TBS or either the candidate 5'UTR-3'UTR gene combination luciferase (fLuc) mRNA-LNP or the control 5'UTR-3'UTR gene combination fLuc mRNA-LNP.

[ Figure 5] : In vivo expression : For all three tested mRNA (muIL-7, muIL-21 and mu41BBL) constructs, UTR V8 and V10 both caused significantly higher protein expression compared to the comparative UTR V5 combination.

[ Figure 6] : In vivo efficacy : The highest efficacy was obtained using V10 UTR (Figure 6D) and V8 UTR (Figure 6C). The comparative V5 UTR (Figure 6B) performed significantly worse than the two UTR combinations of this invention. TBS was used as a counterexample (Figure 6A).

TW202535429A_113150008_SEQL.xml

Claims (15)

An RNA polynucleotide comprising i) at least one 5' untranslated 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' untranslated 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. The RNA polynucleotide of claim 1, wherein the 5'UTR of the RNA polynucleotide contains 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 it. The RNA polynucleotide of claim 1 or 2, wherein the 3'UTR of the RNA polynucleotide contains an RNA sequence corresponding to, or having at least 95% sequence identity with, 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]. The RNA polynucleotide claimed in any of claims 1 to 3, wherein the 5'UTR and 3'UTR of the RNA polynucleotide contain an RNA sequence, fragment or variant thereof corresponding to a DNA sequence selected from any of the following 5' UTR-3' UTR gene combinations: the 5'UTR of ANXA1 and the 3'UTR of HBA1; or the 5'UTR of eno and the 3'UTR of RPS29; or the 5'UTR of eno and the 3'UTR of Ube2d2a; or the 5'UTR of RPS14 and the 3'UTR of HBA1; or the 5'UTR of RPS25 and the 3'UTR of Ube2d2a; or the 5'UTR of TCV and the 3'UTR of UBL5. The RNA polynucleotide of any of claims 1 to 3, wherein the 5'UTR and 3'UTR of the RNA polynucleotide contain an RNA sequence, fragment or variant thereof corresponding to a DNA sequence selected from any of the following 5' UTR-3' UTR gene combinations: the 5'UTR of ANXA1 and the 3'UTR of HBA1; or the 5'UTR of RPS14 and the 3'UTR of HBA1. The RNA polynucleotide of any one of claims 1 to 3, wherein the 5' UTR and 3' UTR of the RNA polynucleotide comprise an RNA sequence corresponding to or having at least 95% sequence identity with a DNA sequence selected from any of the following combinations: SEQ ID NO: 1 [ANXA1] for the 5' UTR and SEQ ID NO: 6 [HBA1] for the 3' UTR; or SEQ ID NO: 2 [eno] for the 5' UTR and SEQ ID NO: 7 [RPS29] for the 3' UTR; or SEQ ID NO: 2 [eno] for the 5' UTR and SEQ ID NO: 8 [Ube2d2a] for the 3' UTR; or SEQ ID NO: 3 [RPS14] for the 5' UTR and SEQ ID NO: 6 [HBA1] for the 3' UTR; or SEQ ID NO: 3 for the 5' UTR. [RPS25] is combined with SEQ ID NO: 8 [Ube2d2a] for the 3'UTR; or SEQ ID NO: 1 [ANXA1] for the 5'UTR is combined with SEQ ID NO: 18 [HBA1] for the 3'UTR; or SEQ ID NO: 3 [RPS14] for the 5'UTR is combined with SEQ ID NO: 18 [HBA1] for the 3'UTR; or SEQ ID NO: 5 [TCV] for the 5'UTR is combined with SEQ ID NO: 9 [UBL] for the 3'UTR. The RNA polynucleotide of any one of claims 1 to 3, wherein the 5' UTR and 3' UTR of the RNA polynucleotide comprise an RNA sequence corresponding to or having at least 95% sequence identity with a DNA sequence selected from any of the following combinations: SEQ ID NO: 1 [ANXA1] for the 5' UTR and SEQ ID NO: 6 [HBA1] for the 3' UTR; or SEQ ID NO: 3 [RPS14] for the 5' UTR and SEQ ID NO: 6 [HBA1] for the 3' UTR; SEQ ID NO: 1 [ANXA1] for the 5' UTR and SEQ ID NO: 18 [HBA1] for the 3' UTR; or SEQ ID NO: 3 [RPS14] for the 5' UTR and SEQ ID NO: 18 [HBA1] for the 3' UTR. The RNA polynucleotide of any of claims 1 to 7, wherein the 5'UTR of the RNA polynucleotide further includes a KOZAK sequence and/or an internal ribosome entry address (IRES). The RNA polynucleotide of any of the requests 1 to 7, wherein the RNA polynucleotide is mRNA or non-coding RNA, preferably mRNA, antisense RNA, siRNA, sgRNA, guide RNA or CRISPR. A pharmaceutical composition comprising one or more RNA polynucleotides as described in any of claims 1 to 9 and at least one pharmaceutically acceptable reagent. An RNA polynucleotide as described in any of claims 1 to 9 or a pharmaceutical composition as described in claim 10, for use in human and/or veterinary medicine. An RNA polynucleotide as described in any of claims 1 to 9 or a pharmaceutical composition as described in claim 10, used as a vaccine or for use in gene therapy. Use of an RNA polynucleotide such as any one of claims 1 to 9 or a pharmaceutical composition such as claim 10, for enhancing the expression and/or translation of a gene product, particularly enhancing translation initiation. Use of an RNA polynucleotide as described in any of claims 1 to 9 or a pharmaceutical composition as described in claim 10, for enhancing the stability and/or duration of expression of a gene product, particularly enhancing its biological distribution. A method for inducing an enhanced immune response in an individual, comprising: administering to the individual a therapeutically effective amount of an RNA polynucleotide as described in any of claims 1 to 9 or a pharmaceutical composition as described in claim 10.